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Biomol GmbH histone h4
Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Histone H4, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

2) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

3) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

4) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

5) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

6) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

7) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

8) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

9) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

10) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

11) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

12) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

13) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

14) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

15) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

16) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

17) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

18) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

19) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

20) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

21) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

22) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

23) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

24) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

25) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

26) Product Images from "New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma"

Article Title: New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2008.00271.x

Effect of ACS33 on prostate cancer xenografts. PC3 xenografts were established in male athymic mice. Animals in the treatment arm received 20 mg/kg bw ACS33 each day, or 2 mg cisplatin/kg/day (cis-pt). *Indicates significant difference to the control animals. Western blot analysis of H3 and H4 histone expression (total and acetylated), of bcl-2, bax, ILK and Fak (total and activated) was carried out on the tissue specimens using specific antibodies as listed in materials and methods (Fig. 6 , right). β-actin served as the internal control. One representative western blot data of three are shown.
Figure Legend Snippet: Effect of ACS33 on prostate cancer xenografts. PC3 xenografts were established in male athymic mice. Animals in the treatment arm received 20 mg/kg bw ACS33 each day, or 2 mg cisplatin/kg/day (cis-pt). *Indicates significant difference to the control animals. Western blot analysis of H3 and H4 histone expression (total and acetylated), of bcl-2, bax, ILK and Fak (total and activated) was carried out on the tissue specimens using specific antibodies as listed in materials and methods (Fig. 6 , right). β-actin served as the internal control. One representative western blot data of three are shown.

Techniques Used: Mouse Assay, Western Blot, Expressing

Western blot analysis of H3 and H4 histone expression (total and acetylated) in ACS-treated- and non-treated cells. DU-145 or PC3 cells were incubated with ACS2 or ACS33 for 24 hrs. Cell lysates were then analysed by specific antibodies as listed in materials and methods. β-actin served as the internal control. One representative experiment of three is shown.
Figure Legend Snippet: Western blot analysis of H3 and H4 histone expression (total and acetylated) in ACS-treated- and non-treated cells. DU-145 or PC3 cells were incubated with ACS2 or ACS33 for 24 hrs. Cell lysates were then analysed by specific antibodies as listed in materials and methods. β-actin served as the internal control. One representative experiment of three is shown.

Techniques Used: Western Blot, Expressing, Incubation

27) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

28) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

29) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

30) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

31) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

32) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

33) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

34) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

35) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

36) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

37) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Journal: Nature

doi: 10.1038/s41586-019-1167-6

Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

Techniques Used:

Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

Techniques Used: Neutralization, Mouse Assay

NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

Techniques Used: Derivative Assay, Lysis

Membrane-pore-forming activity of histone H4.
Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

Techniques Used: Activity Assay

Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant