Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: (A and B) C57BL/6J mice received an intranigral injection of sterile normal saline (2 μL) or LPS(3 μg in 2 μL of saline; 1.8×10 3 endotoxin unit [EU]). At 24 h after the injection, representative confocal double-labeling fluorescence images showed active morphology of CD11b-IR microglia/macrophages (M) and reduction in nuclear HMGB1 (n-HMGB1) after LPS injection. Arrowheads show strong staining of n-HMGB1 in ramified (resting) microglia/macrophages in saline-injected SN. Arrows show faint n-HMGB1 in active microglia/macrophages with amoeboid-like morphology in LPS-injected SN. H, HMGB1 (A). Quantification of n-HMGB1 staining of the maximum intensity projection of a z stack of 30 confocal images from a brain slice taken at 1 mm step size, which displayed 3D structure in a 2D image. We measured 431 and 863 CD11b-IR microglia/macrophages (M) as well as 1,196 and 1,026 CD11b-negative and DAPI-positive non-microglia/macrophage cells (non-M) in saline- and LPS-injected SN, respectively. Fluorescence intensity of n-HMGB1 staining per cell was calculated and normalized to each respective saline-injected control. n = 3 mice for each group. *p < 0.05; unpaired two-tailed Student’s t test (B). (C) Microglia-enriched cultures were treated with LPS (15 ng/mL), NO donor SNP (20 μM), or vehicle with or without 30-min pretreatment with the iNOS inhibitor 1400W (10 μM). Immunoblotting and densitometry analysis showed the levels of HMGB1 and iNOS in whole-cell lysates and the levels of extracellular HMGB1 in the concentrated culture medium 24 h after the treatment. n = 4. (D and E) Nuclear fractionation and immunoblotting analysis showed blockage of LPS-elicited HMGB1 secretion by 1400W at 24 h after LPS treatment of BV2 microglial cells. The nuclear marker histone H3 was examined to monitor loading errors of nuclear proteins (D). Densitometry quantification of HMGB1 level (E). n = 3. (F) The level of nitrite (an indicator of NO production) in the culture medium was measured at 24 h after microglia-enriched cultures were treated with LPS, poly(I:C), or SNP with or without 1400W pretreatment for 30 min. n = 3. (G) MTT assay revealed no cytotoxicity after microglial cultures were treated with LPS, SNP, and/or 1400W for 24 h. n = 3. *p < 0.05 compared with the corresponding control. # p < 0.05 compared with LPS-treated cultures; one-way ANOVA with Sidak’s multiple comparisons (C, E, F) and one-way ANOVA with Dunnett’s multiple comparisons test (G). Ctrl, control; ns, not significant.

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Injection, Labeling, Fluorescence, Staining, Slice Preparation, Two Tailed Test, Western Blot, Fractionation, Marker, MTT Assay

Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: Mac1 −/− and WT (C57BL/6) mice received an intranigral injection of endotoxin-free HMGB1 (2 μg) or BSA (2 μg; as a control). One month later, PD-like phenotypes were examined. (A and B) HMGB1-elicited nigral microglia/macrophage activation was shown by elevated immunoreactivity of Iba1, CD11b, and TMEM119; enlarged size; and irregular shape in WT mice but not Mac1 −/− mice. Decreased number of TH-IR neurons and damaged integrity of TH-IR fibers indicated HMGB1-elicited dopaminergic neurodegeneration only in WT mice (A). Eight and four evenly spaced brain sections from a series of 24 sections that covered the entire SN were used for the count of TH-IR neurons and the measurement of the optical density of Iba1 immunoreactivity in nigral microglia/macrophages, respectively, by two individuals blind to the treatment (B). n = 3 mice for each group. (C) The rotarod behavior test showed HMGB1-elicited impairment of locomotor activity in WT mice but not Mac1 −/− mice. n = 5 mice for each group. (D) The open field test did not show alteration in exploratory activity or anxiety-related activities in HMGB1- or BSA-injected WT or Mac1−/− mice. n = 5 mice for each group. *p < 0.05 compared with BSA-injected controls; two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Injection, Activation Assay, Activity Assay

Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Recombinant, Protease Inhibitor, Western Blot, Transfection, Modification, Plasmid Preparation, Clone Assay, Mutagenesis, Software

Journal: Science translational medicine

Article Title: RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration

doi: 10.1126/scitranslmed.abq6225

Figure Lengend Snippet: (A) RNF41 expression in CD11b+ macrophages isolated from the livers of patients with liver cirrhosis (n = 12) and healthy participants (n = 8). (B) USP8 expression in CD11b+ macrophages isolated from the livers of patients with liver cirrhosis (n = 12) and healthy participants (n = 8). (C) RNF41 expression in CD11b+-macrophages isolated from the livers of healthy and fibrotic mice (n = 6 per group). (D) USP8 expression in CD11b+-macrophages isolated from the livers of healthy and fibrotic mice (n = 6 per group). (E) RNF41 expression in THP-1 macrophages stimulated with TNF-α for 7 days. (F) RNF41 expression in RAW 264.7 macrophages stimulated with TNF-α for 7 days. (G) RNF41 expression in freshly isolated primary hepatic CD11b+ macrophages stimulated with TNF-α for 3 days. (H) USP8 expression in THP-1 macrophages stimulated with TNF-α for 7 days. (I) USP8 expression in RAW 264.7 macrophages stimulated with TNF-α for 7 days. (J) USP8 expression in freshly isolated primary hepatic CD11b+ macrophages stimulated with TNF-α for 3 days. Experiments in (E) and (F) to (J) were performed in triplicates in two independent experiments. (K) Western blot analysis of phospho-Akt, total Akt, phospho-Erk, total Erk, and β-actin in RAW 264.7 macrophages stimulated with TNF-α for 7 days and relative protein abundance (%) of phospho-Akt and phospho-Erk relative to β-actin (n = 3). For (A) to (D), Student’s t test. For (E) to (J), versus day 0 using Student’s t test with Benjamini-Hochberg correction for multiple comparisons. For (K), comparison between pAKT protein abundance and pERK protein abundance in each time point using Student’s t test. Data are shown as means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. ns, not significant.

Article Snippet: Then, the remaining eluted fraction was incubated with CD11b magnetic beads (Miltenyi Biotec, reference: 130-049-601), and MACS-purified CD11b + macrophages were resuspended in TRIzol (Gibco-Invitrogen) for total RNA extraction or in Dulbecco’s modified Eagle’s medium supplemented with FBS (1%) for prolonged inflammation assays.

Techniques: Expressing, Isolation, Western Blot, Quantitative Proteomics, Comparison

Journal: Science translational medicine

Article Title: RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration

doi: 10.1126/scitranslmed.abq6225

Figure Lengend Snippet: (A) Schematic figure illustrating the time points of fibrosis induction with CCl4 and the administration schedule of dendrimer-graphite nanoparticles linked to plasmid pRNF41 (pRNF41-DGNPs) or scrambled pSCR (pSCR-DGNPs). (B) Relative fluorescence units (RFU) of EGFP per milligram of protein of liver isolated CD11b+ macrophages, hepatocytes, hepatic stellate cells, and liver endothelial cells from mice treated with pSCR-DGNPs (n = 6). (C) Immunofluorescence (IF) staining for Ly6C and simultaneous detection of EGFP fluorescence in the livers of fibrotic mice treated with pSCR-DGNPs. (D) High-power image for Ly6C and simultaneous detection of EGFP fluorescence in the livers of fibrotic mice treated with pSCR-DGNPs. (E) IF staining for CD206 and simultaneous detection of EGFP fluorescence in the livers of fibrotic mice treated with pSCR-DGNPs. (F) High-power image for CD206 and simultaneous detection of EGFP fluorescence in the liver of fibrotic mice treated with pSCR-DGNPs. (G) IF staining for CD206 and simultaneous detection of EGFP fluorescence in the livers of fibrotic mice treated with pRNF41-DGNPs. (H) High-power image for CD206 and simultaneous detection of EGFP fluorescence in the livers of fibrotic mice treated with pRNF41-DGNPs. (I) EGFP fluorescence in the kidneys of fibrotic animals treated with pSCR-DGNPs. (J) EGFP fluorescence in the spleens of fibrotic animals treated with pSCR-DGNPs. (K) EGFP fluorescence in the lungs of fibrotic mice treated with pSCR-DGNPs. All plasmids constitutively expressed EGFP under the control of a CMV promoter. Scale bars, 200 μm (C, E, G, I, J, and K) and 20 μm (D, F, and H). For (B), ****P ≤ 0.0001 versus CD11b+ macrophages using a one-way analysis of variance (ANOVA) with the posthoc Newman-Keuls test. i.p., intraperitoneal; i.v., intravenous.

Article Snippet: Then, the remaining eluted fraction was incubated with CD11b magnetic beads (Miltenyi Biotec, reference: 130-049-601), and MACS-purified CD11b + macrophages were resuspended in TRIzol (Gibco-Invitrogen) for total RNA extraction or in Dulbecco’s modified Eagle’s medium supplemented with FBS (1%) for prolonged inflammation assays.

Techniques: Plasmid Preparation, Fluorescence, Isolation, Immunofluorescence, Staining, Control

Journal: Science translational medicine

Article Title: RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration

doi: 10.1126/scitranslmed.abq6225

Figure Lengend Snippet: (A) RNF41 abundance in healthy and fibrotic mice treated with dendrimer-graphite nanoparticles linked to plasmid pRNF41 (pRNF41-DGNPs) or scrambled pSCR (pSCR-DGNPs). (B) Macroscopic aspect of fibrotic liver after treatment with pRNF41-DGNPs. (C to G) Sirius Red staining and quantification of liver fibrosis area (C), hydroxyproline measurements (D), serum liver injury parameters [ALT (alanine aminotransferase), AST (aspartate aminotransferase), serum albumin, and serum total protein] (E), hepatic PCNA immunofluorescence staining (F), and hepatic expression of HGFand IGF-1 (G) in fibrotic mice treated with pSCR-DGNPs or pRNF41-DGNPs. (H) Cell proliferation in isolated mouse hepatocytes treated for 24 hours with conditioned medium from RAW 264.7 cultures treated with FBS, TNF-α, pSCR-DGNPs, pRNF41-DGNPs, or IGF-1 antibody for 3 days. Experiments were performed in triplicates in two independent experiments. (I) Expression of proinflammatory and anti-inflammatory genes in liver tissue and in CD11b+ macrophages isolated from the livers of fibrotic mice treated with pSCR-DGNPs or pRNF41-DGNPs. n = 6 animals per group. Student’s t test was used for (A), (C) to (G), and (I), and a one-way ANOVA with posthoc Newman-Keuls test was used for (H). Data are shown as means ± SD. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Article Snippet: Then, the remaining eluted fraction was incubated with CD11b magnetic beads (Miltenyi Biotec, reference: 130-049-601), and MACS-purified CD11b + macrophages were resuspended in TRIzol (Gibco-Invitrogen) for total RNA extraction or in Dulbecco’s modified Eagle’s medium supplemented with FBS (1%) for prolonged inflammation assays.

Techniques: Plasmid Preparation, Staining, Immunofluorescence, Expressing, Isolation

Journal: bioRxiv

Article Title: Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease

doi: 10.1101/802694

Figure Lengend Snippet: A Workflow summarizing isolation and purification of CD11b + brain immune cells from 4-month-old male C57Bl6/J wild-type mice ( N = 10). Following mechanical dissociation of fresh, whole mouse brains and percoll density centrifugation, mononuclear cells were enriched for CD11b + microglia cells by magnetic activating cell sorting (MACS, N = 5 mice) or isolated via fluorescent activated cell sorting (FACS, N = 5 mice). Representative flow cytometry gating strategy and antibody separation using CD11b-APC/Cy7 antibody for isolation of microglia. B Proteomic workflow for tandem mass tag (TMT) mass spectrometry (MS) based quantification. All 10 microglia samples were lysed in 8M urea, digested with LysC and Trypsin, and peptides were labeled using one 10-plex TMT kit. A total of 5 individual MACS-enriched microglia samples were dedicated to the first five channels (126, 127N, 127C, 128N, 128C) and 5 individual FACS-isolated microglia samples were dedicated to the last five channels (129N, 129C, 130N, 130C, 131). After labeling, the samples were combined and fractionated by off-line high pH fractionation ( n = 9 fractions [fx]). Each fraction was analyzed and quantified by synchronous precursor selection (SPS)-MS3 on an Orbitrap Fusion mass spectrometer. Peptide search on raw files were conducted with Proteome Discoverer (v2.1) in order to obtain the MACS microglia proteome and FACS microglia proteome.

Article Snippet: The cell pellet was washed with DMEM/10% FBS followed by CD11b positive selection (Miltenyi Biotec, Cat. No. 130–093-636) using mini-MACS (Miltenyi Biotec, Cat. No. 130-042-201) columns.

Techniques: Isolation, Purification, Centrifugation, FACS, Flow Cytometry, Mass Spectrometry, Labeling, Fractionation, Selection