Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Antihuman Fractalkine Reagents Used in this Study

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Control

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: Samples of Western lysates from WT CHO-K1 and CHO-K1 cells transfected with a human fractalkine expression vector 1 along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R&D Systems) (lanes 1–3) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd (lane 2, asterisk). These two bands are also detected by the chicken α-C-pep reagent (lane 5, asterisk). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant (lane 3, 85 to 90 kd), whereas the chicken α-C-pep does not (lane 6). Furthermore, the goat α-Fkn detects one larger (lane 2, 100 kd) and two smaller bands (lane 2, 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep (lane 5). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG1 control as a control for C. C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F. E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I. H: 3T3-Fkn stained with rabbit α-C-peptide. I: 3T3-Fkn stained with rabbit α-N-pep polyclonal reagent. 1 Note that although there is light nonspecific staining of the nucleus within the control sections (B, D, and F) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Membrane, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunohistochemistry, Staining, Control, Immunofluorescence

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R&D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 (A–D). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG1 mAb (Serotech), ii) goat α-Fkn polyclonal or 10% goat serum, iii) α-C-pep or rabbit IgG, iv) α-N-pep polyclonal 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Staining, Immunofluorescence, Labeling, Control, Incubation, Recombinant, Cell Culture, Reverse Transcription, Amplification, Expressing, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: Chemokine profile of the control and LPS-induced Raw264.7 EVs. (A) The chemokine profile of EVs from control and LPS-induced Raw264.7 cells. (B) The CXCL2 level (mean ± SD) in the control-Raw-EV and LPS-Raw-EV groups. The experiments were repeated three times. **p < 0.01 versus the control-Raw264.7-EV group. (C) PKH67-labeled control-Raw264.7-EVs and LPS-Raw264.7-EVs were injected into wild-type C57BL/6 mice (n = 6 per group) via the tail vein. The colocalization of PKH67-EVs and CXCL2 in the liver was detected by immunofluorescence (original magnification × 200). (D and E) GO analysis and KEGG analysis of various chemokines expressed in EVs from control and LPS-induced Raw264.7 cells.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: Control, Labeling, Injection, Immunofluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: LPS-induced CXCL2 of Raw264.7 EVs attracts neutrophils in vitro and in vivo and activates neutrophils via the CXCR2/PKC/NOX4 pathway. (A) The CXCL2 level (mean ± SD) of Raw264.7 in the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups detected by Western blotting (WB). The experiments were repeated three times. *p < 0.01 versus the control group, &p < 0.01 versus the LPS group, #p < 0.01 versus the LPS group. (B) The CXCL2 level (mean ± SD) in Raw264.7 EVs of the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups detected by WB. The experiments were repeated three times. (C) Raw264.7 EVs derived from the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups induced BMDN chemotaxis (mean ± SD) (n = 5 fields per group). (D) The expression of CXCR2, PKC, and NOX4 (mean ± SD) of BMDNs in the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EVs groups was detected by WB. The experiments were repeated three times. (E) H&E staining, MPO+ cells, and Ly-6G+ cells (mean ± SD) in the liver of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice (n = 6 per group) detected by immunohistochemistry. Scale bars, 100 μm. (F–H) ALT, AST, and LDH levels (mean ± SD) in the serum of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice (n = 6 per group). (I) The number of systemic circulating neutrophils (mean ± SD) in wild-type C57BL/6 mice treated with control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EVs (n = 3 per group). (J) The expression of CXCR2, PKC, and NOX4 (mean ± SD) in the liver of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice was detected by WB. The experiments were repeated three times. *p < 0.01 versus the control-Raw264.7-EV group, &p < 0.01 versus the LPS-Raw264.7-EV group, #p < 0.01 versus the LPS-Raw264.7-EV group.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: In Vitro, In Vivo, Control, Western Blot, Derivative Assay, Chemotaxis Assay, Expressing, Staining, Immunohistochemistry

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: EVs from the serum of patients with sepsis attract neutrophils and activate the neutrophil CXCR2/PKC/NOX4 pathway in vitro. (A) The chemokine profile of EVs from the serum of healthy individuals and patients with sepsis. (B–G) CXCL2, CXCL7, CXCL17, CCL2, CCL3, and CCL5 levels (mean ± SD) in EVs from the serum of healthy individuals (n = 8) and patients with sepsis (n = 26) detected using ELISA. *p < 0.05 versus the healthy-EV group, ***p < 0.01 versus the healthy-EV group. (H) sEVs derived from healthy control (HC) and patients with sepsis-induced PMN chemotaxis (mean ± SD) (n = 5 fields per group). (I) The expression of CXCR2, PKC, and NOX4 (mean ± SD) in the PMNs of the HC-sEV– and sepsis-sEV–treated groups was detected by Western blotting. The experiments were repeated three times. **p < 0.01 versus the healthy-EV group, #p < 0.05 versus the healthy-EV group, &p < 0.05 versus the healthy-EV group.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Chemotaxis Assay, Expressing, Western Blot

Journal: Protein & Cell

Article Title: Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit

doi: 10.1093/procel/pwae013

Figure Lengend Snippet: IFN-β responsive MICFs recruit macrophages via a positive feedback loop. (A) Violin diagrams showing the expression of Ccl2 / 7 / 12 in cardiac fibroblasts under sham or injury condition. (B) Quantification of migration ratio from the monocytes/macrophages transwell migration assay with various chemokines, n = 3. (C–E) Experimental workflow (C) and representative IF images for CD68 and mScarlet-H2B on in vivo MICFs knockdown experiment (D), with quantification of CD68 + macrophages from in vivo MICFs knockdown experiment (E), n = 3. (F) RT-qPCR analyses for Ccl2 / Ccl7 / Ccl12 genes expression at 3 days after treatment of 25 IU/mL IFN-β or BSA in MICFs, n = 3. (G) qPCR-based quantification of Ccl2 / Ccl7 / Ccl12 in Ifnar2 KD MICFs compared with the sh NT group, n = 3. (H) Dual luciferase assay in MICFs with indicated promoters, n = 4. (I) Representative IF images and density plot images of BMDMs co-cultured with MICFs expressing sh Ifnar2 or sh NT , n = 3. (J) Quantification of the BMDMs transwell migration assay using MICFs expressing sh Ifnar2 or the sh NT, n = 3. (K) Representative IF images for CD68 and mScarlet-H2B on in vivo MICFs knockdown experiment, n = 3. (L) Quantification of CD68 + macrophages from in vivo MICFs knockdown experiment. (M) Schematic diagram of the self-stimulating positive feedback loop between MICFs and macrophages. All data are presented as the means ± SD. The unpaired t -test (B, E, F, G, J and L) or one-way ANOVA (B and H) was used to determine the significance of differences between two groups. ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Scale bars, 100 μm (F = 200 μm). See also .

Article Snippet: To test the ability of secreted chemokines to promote monocyte migration, CCL2, CCL7, and CCL12 (MCE) at concentrations of 0, 0.5, 1, and 2 μg/mL were added to the culture medium to induce monocyte migration, the migrated monocytes were quantitatively analyzed in 24 h. In wound healing assay, MICFs infected with EGFP and sh Ifnar2 / NT and BMDMs isolated from Rosa26 -tdTomato +/+ mice were seeded in culture-inserts (ibidi, #81167) at a density of 50,000 cells per well in the presence or absence of Dox (2 μg/mL).

Techniques: Expressing, Migration, Transwell Migration Assay, In Vivo, Knockdown, Quantitative RT-PCR, Luciferase, Cell Culture

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Mural cells sustain a vascular MΦ niche (A) In vivo multi-photon imaging of Ca 2+ signal and morphological changes of MΦs in Cx3cr1-MΦ Ca-rep mice in an environment of laser-induced microinjuries. MΦs are depicted in red; Ca 2+ signal and vascular flow are depicted in green. MΦs have been rendered additionally below, with a pseudocolored depiction of the Ca 2+ signal. Images are derived from . (B) In vivo and ex vivo confocal and airy-scan imaging of MC-MΦ contacts across organs in MC RFP-rep ; Cx3cr1-MΦ GFP-rep mice, arrows depicting cell-cell contacts: top left: intravital imaging of the microvasculature in the mesentery, the dashed line is depicting MCs (scale bars, 5 μm); top middle: ex vivo imaging of the heart microvasculature (scale bars, 50 μm); top right: en face ex vivo imaging of the aortic atherosclerotic intima after 3 months of western-diet feeding (macrovasculature), the dashed line is subdividing the plaque core from the shoulder region (scale bars, 10 μm); bottom left: ex vivo imaging of the kidney microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 7 μm); bottom middle: ex vivo imaging of the lung microvasculature (scale bars, 20 μm), including higher magnification below (Cx3cr1 hi CD68 lo interstitial MΦs (iMΦs) in green, CD68 hi Cx3cr1 lo alveolar MΦs (aMΦs) in white, MCs in red) (dashed line depicting MCs) (scale bars, 5 μm); bottom right: ex vivo imaging of the stomach microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 10 μm). Interstitial MΦs are shown in green, and MCs are shown in red for all organs, with further subdifferentiation of MΦs in the lung (as depicted above). (C) Analysis of the time until MΦs form their first dendrites (left) and time which MΦs require to reach injury (right), as the time in minutes after laser injury, in MΦ GFP-rep mice treated locally (subcutaneously) and systemically with isotype or CCL2-neutralizing antibody (n = 17–37 individual cells analyzed from 3 to –4 mice/group). (D) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . Uniform Manifold Approximation and Projection (UMAP) based dimensionality reduction of analyzed cells. (E) Highly expressed cytokines and chemokines in human SMCs from coronary arteries analyzed from cells shown in (D) CCL2 is highlighted as the most prominently expressed chemokine. (F) Percentage of peritoneal macrophage survival upon CCL2 stimulation at different time points under starvation stress conditions (n = 3 experiments). (G) Quantification of CD68 + perivascular macrophage content in Ccl2 MC+/+ and Ccl2 MCΔ/Δ mice in percentage of total perivascular area (15 μm radius around the vessel) in the kidney (n = 5–6 mice/group). (H) Quantification of cell proliferation as EdU + cells relative to CD68 + area (as number of proliferating cells/μm 2 ). (I) Quantification of blood monocyte counts by automated blood counter (n = 5–6). (J) Representative images from immunofluorescence staining of kidney sections in Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice for ACTA2 (red), CD68 (green). Scale bars, 50 μm (left: Ccl2 MC+/+ ; right: Ccl2 MCΔ/Δ ). (C, G, H, and I) Student’s t test was used. (F) Repeated measures two-way ANOVA was -808990139890500used. ∗ p < 0.05. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: In Vivo, Imaging, Derivative Assay, Ex Vivo, Western Blot, Single Cell, RNA Sequencing, Immunofluorescence, Staining

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: MC-derived CCL2 sustains a homeostatic MΦ phenotype across the vascular tree (A and B) UMAP based dimensionality reduction of single-cell RNA-seq of FACS-sort enriched CD45 + CD11b hi CD64 hi F4/80 hi cells in kidney (A) and lung (B) of Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice (n = 4/group). (C–G) Volcano and violin plots depicting selected significantly differentially regulated genes in (C) kidney resident MΦ cluster 2, (D) kidney resident MΦ cluster 0, (E) lung monocyte cluster 1, (F) lung alveolar MΦ cluster 2, (G) lung Folr2 hi Mrc1 hi interstitial MΦ cluster 3. (H) Frequency of lung Zeb2 hi interstitial MΦ cluster 4 cells among all analyzed cells. (I) Significantly differentially regulated genes, associated with a functionally differentiated, efferocytotic MΦ phenotype in Ccl2 MC+/+ and Ccl2 MCΔ/Δ chimera mice. Low-input RNA-seq of FACS-sorted Cx3cr1 + MΦs from Ccl2 MC+/+ or Ccl2 MCΔ/Δ chimera mice with MC RFP-ep ; Cx3cr1-MΦ GFP-rep bone marrow after 20 weeks western diet (experimental setup further depicted in Figure S3 ) (n = 3–4 chimera mice). Student’s t test was used. Expression levels of depicted genes normalized to sample with highest expression (set as 1) across all samples. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression. ∗ p < 0.05.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Derivative Assay, Single Cell, RNA Sequencing, Western Blot, Expressing

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Distinct chemotactic SMCs express high levels of MΦ chemoattractants, ameliorating atheroprogression (A–C) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . (A) Violin plots (calculated on all cells expressing detectable baseline levels of the respective gene) of highly expressed cytokines and chemokines in chemotactic SMCs. Dots represent single cells, only cells exhibiting detectable expression of the particular gene are included (B) interactome depicting cell-cell interactions between MΦ and SMC subsets, prominent SMC → MΦ interactions are depicted in red. Intensity of red color depicts the respective portion of the CCL2-CCR2 axis for the concrete interaction (the darker the red color, the more the CCL2-CCR2 axis accounts for the respective inter-cluster interplay among all detected chemokine-receptor interactions). (C) Heatmap further unraveling SMC → MΦ chemokine:chemokine-receptor interactions. Blue box depicts interactions of chemotactic SMC subset, red box depicts CCL2-mediated interactions between SMC and MΦ subsets. (D) Ccl2 and Mif expression in Ng2 + SMCs FACS-sorted from western-diet fed atherosclerotic MC RFP-rep mice compared to chow-diet fed non-atherosclerotic control mice. n = 3–4 mice per group. (E) Representative images of BCA sections from Ccl2 SMC +/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet stained for ACTA2 (green), LGALS3 (red), and Hoechst (blue). Scale bars, 100 μm. (F and G) Morphometric analysis of plaque size (F) and vascular remodeling (G) from BCA sections at three consecutive locations from Ccl2 SMC +/+ (n = 11) and Ccl2 SMCΔ/Δ (n = 10) littermates. (H and I) Quantification of ACTA2 + smooth muscle cell content as ACTA2 + area in percentage of total plaque area and percentage of 30 μm plaque surface area in valves (H) and in the BCA at three consecutive locations (I). (H and I) n = 10–11 mice per group. (J and K) Analysis of intimal LGALS3 + area as percentage of plaque size in BCA sections at three consecutive locations (J) and in plaques from aortic valves (K) (n = 10–11 each). (L) Schematic illustration of media and intima processing from aortae of Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet (left). Heatmap displaying expression of differentially regulated genes in bulk RNA-seq of Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4). Rows represent individual replicates, differentially expressed genes are illustrated in columns (right). (M) Volcano plots of intima/media RNA-seq showing differentially expressed genes in Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4), x-axis depicts Log2FC, y-axis depicts -Log10(adj. p-value). Data are shown as mean and SEM. (H and K), Student’s t test was used. (F, G, I, and J) Repeated measures two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; NS, not significant. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Control, Staining

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: SMCs within the fibrous cap preserve a strategic positioning of plaque MΦs and secure homeostatic MΦ functions (A and B) Reanalyzed single-cell RNA-seq data from mouse aortic roots from atherosclerotic SMC lin mice from Wirka et al., GEO: GSE131780 . (A) UMAP based dimensionality reduction of analyzed cells (left), heatmap illustrating cytokine and chemokine expression of different SMC subsets (right). (B) Marker genes of SMC clusters illustrated in a heatmap, composed by ClustVis. (C) Representative confocal image depicting the spatial distribution of the key cSMC marker PDGFRβ within an atherosclerotic valve in SMC lin ; Cx3cr1-MΦ GFP-rep mice after 22–24 weeks of western diet, SMC lin cells in red, MΦs in green, and PDGFRβ in white. Scale bars: 30 μm (left) and 15 μm (right images). (D) Illustration of the experimental setup of the migration assay: macrophages undergo a migratory decision either moving toward the artificially composed SMC-rich fibrous cap below or residing at the artificially composed, necrotic cell rich, necrotic core. SMCs (representing the fibrous cap) are located in the lower chamber, whereas peritoneal macrophages have been attached on the transwell of the upper chamber. Necrotic Jurkat cells (representing the necrotic core) have been added to the upper chamber. (E) Number of peritoneal MΦs from Lyz-MΦ GFP-rep mice that transmigrated toward the lower chamber per field of view (FOV). Isotype or anti-CCL2 blocking antibody was simultaneously added to the lower chamber. MΦ numbers per FOV counted at 4 subsequent time points (n = 4 independent experiments). (F) Distribution of macrophages as percentage of LGALS3 + area in 30 μm plaque surface area in percentage of total plaque LGALS3 + area at three subsequent locations (n = 10 each). (G) Left: quantification of LGALS3 + surface macrophage content as relative LGALS3 + area in percentage of total plaque surface area (defined as the upper 30 μm of the plaque) from BCA sections at three consecutive locations (n = 10 each). Right: representative immunofluorescent images of BCA sections for ACTA2 (green), LGALS3 (red), and Hoechst (blue) with highlighted 30 μm plaque surface area from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western-diet feeding. Scale bars, 100 μm. (H) Volcano plot depicting differentially regulated genes analyzed by RNA-seq of FACS-sort enriched peritoneal MΦs, coincubated either with live or dead Jurkat cell supernatant for 12 h. (I) Quantification of peritoneal macrophages 12 h after addition of live or dead Jurkat cell supernatant (n = 6). (J–L) Efferocytosis assay, analyzing the efferocytotic capacity of the MΦ population, isolated from Lyz-MΦ GFP-rep mice. Apoptotic Jurkat cells were added for 1 h after 6 h incubation either with or without CCL2. (J) Quantification of MΦs with engulfed apoptotic cells upon presence or absence of CCL2 (n = 5 independent experiments). (K) Quantification of the total number of engulfed apoptotic cells upon CCL2 presence of absence. (L) Representative epifluorescence images of the efferocytosis assay with peritoneal macrophages (green) and apoptotic Jurkat cells (red), 1 h after Jurkat cell addition. Scale bars, 50 μm. (M–O) Necrotic core analysis as total necrotic area in μm 2 (M) and in percentage of plaque area (N), assessed with Masson Trichrom’s staining of valve sections, from Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) littermates after 14 weeks of western diet. (O) Left: representative images of necrotic core content analyzed by Masson Trichrom’s staining of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet. ∗ indicates necrotic areas. Scale bars, 100 μm. Right: representative images of immunofluorescence stainings of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet for ACTA2 (green), LGALS3 (red), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (yellow), and DAPI (blue). Scale bars, 100 μm. (P and Q) Quantification of cell apoptosis as total amount of TUNEL + LGALS3 + Hoechst + MΦs in plaque (P) and as total amount of TUNEL + Hoechst + apoptotic cells (Q) in Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) individual littermates in total after 14 weeks of western diet, only including plaques at the proximal and intermediate BCA, without distal BCA areas with its early lesions. (R) Quantification of valve atherosclerotic plaques for (left) total and relative TUNEL + cells. Data are shown as mean and SEM. (I, J, K, M, N, and R) Student’s t test was used for normally distributed data and Wilcoxon matched-pairs signed rank test for not normally distributed data. (E, F, G, P, and Q) Repeated measure two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; ∗∗ p < 0.01; NS, not significant. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Western Blot, Migration, Blocking Assay, Isolation, Incubation, Staining, Immunofluorescence, End Labeling, TUNEL Assay

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Short-term CCL2 inhibition in advanced atherosclerosis triggers detrimental changes in plaque phenotype (A) Acute pharmacological CCL2 inhibition in ApoE −/− mice after 6 months of western type diet. The anti-CCL2 or isotype control antibody was injected intravenous (i.v.) 2 weeks before sacrifice every 48 h (n = 7–8 / group). (B) Quantification of fibrous cap coverage as continuity (percentage of fibrous cap covered plaque surface length relative to complete plaque surface length) at three subsequent BCA locations. (C) Quantification of ACTA2 + area within plaque surface as % of plaque surface area (defined as the top 30 μm stripe of the lesion) at three subsequent BCA locations. (D) Quantification of absolute ACTA2 + area in μm 2 at three subsequent BCA locations. (E) Quantification of macrophage area as LGALS3 area in μm 2 at three subsequent BCA locations. (F) Quantification of total plaque size as absolute plaque area in μm 2 at three subsequent BCA locations. (G) Quantification of cell apoptosis as total amount of TUNEL + cells in plaque at three subsequent BCA locations. (H) Representative images of BCA sections from ApoE −/− mice after 6 months of western diet stained for ACTA2 (green), LGALS3 (far red), TUNEL (red), and Hoechst (blue). Scale bars, 50 μm. (I) Quantification of blood leukocytes, neutrophils, lymphocytes, monocytes, and plasma cholesterol (n = 7–8). (I) Student’s t test was used. (B–G) Repeated measures two-way ANOVA or mixed-effects model, with subsequent Šídák’s multiple comparisons test in (B)–(D), was used. ∗ p < 0.05; ∗∗ p < 0.01 NS, not significant. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Inhibition, Western Blot, Control, Injection, TUNEL Assay, Staining, Clinical Proteomics

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet:

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Control, Blocking Assay, Selection, Recombinant, Western Blot, In Situ, Staining, Single Cell, DNA HS Assay, Picogreen Assay, cDNA Synthesis, Clinical Proteomics, SYBR Green Assay, Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Software