Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 1. CCR2 is required for efficient SFTSV infection in cells. (A to C) RT-qPCR (A), flow cytometry (B), and Western blot (C) analysis of SFTSV infection at 24 hours after infection in CCR2- or ATF6-knockdown THP-1 cells. n = 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) RT-qPCR analysis of virus RNA in CCR2-KO THP-1 cells inoculated with four phylogenetically distinct SFTSV strains, including HBMC16, HNXY2017-50, HNXY2017-66, and WCH, for 24 hours. n = 4. (E) Multistep growth curves of four SFTSV strains in CCR2-KO THP-1 cells. n = 4. (F) Surface expression of CCR2 on BMDMs from CCR2−/−and WT C57BL/6J mice. A representative of three replicates is shown. (G and H) Statistical results (G) and scanned images (H) of the immunological focus assay of SFTSV titers at 24 hours after infection in BMDMs with deletions in CCR2. n = 6. (I) Microscopy of BMDMs immunostained for F4/80, SFTSV NP, and DAPI at 24 hours after infection. (J) SFTSV infection rates at 24 hours after infection determined by flow cytometry analysis in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (K) Representative flow plot of SFTSV infection in Huh7 cells. (L) Supernatant viral titers measured by immunological focus assay in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. One-way ANOVA followed by Tukey’s multiple comparisons test was per- formed for comparison of variables among three groups [(J) and (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Flow Cytometry, Western Blot, Knockdown, Virus, Expressing, Microscopy, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 2. Effect of CCR2 inhibitor and antibody on SFTSV infection in cells. (A to F) Effects of CCR2 antagonist RS102895 and CCR2 antagonist 1 on SFTSV infection in THP-1 (A) to (C) and Huh7 cells (D) to (F). At 24 hours after infection, relative vRNA levels were measured via RT-qPCR [(A), (B), (D), and (E), n = 3], and relative intracellular SFTSV NP levels were measured by Western blotting (C) and (F). Cell viability was measured using CCK-8 [(D) and (E), n = 3]. (G to I) Effects of CCR2 antibody on SFTSV infection in THP-1 cells. The dose-dependent inhibitory effects of the CCR2 antibody were analyzed by detecting virus loads in THP-1 cells at 24 hours after infection [(G), n = 6]. SFTSV infection rates were measured at 24 hours after infection with MOIs of 1 and 5 [(H), n = 6]. Representative flow plot of SFTSV infection in THP-1 cells treated with anti-human CCR2 or isotype control antibody (I). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), (E), (G), and (H)]. R2 [(A), (B), (D), and (E)] was estimated by a nonlinear regression model (curve fit).

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Virus, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 3. CCR2 mediates SFTSV binding and internalization. (A and B) Effects of CCR2 deletions on the internalization (A) and binding (B) of SFTSV in BMDMs. Relative vRNA levels were measured via RT-qPCR. n = 6. (C) Fluorescence microscopy analysis of the effects of CCR2 deletions on the binding of SFTSV. BMDMs were immunos- tained with F4/80 (green), SFTSV NP (red), and DAPI. A representative of three replicates is shown. (D and E) SFTSV binding assay in CCR2-KO (KO) THP-1 cells by using RT- qPCR (D) and immunofluorescence staining (E). n = 4. Cellular membranes were labeled with WGA. (F) Flow cytometry analysis of SFTSV infection at 2 hours after infection in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (G to I) Effects of the CCR2 antagonist RS102895 (G), CCR2 antagonist 1 (H), and favipiravir (I) on the binding and internalization of SFTSV in THP-1 cells. n = 3. (J) Binding assay of HRTV, RVFV, and AMRV for control and CCR2-KO THP-1 cells. n = 3. (K and L) Effects of CCR2 antagonist RS102895 (K) and CCR2 antagonist 1 (L) on the binding of HRTV, RVFV, and AMRV in THP-1 cells. n = 3. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (J)]. One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(F) to (I), (K), and (L)]. ns, no significance; p.i., post-infection.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling, Flow Cytometry, Infection, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 4. The CCR2 N-terminal extracellular domain mediates SFTSV binding to cells. (A) Coimmunoprecipitation of co-overex- pressed Gn-strep protein and CCR2-flag proteins in HEK293T cells using strep-tag binding beads or anti-Flag antibody beads. Vector-flag, GFP-flag, and SCARB1-flag pro- teins were used as negative controls. (B to D) Effect of CCR2 overexpression on the in- fectivity of SFTSV in Huh7 (B), HeLa (C), and Jurkat (D) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry analysis in control-, CCR2A-, CCR2B-, CCR2A-ΔN–, and CCR2B-ΔN–over- expressing cells. N = 6. (E) Surface expres- sion of CCR2 and representative flow plot of SFTSV infection in Jurkat cells. (F and G) Effect of the CCR2 N-terminal extracellular domain on the infectivity of SFTSV in HeLa (F) and Jurkat (G) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry in control-, CCR2A-, CCR2A-N14Q–, CCR2A-Y26F–, CCR2B-, CCR2B-N14Q–, and CCR2B-Y26F–overex- pressing cells. n = 6. (H) Binding of CCR2 N- terminal–derived Y26 sulfated peptide (p2), peptide without tyrosine sulfation at Y26 (p1), or the scrambled peptide to SFTSV virions determined by PRM assay. Statistical analysis is shown in the left panel, and PRM transitions are shown in the right panel. (I and J) Effect of CCR2 N-terminal–derived peptides on the infectivity of SFTSV in THP-1 cells. Relative intracellular vRNA levels and supernatant viral titers were measured at 24 hours after infection via RT-qPCR. n = 4. (K) Effect of CCR2 N-terminal–derived peptides on the binding of SFTSV in THP-1 cells. Rel- ative levels of bound virions were measured via RT-qPCR. n = 4. One-way ANOVA fol- lowed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(B) to (D) and (F) to (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Strep-tag, Plasmid Preparation, Over Expression, Infection, Flow Cytometry, Control, Expressing, Derivative Assay, Quantitative RT-PCR, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 5. CCR2 contributes to SFTSV pathogenesis in mouse models. (A) Serum and spleen viral titers in SFTSV-infected CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (B and C) Survival probability (B) and relative body weight (C) in anti-IFNAR1 antibody–pretreated CCR2−/−(n = 11) and WT (n = 11) C57BL/6J mice after intraperitoneal infection with SFTSV. (D) Viral titers in serum, spleen, liver, and lung samples from anti-IFNAR1 antibody–pre- treated CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (E and F) Representative images of spleen, liver, and lung sections collected at 5 dpi from control and SFTSV-challenged CCR2−/−and WT C57BL/6J mice stained with a rabbit polyclonal antibody against SFTSV NP (E) or with hematoxylin and eosin (F). (G) Viral titers in serum from anti-IFNAR1 antibody–pretreated C57BL/6J mice tested by immunological focus assay at 3 (n = 13 for each group) and 5 (n = 10 for nontreated group and n = 13 for treated group) dpi. (H) Survival probability in anti-IFNAR1 antibody–pretreated C57BL/6J mice with SFTSV infection in the absence (n = 13) or presence (n = 13) of CCR2 antagonist RS102895 and without SFTSV infection (n = 5). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. The Kaplan-Meier method was used to analyze time-to-event data [(C) and (H)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Control, Staining, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 6. CCR2 contributes to SFTSV infectivity in primary human monocytes. (A and B) Comparisons of surface CCR2 expression levels on primary human mono- cytes (left) and the ability of SFTS binding (right) between donors aged <60 (n = 10) and ≥60 (n = 10) years old (A), as well as between healthy donors (n = 8) and donors with DM (n = 8) (B). MFI, mean fluorescent intensity. (C) Association of surface CCR2 expression level on primary human monocytes with virus binding ability or with the age of donors (n = 20). Ra 2 indicates the correlation between the CCR2 expression level and individual age, and Rb 2 indicates the correlation between the CCR2 expression level and the binding ability of SFTSV. (D) Associa- tion of surface CCR2 expression level on primary human monocytes with the peak viral load in serum that was consecutively collected from SFTS patients (n = 45) during the clinical course. Two-tailed Student’s t test was performed for compar- ison of variables between two groups [(A) and (B)]. R2 [(C) and (D)] was estimated by a linear regression model. HC, healthy control; DM, diabetes mellitus; CT, cycle threshold.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Expressing, Binding Assay, Virus, Two Tailed Test, Control

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

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: Effects of MPs on teloHAEC. Cell viability evaluated in teloHAEC exposed to different concentrations of A PE and B PVC (0–70 µg/mL) for 24, 48, and 72 h. C , D Cell viability assessed after 48 h of treatment with PE or PVC (30 µg/mL) and increasing concentration of PVC or PE (0–70 µg/mL), respectively. Evaluation of E MCP-1, F VCAM1, and G ICAM1 content in teloHAEC treated with PE (70 µg/mL), PVC (70 µg/mL), or PE + PVC (30 µg/mL + 30 µg/mL) for 48 h. H , I Representative cell cycle detection by FACS analysis in EC exposed to MPs. Data expressed as mean ± standard deviation (SD) of n = 4 independent experiments. * p < 0.05 versus 0 µg/mL or Ctr; ** p < 0.01 versus 0 µg/mL or Ctr; • p < 0.001 versus 0 µg/mL or Ctr; † p < 0.05 versus PE; ‡ p < 0.05 versus PVC; ns, not significant versus PE and PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Concentration Assay, Standard Deviation

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: iPCSK9 opposed the MP-related inflammation. A TeloHAEC viability evaluated after treatment with PE and PVC alone or combined PE + PVC, or pretreated with iPCSK9 (100 ng/mL) for 8 h and then exposed to PE, PVC, and PE + PVC. B Immunoblotting analysis of SIRT6 protein levels and ELISA assays of C MCP-1, D VCAM1, and E ICAM1. F Representative annexin V-FITC and PI-staining detected by FACS analysis and G cell cycle investigation. Data expressed as mean ± SD of n = 4 experiments. M, molecular weight markers; lane 1, Ctr; lane 2, PE; lane 3, PVC; lane 4, PE + PVC; lane 5, iPCSK9; lane 6, PE + iPCSK9; lane 7, PVC + iPCSK9; lane 8, PE + PVC + iPCSK9. * p < 0.05 versus Ctr; ** p < 0.01 versus Ctr; • p < 0.001 versus Ctr; † p < 0.05 versus PE; ‡ p < 0.05 versus PVC; + p < 0.05 versus PE + PVC; ¶ p < 0.01 versus PE; § p < 0.01 versus PVC; ▲ p < 0.01 versus PE + PVC; ns, not significant versus PE and PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Molecular Weight

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: SIRT6 silencing opposed the effects of iPCSK9 on inflammation and apoptosis. A Immunoblotting analysis of SIRT6 protein levels in teloHAEC treated with the empty transfection reagent (Vehicle) or transfected with negative control siRNA (NT) or with SIRT6 siRNA (siSIRT6). M, molecular weight markers; lane 1, Ctr; lane 2, Vehicle; lane 3, NT; lane 4, siSIRT6. B Immunoblotting analysis of PCSK9 protein levels and ELISA assays of C MCP-1, D VCAM1 and E ICAM1. F , G Representative dot plots and FACS analysis of annexin V-FITC and PI-staining of EC transfected with NT or siSIRT6 and exposed to PE + PVC alone or pretreated with iPCSK9 (100 µg/mL) before MP stimulation. M, molecular weight markers; lane 1, Ctr; lane 2, NT; lane 3, NT + PE + PVC; lane 4, NT + iPCSK9; lane 5, NT + PE + PVC + iPCSK9; lane 6, siSIRT6; lane 7, siSIRT6 + PE + PVC; lane 8, siSIRT6 + iPCSK9; lane 9, siSIRT6 + PE + PVC + iPCSK9. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Data are expressed as mean ± SD of n = 3 experiments. ° p < 0.01 versus NT; # p < 0.001 versus NT; + p < 0.05 versus PE + PVC; ▲ p < 0.01 versus PE + PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Western Blot, Transfection, Negative Control, Molecular Weight, Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Disruption of tumor-intrinsic PGAM5 increases anti-PD-1 efficacy through the CCL2 signaling pathway

doi: 10.1136/jitc-2024-009993

Figure Lengend Snippet: PGAM5 reduced CCL2 secretion in HCC cells by downregulating DRP1. ( A ) Schematic diagram of the in vitro co-culture system for Huh7 shctrl or shPGAM5 cells and phorbol-12-myristate-13-acetate-induced THP-1 cells. ( B ) Quantitative PCR analysis of macrophage polarization maker genes of THP-1 cells co-cultured with indicated HCC cells. ( C ) cytokine antibody array incubated with culture medium of Huh7 shctrl or shPGAM5 cells. ( D ) Quantitative PCR analysis of macrophage M1/M2 polarization maker genes of THP-1 in the co-culture system, culture medium of Huh7 shPGAM5 cells were supplemented with recombinant CCL2 and HCCLM3 PGAM5 OE cells were supplemented with neutralizing-CCL2 antibody. ( E ) TOP20 GO enrichment of differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( F ) Volcano plot of significantly differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( G ) Western blot analysis of protein expression of PGAM5 and mitochondria dynamic proteins DRP1, MFN1, MFN2, OPA1 in HCC cells. ( H ) Western blot analysis of DRP1 overexpression efficiency in Huh7 shPGAM5 cells by lenti-virus infection. ( I ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA. ( J ) Western blot analysis of DRP1 silencing efficiency in HCCLM3 PGAM5-OE cells by transient transfection of SiRNA. ( K ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA kit. *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001, ns: not significant. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; UMAP, uniform manifold approximation and projection.

Article Snippet: Concentrations of CCL2 in the supernatant were examined by ELISA kit (CSB-E04655h, cusabio) following manufacturer’s instructions.

Techniques: In Vitro, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Ab Array, Incubation, Recombinant, Western Blot, Expressing, Over Expression, Virus, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Immunohistochemistry