IL-1–Activated Inflammatory Cardiac Fibroblasts Exacerbate Acute Coxsackievirus B3 Myocarditis

Figures

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  Figure 1. Inflammatory Fibroblast Activity Is Detected Early During CVB3 Myocarditis (A) Schematic of experimental paradigm. CCL2-mCherry mice were infected with CVB3 and sacrificed at days 3, 7, 10, and 14 with naïve mice used as a control (n = 4-5 mice per group). Flow cytometry was used to identify CCL2-expressing cells in the heart. (B) Percentage of total live cells that express CCL2. (C) Stacked bar graph showing relative proportions of various cardiac cells as a percentage of total CCL2-expressing cells. (D) Percentage of PDGFRα+ FEEDER+ cardiac fibroblasts that express CCL2. (E) Median fluorescence intensity of mCherry (CCL2) signal in PDGFRα+ FEEDER+ cardiac fibroblasts. (F) Pie charts showing proportions of CCL2+ PDGFRα+ FEEDER + cardiac fibroblasts that are Ly6C–, Sca-1+; Ly6C+, Sca-1+; and Sca-1–. Quantification of percentage of CCL2+ fibroblasts that are either (G) Sca-1+ or (H) Ly6C+. (B, D, E, G, H) Brown-Forsythe and Welch ANOVA with Dunnett's multiple comparisons test. The line represents the mean of each group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CVB3 = coxsackievirus B3; MFI = median fluorescence intensity.

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  Figure 2. Gene Expression Analysis Reveals That CCL2+ Inflammatory Fibroblasts Promote Th1, Th2, and Th17 Immune Responses While Downregulating Fibrosis-Related Functions CCL2-mCherry mice were infected with CVB3, and single-cell suspensions from hearts on day 3 of CVB3 myocarditis were produced (n = 3 biological replicates each consisting of 4-5 mice pooled together). Bulk RNA sequencing was performed from FACS-sorted CD45−CD31−FEEDER+mCherry+ and CD45−CD31−FEEDER+mCherry− populations. (A) Principle component analysis showing the variability of the data. (B) Heatmap of top 25 differentially upregulated and downregulated genes between mCherry-positive and -negative cardiac fibroblasts. (C) Volcano plot showing which genes were differentially upregulated (blue) or downregulated (red) in mCherry-positive fibroblasts compared to mCherry-negative fibroblasts. (D) Pathway activity inference analysis. Pathways that are upregulated in mCherry-positive CFs are shown in red. (E) Gene set enrichment activity analysis showing which GO terms are upregulated or downregulated in mCherry-positive fibroblasts. Adjusted P-values were determined by using the Benjamini-Hochberg false discovery rate method.…

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  Figure 3. IL-1 Signaling Potently Induces the Inflammatory Cardiac Fibroblast Phenotype (A) Primary murine cardiac fibroblasts were isolated from C57BL/6 mice and stimulated with IFNγ (50 ng/mL), IL-1β (10 ng/mL), IL-4 (10 ng/mL), IL-17a (50 ng/mL), IL-22 (10 ng/mL), and IL-13 (50 ng/mL) (n = 3 biological replicates per group). Heatmap representation of qPCR data with stimulants on the horizontal axis and readouts on the vertical axis. Bar graphs of relative normalized expression of (B) Ccl2, (C) Csf2 (GM-CSF), (D) Il6, (E) Cxcl1, (F) Cxcl10, (G) and Ccl11 (n = 3 each group). Results were analyzed by 1-way ANOVA with Tukey's post hoc test. Multiplex results from supernatant of unstimulated or IL-1β–stimulated primary cardiac fibroblasts showing levels of (H) CCL2, (I) GM-CSF, (J) CXCL1 and (K) CXCL2. Results were analyzed by Student's t-test. Data are plotted as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. GM-CSF = granulocyte-macrophage colony-stimulating factor; qPCR = quantitative polymerase chain reaction.

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  Figure 4. Examining Inflammatory Fibroblast Plasticity (A to C) Primary murine cardiac fibroblasts were stimulated with IL-1β for 72 hours followed by either keeping IL-1β in the media or replacing the media with IL-1β–deficient media for 24 hours (n = 3 biological replicates). Quantitative PCR was performed for A Ccl2, (B) Cxcl1, or (C) Csf2 (GM-CSF). (D to G) Primary murine cardiac fibroblasts were stimulated with IL-1β for 24 hours and either IL-13 or IL-10 was spiked into the media for the subsequent 24 hours (n = 4 biological replicates). Quantitative PCR was performed for D Il6, E Ccl2, (F) Cxcl1, or G Ccl11. Data were analyzed by 1-way ANOVA with Dunnett's post hoc test. A to C Data are plotted as mean ± SD. D to G The line represents the mean of each group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. GM-CSF = granulocyte-macrophage colony-stimulating factor; PCR = polymerase chain reaction.

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  Figure 5. Loss of IL-1 Signaling to Cardiac Fibroblasts Protects Against Severe Acute CVB3 Myocarditis (A) Relative normalized expression of Il1β in heart homogenates at indicated time points of CVB3 myocarditis (n = 4-5 mice per group). (B) Schematic of experimental paradigm. PDGFRαcre or PDGFRαcreIL1r1fl/fl mice were infected with CVB3 and sacrificed on day 10 postinfection. (n = 14-16 mice per group; data shown in (B-F) are from 1 experiment and are representative of 3 independent experiments. (C) Weight-loss curves of Cre control and PDGFRαcreIL1r1fl/fl mutant mice. Two-way repeated-measures ANOVA with Šídák's post hoc test. Data are displayed as mean ± SD. The P value displayed is the genotype × time interaction (P = 0.008). (D) Representative H&E-stained heart sections. Top panels: 1× whole heart. Bottom panels:10×; scale bar = 50 μm. (E) Myocarditis was scored on H&E-stained sections using a quantitative approach as described in the Methods section. (F to M) Flow cytometry was used to quantify different immune cell subsets in the heart. (F) Enumeration of total CD45+ immune cells per milligram of cardiac tissue. (G) Pie charts representing composition of CD45+ immune cells for each…

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  Figure 6. Human Cardiac Fibroblasts Recapitulate Inflammatory Fibroblast Features Human ventricular cardiac fibroblasts were stimulated with IL-1β (100 ng/mL), TNFα + IL17A (100 ng/mL each), poly I:C (10 μg/mL), lipopolysaccharide (10 ng/mL), imiquimod (5 μg/mL), or ODN2006 (1 μM) and assessed expression of select genes by qPCR (n = 3 technical replicates). (A) Ccl2, (B) Cxcl1, (C) Cxcl2, (D) Csf2 (GM-CSF), (E) Il1b, (F) Il6. A to F Data are presented as mean ± SD. One-way ANOVA with Dunnett's post hoc test. (G) Human cardiac biopsies from 12 lymphocytic or 9 eosinophilic myocarditis hearts underwent immunohistochemical staining for vimentin (Top Left), CCL2 (Bottom Left) and the overlay of pixel classification identifying vimentin staining (red) and CCL2 staining (green) arrows refer to overlap between vimentin and CCL2 staining (Right). Quantification of (H) vimetin+CCL2+ or (I) vimentin+eotaxin+ costaining from lymphocytic or eosinophilic myocarditis. (J) UMAP projection of 245,353 cells from integrated dilated cardiomyopathy (GSE145154), ischemic cardiomyopathy (GSE145154), hypertrophic cardiomyopathy (GSE181764), and cardiac sarcoidosis patients (GSE 205734). (K) UMAP projection of…