Inhibitor of CD147 Suppresses T Cell Activation and Recruitment in CVB3-Induced Acute Viral Myocarditis

Figures

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  Figure 1. Effect of AC-73 on the expression of CD147 mRNA and virus replication in the myocardium of CVB3-infected mice and body weight. (A,B) The relative expression of CD147 mRNA and viral load in the myocardium on the 7th day post-infection was detected using q-PCR. The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests (n = 5). (C) The weight change in the four groups. The data on weight were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests (n = 5). ** p < 0.01, **** p < 0.0001.

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  Figure 2. The effect of AC-73 on the expression of CD147 and CyPA in myocardium. The sections of the hearts on the 7th dpi were stained with rabbit anti-CD147 and rabbit anti-CyPA antibodies. (A) The immunofluorescence assays of CD147 in myocardium. Red indicates CD147 and blue indicates DAPI-stained cellular nuclei. Scale bar = 50 μm. (B) The immunohistochemical staining of CyPA in myocardium. Scale bar = 100 μm. (C,D) The histograms for the expression of CD147 and CyPA in myocardium. The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. The histopathological change and the pathological score of the myocardium in the CVB3-infected mice after the administration of AC-73. Scale bar = 50 μm. The arrows represent inflammatory infiltration, and the triangles represent myocardial necrosis. All the mice were sacrificed on the 7th day post-infection and the hearts were taken for H&E staining: (A) control group; (B) 50 mg/kg AC-73 group; (C) CVB3-infected group; (D) CVB3-infected mice administered 25 mg/kg of AC-73; and (E) CVB3-infected mice administered 50 mg/kg of AC-73. (F) The pathological score of the myocardium (n = 5). Three visual fields were chosen randomly from each section, and the pathological score obtained using the three analyzed images from each heart tissue was statistically analyzed. The data were analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparison test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 4. The proportion of total T cells, CD4+ cells or CD8+ T cells in the spleen and heart. After treatment with AC-73 on the 4th day after CVB3 infection, the mice were sacrificed on the 7th day post infection, and their spleen and heart were taken for flow cytometry. (A) The proportion of CD45+ CD3+, CD3+ CD4+ and CD3+ CD8+ T cells in the spleen. (B) The proportion of CD45+CD3+, CD3+CD4+ and CD3+CD8+ T cells in the heart. (C) The histograms for the percentage of T cells in the spleen (n = 5). (D) The histograms for the percentage of T cells in the heart (n = 3). The hearts from 15 mice in each group were obtained, and mononuclear cells of the hearts of every 5 mice were mixed into one sample (5 mice/sample); thus, we obtained 3 datasets in each group for statistical analysis. The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 5. The proportion of activated T cells in the spleen. After treatment with AC-73 on the 4th day after CVB3 infection, the mice were sacrificed on the 7th day post-infection, and then spleen cells were taken for flow cytometry. (A) The proportion of CD69+/CD38+ CD4+ T cells in the spleen. (B) The proportion of CD69+/CD38+ CD8+ T cells in the spleen. (C) The histograms for the percentage of activated T cells in the spleen (n = 5). The data was analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. The proportion of Th17, Th1, Th2 and Treg cells in the spleen was detected using flow cytometry on the 7th day after CVB3 infection (n = 5): (A) the proportion of CD4+ IL17A+ T cells; (B) the proportion of CD4+ IFN-γ+ T cells; (C) the proportion of CD4+ IL-4+ T cells; and (D) the proportion of CD4+ CD25+ Foxp3+ T cells. (E) The histograms for the percent of Th17, Th1, Th2 and Treg cells in the spleen. The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 7. The effect of AC-73 on the expression of CD69 in myocardium. The sections of the hearts harvested on the 7th dpi were stained with rabbit anti-CD69 antibodies. Green indicates CD69, and blue shows DAPI-stained cellular nuclei. Scale bar = 50 μm. The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests (n = 5). * p < 0.05, *** p < 0.001.

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  Figure 8. The effect of AC-73 on the macrophages in myocardium. The sections of hearts were stained with rabbit anti-F4/80 antibodies on 7 dpi. Representative images of macrophages stained with anti-F4/80 in myocardium. Green indicated F4/80 and blue showed DAPI-stained cellular nuclei. Scale bar = 50 μm. The data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post hoc tests (n = 5). * p < 0.05, *** p < 0.001.

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  Figure 9. The effect of AC-73 on cytokine expression in the CVB3-infected mice. (A) Heat map of the expression of plasma cytokines and chemokines after the administration of AC-73 (n = 5). The 15 cytokines and chemokines were detected using Luminex multiplex immunoassay on the 7th day post-infection. In the heatmap, red and blue represent high and low expression, respectively. (B) The mRNA expression of IFN-γ, IL-10 and IL-17 in the heart was detected using q-PCR on the 7th day post-infection (n = 5). The data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.