Myocardial Mitochondrial DNA Drives Macrophage Inflammatory Response through STING Signaling in Coxsackievirus B3-Induced Viral Myocarditis

Figures

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  Figure 1. Myocardial mtDNA drives macrophage inflammatory responses. (A) Schematic diagram of the experimental design. (B–D) BMDMs were cultured with a conditioned medium using a culture supernatant (CS) from CVB3-infacted cardiomyocytes, and IL-6, TNF-α, and MCP-1 production was detected with ELISA. Mean ± SEM from six independent experiments in each group. (E) Ct values of mitochondrial gene 16S rRNA, ND1, and nuclear gene HK2 in the culture supernatant of CVB3-infected cardiomyocytes. Mean ± SEM from five independent experiments in each group. (F) BMDMs were cultured with or without CS from CVB3-infacted cardiomyocytes, and the intracellular DNA (green) content was detected using immunofluorescence. (G) RAW264.7 cells were cultured with or without mtDNA from CVB3-infacted cardiomyocytes; then, the intracellular DNA (green) content was detected. Scale bar in (F,G) 20 μm. (H–J) CS from CVB3-infected cardiomyocytes were pretreated with or without DNase I, and they were subsequently incubated with BMDMs when analyzing the inflammatory responses. Mean ± SEM from five independent experiments in each group. *** p < 0.001 with t-test (B–D,H–J) and ANOVA (E).

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  Figure 2. CVB3 infection promotes STING expression in myocardial macrophages. (A) Myocardial mRNA levels of STING in CVB3-infected mice were detected using qPCR at the indicated time. Mean ± SEM from six mice in each group. (B). Myocardial protein levels of STING in CVB3-infected mice were detected using immunoblots at the indicated time. Mean ± SEM from five mice in each group. (C) Immunohistochemical staining for the myocardial STING protein in CVB3-infected mice. (D–F) Myocardial infiltration of immune cells were characterized using flow cytometry, as indicated. Mean ± SEM from three mice in each group. (G,H) Cardiomyocytes and macrophages from CVB3-infected mice were assessed in order to find the STING protein using immunoblots. * p < 0.05, ** p < 0.01, *** p < 0.001 with ANOVA (A,B,F) and t-test (E,G,H), ns: no significant difference.

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  Figure 3. CVB3 infection activates myocardial STING signaling in vivo. The protein expressions of cGAS, STING, p-TBK1, total TBK1, p-NF-kB, and total NF-kB in myocardial tissues from CVB3-infected mice were detected using immunoblots at the indicated time. Representative (A) and mean ± SEM (B) from three mice in each group. * p < 0.05, *** p < 0.001 with ANOVA, ns: no significant difference.

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  Figure 4. Myocardial mtDNA induces macrophage inflammatory responses in a STING-dependent manner. (A–F) Wild-type (WT) and STING-deficient BMDMs were cultured in a conditioned medium, using the CS from CVB3-infected cardiomyocytes, and STING activation was detected by analyzing the expressions of the indicated proteins. Mean ± SEM from three independent experiments in each group. (G) WT and STING-deficient BMDMs were cultured in a conditioned medium, using the CS from CVB3-infected cardiomyocytes, and inflammatory responses were detected. Mean ± SEM from six independent experiments in each group. (H–M) WT and STING-deficient BMDMs were cultured with or without mtDNA from CVB3-infected cardiomyocytes, and STING signaling was detected. Mean ± SEM from three independent experiments in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 with ANOVA (B–F,I–M) and t-test (G). ns, no significant difference. N.D, not detected.

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  Figure 5. STING expression correlates with myocardial function in CVB3-infected mice. Correlation analyses of the myocardial mRNA level of STING and body weight loss (A), serum cTnI level (B), serum CK-MB activity (C), and myocarditis score (D) in CVB3-infected mice (n = 16). Pearson correlation analysis.

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  Figure 6. Deficiency of STING abrogates the development of CVB3-induced VMC. WT and STING-deficient mice were infected with CVB3 for 7 days, and the myocardial histology (100×, (A), black arrow: inflammation site, n = 5), myocardial CD45 (200×, (B), black arrow: CD45+ infiltrated leukocytes), F4/80 expression (200×, (C), black arrow: F4/80+ infiltrated macrophages), myocardial levels of the indicated pro-inflammatory cytokines ((D), n = 5), serum levels of the indicated pro-inflammatory cytokines ((E), n = 5), body weight loss ((F), n = 7), serum cTnI levels ((G), n = 5), serum CK-MB activity ((H), n = 5), myocardial function ((I,J), n = 5), survival rates (K), and CVB3 RNA (L) were analyzed. * p < 0.05, ** p < 0.01, *** p < 0.001 with t-test (A,D,E,L) and ANOVA (G–J). ns: no significant difference.

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  Figure 7. Schematic diagram of CVB3-induced myocardial inflammation. Upon CVB3 infection, cardiomyocytes release mtDNA into the extracellular myocardial environment, promoting cGAS/STING/NF-kB signaling in myocardial macrophages, and thus, inducing pro-inflammatory cytokines.