IL-37 alleviates Coxsackievirus B3-induced viral myocarditis via inhibiting NLRP3 inflammasome-mediated pyroptosis

Abstract

Our study aims to verify the potential effects and underlying mechanisms of IL-37 in Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC). VMC model was established by intraperitoneal injection of CVB3 into 6-week-old male balb-c mice on day 0. Each mouse of the IL-37-control group and IL-37-VMC CVB3 groups was intraperitoneally injected with IL-37 on day 4 and day 7. The cardiac function was evaluated by transthoracic echocardiography including LVEF, LVFE, IVSs and IVSd. Myocardial injury was measured by Elisa for serum cTnI. The inflammation infiltration and fibrosis were evaluated by hematoxylin and eosin (HE) staining and Masson staining. The expression levels of NLRP3 inflammasome components in pyroptosis were determined by western blot, Elisa, and immunofluorescent analysis. We also detected the expression of IL-37-IL-1R8 in PBMCs by immunofluorescence after injection with CVB3 and IL-37. Compared with the VMC group, mice received CVB3 and IL-37 have improved cardiac function, reduced inflammation infiltration and fibrosis, and with lower expression of cTnI, IL-1β, IL-18 and NLRP3 inflammasome component. IL-37 weakened the upregulation of GSDMD and phosphorylation of NF-κB p65 induced by CVB3. Exogenous addition of IL-37 with CVB3 further increases the production of IL-37-IL-1R8 -IL-18RA complex in vitro. Our findings indicate that IL-37 alleviates CVB3-induced VMC, which may be produced by inhibiting NLRP3 inflammasome-mediated pyroptosis, NF-κB signaling pathway, and IL-37-IL-1R8 -IL-18RA complex.

Figure 1

Establishment of the VMC model. (A) M-mode echocardiographic images of mice in control group and VMC group and the LVEF, LVFS, IVSs, IVSd from the echocardiographic data. (B) Hearts of mice in control group and VMC group. (C) The levels of cTnI in serum of mice in control group and VMC group were measured by ELISA. (D, E) Histopathological changes in heart tissue of control group and VMC group were examined by H&E staining and Masson staining. The pathologic score of mice in control group and VMC group. (*P < 0.01, **P < 0.001, ****P < 0.00001, ns: not significant.)

Figure 2

Pyroptosis is overactivated in the VMC group. (A, B) The levels of IL-18 and IL-1β in serum of mice in control group and VMC group were measured by ELISA. (C) Analysis of the expression of GSDMD in heart tissue from the immunofluorescent data. (D) The representative pictures of immunofluorescent analysis of GSDMD expression. (*P < 0.01, **P < 0.001.)

Figure 3

IL-37 alleviates myocarditis in CVB3-induced VMC mice. (A, B) M-mode echocardiographic images of mice in control group and VMC group and the LVEF, LVFS, IVSs, IVSd from the echocardiographic data. (C) The levels of cTnI in serum of mice in four groups were measured by ELISA. (Con: control group, *P < 0.01, **P < 0.001, ****P < 0.00001, ns: not significant.)

Figure 4

IL-37 attenuated inflammatory cell infiltration and collagen tissue deposition. (A) Hearts of mice in all groups. (B, D) Histopathological changes in heart tissue of four groups were examined by H&E staining and Masson staining. (C, E) The pathologic score of mice in control group and VMC group. (*P < 0.01, **P < 0.001, ****P < 0.00001, ns: not significant.)

Figure 5

IL-37 attenuated the activation of NLRP3 inflammasome and the production of IL-1β and IL-18. (A) The expression of NLRP3, ASC dimer and cleaved caspase 1 in heart tissues were measured by western blot. (B–D) Bar graphs of quantitative analysis of NLRP3, ASC dimer and cleaved caspase 1. (E, F) The levels of IL-18 and IL-1β in serum of mice in all groups were measured by ELISA. (*P < 0.01, **P < 0.001, ***P < 0.0001, ns: not significant.)

Figure 6

The representative pictures of immunofluorescent analysis of GSDMD expression in four groups.

Figure 7

IL-37 may decrease pyroptosis via inhibiting the expression of NF-κB. (A) The representative pictures of NF-κB p65 and phosphorylation of p65 expression of in heart tissues assayed by western blot. (B, C) Bar graphs of quantitative analysis of NF-κB p65 and phosphorylation of p65 protein. (D) The mRNA levels of NF-κB were detected by real time PCR. (*P < 0.01, **P < 0.001).

Figure 8

Binding of IL-37 to IL-1R8 in the PBMCs. Exogenous supplementation of IL-37 enhanced the combination of IL-37 and IL-1R8. Both injection of IL-37 and CVB3 virus increase the binding of IL-37-IL-1R8 complex respectively. CVB3 infection with exogenous IL-37 enhanced the mutual binding of IL-37 and IL-1R8 (P < 0.01 VS Control and IL-37 alone). Original blots are presented in Supplementary Figures.