Stat3Acts Together With Roryt To Induce The Differentiation Of Th17Cells In Viral Myocarditis Mice

Objective:This study was designed to explore whether STAT3and RORyt are both involved in the differentiation of Thl7cells in viral myocarditis (VMC) induced by Coxsackievirus B3(CVB3) and the interaction between STAT3and RORyt.Methods:VMC was induced with6-week old male BALB/c mice by CVB3peritoneal injection(I.P)(VMC group, n=40), mice I.P with phosphate-buffered solution (PBS) were taken as control group(n=25), mice of each group were divided into5subgroups according to the different time (0、1、2、4、6weeks) after peritoneal injection. Flow cytometric analysis was used to evaluate the percentages of Th17cells; Th17cell-related transcription factors STAT3and RORyt mRNA expression in the myocardium of mice were assessed by semi-quantitative RT-PCR; phosphorylated-STAT3(p-STAT3) and RORyt protein expression in the myocardium of mice were evaluated by Western-blot.1week after being infected by CVB3, splenic CD4+T cells of VMC mice were isolated by immunomagnetic beads and cultured in vitro with different concentrations of S3I-201(a selective STAT3inhibitor) for48hours, the cultured cells were divided into4groups:a. PHA (phytohemagglutinin); b. PHA+rIL-23(recombinant mouse IL-23); c. PHA+rIL-23+S3I-201(200μM); d. PHA+rIL-23+S31-201(500μM). The cultured CD4+T cells were collected after 48hours, p-STAT3protein expression, RORγt mRNA expression, the percentages of Thl7cells in the cultured CD4+T cells were detected by Western-blot, semi-quantitative RT-PCR, flow cytometric analysis, respectively.Results:The percentages of Th17cells in splenic CD4+T cells began to increase markedly1week after CVB3was infected,4-week subgroup were the highest, and6-week subgroup was lower than4-week subgroup, but still higher than0-week subgroup, except the0-week subgroup, the percentages of Th17cells in all of the other VMC subgroups were statistically significant compared with the corresponding control subgroups (P<0.05, respectively), however, the percentages of Thl7cells in control group remained invariable throughout the study (P>0.05). Likewise,1week after CVB3was infected, the expression of STAT3and RORyt in myocardium started to increase too, the highest levels were also observed on4-week subgroup,6-week subgroup was lower than4-week subgroup, but still higher than0-week subgroup, the expression of STAT3and RORyt in1-week,2-week,4-week and6-week subgroups in VMC group were statistically significant compared with the corresponding control subgroups (P<0.05, respectively), whereas there was no significant alteration in the control group throughout the study (P>0.05). In the cultured CD4+T cells in vitro, the expression of p-STAT3protein and RORyt mRNA were greatly inhibited by administration of S3I-201(200μM,500μM), correspondingly, Th17cells proliferation in the cultured CD4+T cells decreased dramatically.Conclusions:STAT3acts together with RORyt to induce the differentiation of Thl7cells in viral myocarditis induced by Coxsackievirus B3.