Relationship Of Th17 Cells And IL17 To Viral Heart Disease

Viral heart disease(VHD) includes viral myocarditis(VMC) and dilated cardiomyopathy(DCM).Viral infection produced not only VMC but DCM in part of suffered patients.VMC and DCM might be the different stages of VHD. In most cases,the etiology of myocarditis was infection of Coxsackievirus B. Autoimmunity injury mediated by viral infection might be a critical role in VMC and the development of DCM.Th17 cell is a recent discovered CD4~+ T helper subset,which can produce IL-17.It produces IL-6,IL-22 and TNF-αalso.STAT3 and RORγt seems to be two critical transcription factors for IL-17 production and differentiation of Th17.IL-17 is an important proinflammatory factor which mainly produced by Th17 cells and plays an important role in inflammation. Now,it has been demonstrated that Th17 cells is critical for autoimmune disease.Recently,it was found that Th17 cells might play an important role in experimental autoimmune myocarditis(EAM).It was found that the percentage of Th17 cells was increased in peripheral blood of acute VMC patients.But there led us some questions:whether Th17 cells differentiate in VHD? How does Th17-related cytokines work with it? So we built murine models of viral myocarditis and cardiomyopathy by CVB and BABL/c mice aiming the mechanism of VMC and DCM,and the new therapy targets of it.Our research included 4 parts below: Part 1:The expression of IL-17 and IL-23 in different stages of murine viral myocarditis.A total of 100 male BALB/c mice,4 weeks old,including 80 in the viral myocarditis(VMC) group and 20 in the control group,were used.Mice in VMC group were peritoneally injected(PI) with Coxsackievirus B3.Mice were sacrificed 0 day,14 days,28 days,42 days and 56 days after PI.For different stages post infection,pathological changes of the mouse were observed,and IL-17 of heart by immunohistochemistry.Splenocytes were collected from the mice and restimulated with phytohemagglutinin(PHA) for 2 days in vitro.IL-17 mRNA and IL-23 mRNA of splenocytes were measured by RT-PCR and compared with those in the control.We found that the IL-17 mRNA extracted from splenocytes increased at 28 days after PI as compared with that in the control and 14 days,and decreased at 42 days and 56 days.All of the VM C group and control mice expressed IL-23 mRNA,but IL-23 mRNA level,which up-regulated at 14 days,increased earlier than IL-17 mRNA,and continued to rise to a high level after that.Heart IL-17 level which examined by IOD (Integrate optical density) also increased at 28 days of viral myocarditis in mouse as compared with that in the control and 14 days.And the protein levels decreased at 42 days and 56 days then.Part 2:Research of Th17 and its relative cytokines in viral dilated cardiomyopathy.There were three researches in this part:(1) Murine viral DCM model and the pathology oberservation;(2) Th17 detection in DCM mice; (3) The expressions of IL-23 and IL-17 in murine DCM model.In this part,forty male BABL/c mice were divided into two groups,the DCM group and the control.We successfully built murine DCM model by monthly peritoneal injection of CVB3 for 180 days in the DCM group.In the DCM mice,the heart weight was higher,and the ventricular wall was thinner than the control,and fibrosis in heart was observed.Then we detected Th17 cells in peripheral blood and splenocytes from DCM mice by flow cytometry,and had a negative result in the control.The expression of IL-23 in DCM mice plasm were significantly higher than those in non-infected.And IL-17 mRNA extracted from splenocytes was detected by RT-PCR,and had a negative result in the control also.Part 3:STAT3 induced Th17 differentiation mechanism in viral DCM.In this part,the same model in part 2 was used,protein was obtained from mice hearts and spleens,and plasm was collected also.With Western blot,we found that the phosphorylation STAT3,the critical transcription factor of IL-17, significantly increased in DCM group.And DCM mice serum IL-6,IL-21,IL-23 levels detected by ELISA were higher than those in the control.Part 4:IL-23/IL-17 axis in DCM patients.In this part,two investigations were included.Firstly,we invested 26 patients with DCM,26 non-DCM heart failure patients and 26 normal volunteers.We found that the plasm IL-23 level of DCM patients was higher than non-DCM and the control.Secondly,we investigated 10 DCM patients,26 non-DCM heart failure patients and 20 normal volunteers.Though we didn't detected IL-17 in DCM patient plasm,but in vitro,the PBMCs from DCM individuals were stimulated by PHA,and then it could be detected IL-17 in the culture supernatant by ELISA,and it was failed neither in non-DCM nor in the control. In summary,our research suggested that:(1) The expressions of IL-17 and IL-23 were up-regulated in mice myocarditis,it might be caused Thl7 cells differentiation in the post stage of viral infection.(2) Monthly peritoneal injection of CVB3 for 180 days could result higher heart weight,thinner ventricular wall in infected mice than that of the control,and fibrosis in heart was observed in infected mice.It seems to be a good model of murine DCM.(3) Th17 cells differentiated in viral DCM mice, and IL-17,IL-23 were up-regulated in vivo.(4) Signal Transduction mediated by p-STAT3 was up-regulated in DCM mice,it might induce the differentiation of Th17.(5) Signal transduction mediated by STAT3 might be induced through the up-regulation of IL-6,IL-21 and IL-23 expression.(6) Expressions of IL-17 and IL-23 were higher in DCM patients,it could be relative with Th17 development in DCM.