The Critical Role Of Th17Cell And Interleukin-17in Coxsackievirus B3-induced Viral Myocarditis And Cardiomyopathy

Myocarditis is an inflammatory disease of the heart frequently resulting from viral infections and/or post-viral immune-mediated responses. It is one of the important causes of dilated cardiomyopathy (DCM) worldwide, which is a common cause of heart failure, especially among adolescents and young adults. Although, most individuals recover from acute myocarditis, genetically susceptible individuals may go on to develop chronic myocarditis and DCM resulting in congestive heart failure. In present, myocarditis is a poorly understood disease because it progresses through stages with distinctly different mechanisms and manifestations. Some observers are to better define myocarditis for both clinicians and clinical scientists by setting it in the framework of3phases of disease. In phase1, the viral stage, they review recent discoveries about the way viruses gain access to target tissue and how they trigger immune responses. In the second, autoimmune phase of disease, they examine the roles of T cells, cytokines, and cross-reacting antibodies and reconsider the relevance of recent therapy trials. In the third phase of the disease, DCM, they consider the remodeling processes. Based on the character of the inflammatory infiltrate in the heart, Rose et al has shown that the types of myocarditis are lymphocytic myocarditis、giant cell myocarditis、eosinophilic myocarditis and DCM. An improved understanding of the immune-mediated mechanisms involved in the pathogenesis linking viral and autoimmune myocarditis is urgently needed to develop better approaches to therapy.CD4+T cells, upon activation and expansion, classically develop into Thl/Th2cell subsets with different cytokine profiles and distinct effector function. Until recently, a newly subset of IL-17-producing effector T helper cells, called Th17cells, has now been discovered and characterized obviously difference from the others subset of T helper cells. The growth and differentiation of Th17cell mainly depends on the local environment of cytokine that they produced. Th17cells produce IL-17A、 IL-17F、IL-22and IL-21. For many self-autoimmune diseases and the pathogen infection were closely associated with Thl7cells, we explore the importance of Th17cells in clearing pathogens during immune defense reactions and in inducing tissue inflammation in autoimmune disease and pathogen infection. Recently, the relationship between Th17cells and many cardiovascular diseases is becoming a hotspot for study in the current research. Moreover, the effects of Th17cells on EAM have also made greatly development. On the other hand, Tregs play a foundamental role in the maintenance of immunological self-tolerance and immune homeostasis. The developmental link between Th17cells and Tregs has led to speculation that these CD4+T cells have evolved to exist in different local immune micro-environment. However, the imbalance of Th17cells and Tregs has played a critical role in the many self-immune diseases.Viral infection is the common cause of viral myocarditis, of which coxsackie B virus (CVB) is the major causative agent. Innate immunity has been shown to play an important role in the development of myocardial damage in acute viral myocarditis. Owing to the discovery of T helper cell subset and the development of immune regulation, we further focus on the causes of viral myocarditis. DCs are highly specialized antigen-presenting cells. Upon encountering a pathogen, DCs undergo maturation, induction of costimulatory activity and migration to lymph nodes, where they prime antigen-specific T cells. Because of DCs also process endogenous antigens, they might trigger autoreactive T cells if activated appropriately. Meanwhile, the mechanisms leading to susceptibility to viral myocarditis are certainly multifactorial, we should further explore the difference of T helper cells (Thl、Th2、Th17and Treg) on the myocardial damage in viral myocarditis, especially DCs on the differences of Thl7/Treg cells subset. Accordingly, the newly findings of our studies will occur in clinical diagnosis and therapeutics of viral myocarditis.Part I:Developing the coxsackievirus3-induced murine viral myocarditis and dilated cardiomyopathyObjective To build coxsackievirus B3-induce animal models of acute and chronic myocarditis and dilated cardiomyopathy. Methods Two hundreds and ten Balb/c mice were randomized to6groups, they were groups of day0(n=35), day7(n=35), day10(n=35), day14(n=35), month2(n=35) and month3(n=35), respectively. Ten mice randomly selected from every group were injected ip with Eagle’s minimal essential medium (EMEM) solution, and served as controls; The remaining of them were inoculated ip with coxsackievirus B3(CVB3) diluted in EMEM solution to establish animal model of acute and chronic myocarditis and DCM. All mice were examined by echocardiography on the end of day7, mon2and mon3postinoculation, respectively and then were sacrificed. Portions of heart were processed for histological and morphological examination. Results Degeneration and necrosis of cardiomyocyte, inflammatory infiltration, collapse of cardiac muscle fibers and little fibrosis located around necrosis were detected in the myocardium of CVB3-infected mice on day7and day10. The pathological scores were higher on day7than control group, then get to the peak on day10, at last were fallen down on day14. Less inflammatory infiltration and manifest interstitial fibrosis were seen in infected mice on the end of2month, left ventricular (LV) function was found decreased in comparison with controls; On the one hand, massive diffused fibrosis and fibrosis scores were higher on3month than2month. Moreover, LV dilation and dysfunction in the3month group were fallen down than2month by echocardiography. Conclusion Viral myocarditis and DCM animal models are available with single or repeated CVB3infection, which closely resemble human myocarditis and DCM. The pathological scores were elevated on day7, and get to peak on day10in the acute myhocarditis. Massive diffused fibrosis and fibrosis scores were higher on3month than2month in the chronic myocardtis models. Part II:Study the role and mechanism of Th17cells in acute viral myocarditisObjective To investigate the role and mechanism of Th17cells in the acute myocardtis. Methods To detect the percentage of CD4+Th17cells and CD4+CD25+Treg cells on days7,10, and14in AVMC using flow cytometric. Specifically, the relationship between mRNA expression of IL-17/TGF-β and CVB3RNA were detected with real time-PCR in the myocardial tissues on day7after viral inoculation. Balb/c mice (n=70) were randomly divided into four groups:(1) Normal control group (n=10) mice were injected intraperitoneally (i.p.) with100μl of EMEM,(2) AVMC group (n=20) mice were injected i.p. with100μl of CVB3diluted in EMEM on day0to induce acute viral myocarditis after days7.(3) Isotype control group (n=20) mice were treated with CVB3i.p. on day0and isotype control IgG1Ab (100μg per mouse) i.p. on days3and5, and (4) IL-17mAb group (n=20) mice were treated with CVB3i.p. on day0and anti-mouse IL-17mAb (100μg per mouse) i.p. on days3and5. Firstly, CVB3RNA was determined with real-time PCR and the pathological scores were performed with H&E staining. Then, the percentages of CD4+, CD8+, CD4+/CD8+, CD4+Perforin+, CD8+Perforin+, CD4+CD25+Foxp3+T cells were investigated by flow cytometry in the control and AVMC groups. Then, the proteins expression of cardiac protein levels of IL-17A, TGF-β, IFN-y and VP1were examined by Western blot on day7in myocarditis upon neutralization of IL-17. Finally, the production of related cytokines of IL-6, IL-17A, IL-21, IL-10, IFN-γ, and IL-2were detected by Flowcytomix bead assay in the serum of different group mice. Results The results showed that the expression of splenic Th17cells and Th17-related cytokines (IL-17A, IL-21) markedly increased. Interestingly, the expression of splenic Treg cells and Treg-related cytokines (TGF-β, IL-10) also significantly increased. Using neutralization of IL-17in the AVMC, we found that Treg cells roughly decreased compared to isotype control mice. However, T cells and perforin dramatically increased, followed by a marked reduction in CVB3replication. On the other hand, the expression of IFN-γ protein was elevated in the myocardium when using neutralization of IL-17. Conclusion The results suggest that blockade of IL-17possibly contributed to viral replication through the action of regulatory T cells in mediating T cells and perforin response in AVMC. Part Ⅲ:In vivo delivery of adenoviral vector containing interleukin-17receptor A reduces cardiac remodeling and improves myocardial function in CVB3-induced chronic myocarditisObjective This study to investigate whether adenoviral transfer of IL-17receptor A would reduce myocardial remodeling and dysfunction in chronic viral myocarditis. Methods Balb/c mice were randomly divided into three groups, then the mice were injected i.p. with100μl of CVB3diluted in EMEM on day0,60,90to establish chronical viral myocarditis models,(a) PBS group (n=20); PBS were injected i.p with25μl at4day old, then with50μl of PBS at60day in the period of chronic viral myocarditis (b)Ad:IL-17R:Fc group (n=20):AdIL-17R:Fc were injected i.p with Ad-IL-17R:Fc in the period of chronic viral myocarditis.(c)Ad:null group (n=20), mice were injected with the Ad-null recombinant adenovirus was injected into the control group at the same time points. The turnover of myocardial injury and fibrosis to assess with hematoxylin&eosin (H&E) and picrosirius red staining in each group of mice, and the cardiac structure and function were determined by transthoracic echocardiography. Th17cells (CD4+IL17+) were detected in spleen of each group by flow cytometry. Expression of ADAMTS-1and MMPs/TIMPs in hearts of each group of mice was confirmed by western blot. In vivo, IL-17induces MMPs/TIMP protein expression, collagen deposition and cell proliferation in cultured cardiac fibroblasts using western blot and real-time PCR. Results In a mouse model of coxsackievirus B3(CVB3)-induced chronic myocarditis, delivery of adenovirus containing IL-17receptor A (Ad-IL17R:Fc) reduced IL-17A production and decreased Th17cells in the spleen and heart, leading to down-regulation of systemic TNF-α and IL-6productions. Cardiac function was significantly improved in Ad-IL17R:Fc compared with Ad-null treated mice3months after first CVB3infection. Ad-IL17R:Fc reduced left ventricle dilation and decreased the mortality in chronic viral myocarditis (56%in Ad-IL-17R:Fc versus76%in Ad-null group). These protective effects of AdIL17R: Fc on remodeling correlated with an attenuation of myocardial collagen deposition and a reduction of fibroblasts in CVB3-infected hearts, which was accompanied by down-regulation of A distintegrin and metalloprotease with thrombospondin type1motifs (ADAMTS-1), MMP-2, collagen subtypes Ⅰ and Ⅲ in the heart. In cultured cardiac fibroblasts, IL-17A induced expressions of ADAMTS-1, MMP-2, collagen subtypes Ⅰ and Ⅲ, and increased the proliferation of fibroblasts. On the other hand, when using the IL-17A induced with the inhibitors of U0126(ERK inhibitor), SB203580(p38inhibitor) and SP600125(JNK inhibitor), the expression of ADAMTS-1mRNA were elevated in ERK and p38inhibitor groups. Conclusions Delivery of IL-17receptor A reduces cardiac remodeling, improves function, and decreases mortality in chronic viral myocarditis possibly by suppressing fibrosis via mediating the balace of MMPs/TIMPs. Thus, adenoviral transfer of IL-17receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM. Moreover, IL-17A induced the expression of ADAMTS-1mRNA mainly via ERK and p38MAPK signaling pathway. Part Ⅳ:Effect of virus-induced DC on differentiation of Th17and Treg cells in acute myocarditis Objective To test the effect of and virus-induced DC on differentiation of Th17and Treg cells in acute myocarditis and its mechanisms. Methods Thirty Balb/c mice were randomized into groups of control(n=15) and AVMC (n=15). The DC was isolated from the spleen in each group. Then, the expression of CD40, CD80, CD86, and MHC-Ⅱ were detected in the bone marrow-derived dendritic cells (BM-DC) of each group by flow cytometry. When CD4+T cell and BM-DC cells were isolated from the Control or/and AVMC group(s) using MACS separation. Finally, these mixed cells were fully cultured for4days with the help of cytokines for Th17and Treg differentiation. We calculated the percent of Th17and Treg cells in each group by flow cytometry. And we detected the concentrations of IL-17and TGF-β in supernatant of each group by ELISA kit. Cell proliferation was examined in each group by CCK-8. Results The markers of BM-DC isolated from AVMC group significantly higher than control group, especially the CD40, CD80and MHC-Ⅱ; While the CD86maker was not significantly changed in the each group. When CD4+T cell and BM-DC cells were isolated from the control or/and AVMC group(s) using MACS separation, the purity of CD4+T cells was more than92%and the BM-DC(CDllc) was no less than95%by flow cytometry. Using the co-cultured with CD4+T cells and DC from Virus-DC and Control-DC groups, we found that the percentage of Th17cells were significantly grown in Virus-DC group by flow cytometry. However, the percentage of Treg cell has no signigicant difference between Virus-DC and Control-DC group. Moreover, the concentrations of IL-17were also evaluated in the supernatant of Virus-DC group by ELISA kit. While the concentrations of TGF-β1showed no obvious variations between Virus-DC and Control-DC groups. Conclusions CVB3was able to induce BM-DC to highly expression the markers of CD40, CD80, and MHC-Ⅱ compared to control group. When using co-cultured CD4+T cells and BM-DC from Virus-DC or Control-DC groups, we found that CVB3-induced BM-DC could promote the differentiation of Th17cells, not Treg cells. On the other hand, the IL-17cytokine was also higher in the Virus-DC group, but TGF-β has not changed in the Virus-DC group when compared to Control-DC.