| Cardiovascular disease is a leading cause of morbidity and mortality. Dilated cardiomyopa-thy (DCM) often results from coxsackievirus B3 (CVB3)-triggered myocarditis and may be present in up to one quarter of all cases of heart failure. Many affected individuals develop heart antigen-specific autoantibody responses. In animal models of cardiac myosin-induced myocarditis, pathogenic roles for CD4+ T cells and autoantibodies have been described. Al- though virus is important in acute myocarditis, the development of chronic myocarditis and cardiomyopathy may require exposure and release of cardiac myosin from damaged myocytes. To further test the autoimmune hypothesis of CVB3-induced myocarditis, we originated virus-free, experimentally induced autoimmune myocarditis (EAM) in the mouse. Suscepti-ble strains immunized with purified cardiac myosin in Complete Freund's Adjuvant (CFA) develop myocarditis. The cardiac lesions resemble the second (virus-independent) phase of CVB3-induced myocarditis. Strains of mice resistant to myocarditis develop minor disease. Therefore, it is likely that the mechanisms leading to genetic predisposition to autoimmune myocarditis are similar in both the virus-induced and antigen-induced experimental models, making EAM an effective tool in the study of the pathogenesis of virus-induced autoimmune disease.The mechanisms leading to susceptibility to autoimmune myocarditis are certainly multi-factorial and genetically complex. The major histocompatibility complex (MHC) genes in-fluence susceptibility to myocarditis. Several myocarditis-related non-MHC loci have been identified. To focus on the role of non-MHC genes on susceptibility to myosin-induced my-ocarditis, we used two prototypic mouse strains-A.SW and B10.S-that share the same MHC genes. A.SW is susceptible and B10.S is resistant to autoimmune myocarditis, respectively.Bone marrow cells from myocarditis-susceptible A.SW mice can render myocarditis-resistant B10.S recipient mice susceptible to myosin-induced myocarditis, indicating that hematopoietic cells express the genetic differences controlling susceptibility to autoimmune myocarditis. T and B lymphocytes were indispensable for transferring the susceptible phe-notype to disease-resistant recipients. T helper cells differentiate towards Thl, Th2, and Th17 cells in response to external signals. The differentiation depends on epigenetically controlled global changes in gene expression and it is regulated by cytokines and chemokines produced by T cells or other hematopoietic cells and the signaling pathways they activate. Thl cells secrete IL-2, IFN-γ, and TNF-αduring cell-mediated immune responses against intracellular pathogens and viruses. Recently, a new subset of helper T cells, Th17, which produces IL-17A, IL-17F, IL-10, IL-21, and IL-22 have been identified. IL-17 is also known to be a critical cytokine for regulating inflammatory responses.EAM represents a CD4+ T cell-mediated disease. Historically, two major CD4+ T cell subsets have been defined according to their cytokine production pattern:IFN-γ-producing Thl cells, and Th2 cells releasing IL-4. Recent data, however, suggest that EAM development critically depends on the IL-23-STAT4 axis promoting the expansion of another autoreactive CD4+ T cell subset characterized by IL-17 production. Our study has found that non-MHC genes influence the susceptibility to EAM by driving the CD4+ T cells towards Th17 cells and resistance to EAM by differentiating towards Thl cells.Our study is composed of four parts:Part one:Isolation of murine heart-specific myosin and induction of experimental autoim-mune myocarditis.We collected 180 murine hearts from different stain of mice, such as Balb/c, A/J, A.SW, C57L/B6 and so on, and isolated the myosin according the protocol. The emulsion was made by CFA and myosin with equal volume. We induced EAM in A.SW and B10.S mice by immunizing with murine cardiac myosin in CFA on day 0 and 7. A.SW displayed severe myocarditis compared to B10.S (2.91±0.84 vs 0.85±0.84, p=0.008) on day 21. The pathogenesis of immune-mediated myocarditis depends on genetic and environmental factors. The MHC genes influence susceptibility to myocarditis. Here, we decided to identify non-H2 loci controlling differential susceptibility to EAM. A.SW and B10.S mice share the same MHC (H-2S) haplotype, yet A.SW are susceptible to EAM while B10.S are resistant. These two inbred mice strains provide an opportunity to study myocarditis susceptibility factors while excluding the effects of MHC-associated genetic compontents. Part two:A.SW CD4+ T cells have higher ability to differentiate toward Th17 cells than B10.S CD4+ T cells.Genetically prone high-pathology A.SW mice have a higher proportion of CD4+ T cells in the heart and spleen (p<0.001) after immunization. Myosin specific T cells proliferation and activation were greater in A.SW mice (p<0.001). A.SW and B10.S had different proportions of T helper (Th) subsets in their hearts after immunization; A.SW had more Th17 cells (7.05±1.25 vs.2.24±1.05, p=0.003) and B10.S had more Thl cells (8.94±1.26 vs.2.9±1.06, p=0.001). In support of these in vivo data we found that CD4+ T cells from naive A.SW have higher potential to differentiate towards the Th17 pathway than CD4+ T cells from naive B10.S (11.93±3.72 vs.5.5±1.14, p=0.04). In contrast, B10.S T cells differentiated towards a Thl phenotype compared with that from naive A.SW (12.73±0.61 vs.8.14±0.85, p=0.002).Part three:The role of bone-marrow derived dendritic cells in the differentiation of Th cells.IL-23p19, which is important in the development and maintenance of Th17 cells, was increased in the heart and spleen of A.SW mice after immunization (p=0.03). IL-23 was secreted mainly from dendritic cells so we cultured bone marrow-derived dendritic cells from A.SW and B10.S mice to measure the cytokines. After dendritic cells were stimulated with LPS for 24 hours, TNF-1αand IL-12 secreated from A.SW dendritic cells were significantly increased compared with B10.S dendritic cells. There were no differences in TGF-β1, IL-6, IL-23 and IL-12 in the supernatant of A.SW and B10.S dendritic cells. The CD40 and CD86 were higher in A.SW dendritic cells than B10.S dendritic cells. Therefore, dendritic cells from A.SW are prone to be matured after stimulation. Part four:IL-6 receptor promotes the production of Th17 cells in A.SW mice regulated by non-MHC genes.Consistently, IL-6 receptor which is a key receptor for Th17 differentiation was higher in naive A.SW mice than B10.S (p=0.0002). The transcription factor of Th17 cells, RORγt, was significantly higher on A.SW Th17 cells than B10.S (p<0.05). We reported previously genetic crosses and linkage analysis revealed loci on Chromosome 1 (Eaml) and 6 (Eam2) were important in determining susceptibility to EAM. Interestingly, IL-17A and IL-17F genes are located on Chromosome 1. B10.A.Eaml congenic mice derive the Eaml locus from A.SW mice, while the remainder of chromosome 1 and the rest of the genome are of B10.S origin. The severity of myocarditis in B10.A.Eaml mice is medium compared with A.SW and B10.S mice. Consistently, the frequencies of CD4+ T cells in splenocytes and IL-6 receptor on naive CD4+ T cells were high in A.SW mice, media in B10.A.Eaml mice, low in B10.S mice.Our data suggest that the tendency of A.SW differentiate towards a Th17 phenotype contributes to their heightened susceptibility of autoimmune myocarditis, which is regulated by non-MHC genes.
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