| Summary: | 碩士 === 國立中正大學 === 分子生物研究所 === 104 === Multiple sclerosis (MS) is a Th1/Th17 CD4+ T cell-mediated chronic autoimmune disease caused by genetic and/or environmental factors. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model to study the MS. Because of myelin antigens presented by antigen presenting cells (APCs) and stimulated T cells that released inflammatory mediators and cytokines such as IFN-γ and IL-17 will destroy the myelin and central nerve system (CNS). In recent study, epigenetic regulation has been shown to involve in distinct T helper lineages differentiation and polarization. In our previous study, EAE mice pre treated with 5-aza-2′-deoxycytidine(5-Aza), an inhibitor of DNA nucleotide methylation transferase (DNMT), inhibited the disease through upregulated the expression of CD4+Foxp3+.In addition to the Treg cell, other T cells epigenetic pattern has not been detailed study in EAE model and MS. Therefore, we aimed to identify the cytokines of IFN-γ and IL-17A in DNA methylation pattern in vitro and in vivo EAE model. First part in this study, we carried out in vitro Th1 and Th17 polarization under 5-Aza treatment. We used bisulfite pyrosequencing to detect the regions of IFN-γ methylation pattern including intron1, promoter-53CG, CNS-6, CNS-22, CNS-54 and CNS2 of IL-17A. The CNS-6 in Th1 polarization and CNS2 site6 and site8 of IL-17A in Th17 polarization had significant difference after treated with 5-Aza. In Th1 polarization, protein level of IFN-γ was increased in CD4+ after treated with 5-Aza, however IL-17A was downregulated in Th17 polarization. The downregulation of Th17 cells were through cell apoptosis because more 7-AAD and annexin-V double positive cells was observed after treated with 5-Aza. Second part, we evaluated the methylation pattern of cytokine in different disease status in peripheral blood mononuclear cells (PBMC). In DNA level, we found the intron1 of IFN-γ was demethylated when EAE disease at peak. Older mice (more than 10 weeks) showed more demethylation and severe disease score than younger mice ( 6-8 weeks). CNS-6 of IFN-γ was demethylated when EAE score=1. Site8 and site10 in CNS2 of IL-17A was demethylated when EAE score at onset and peak. Summary, the above data show 5-Aza treatment will increase IFN-γ expression while inhibit IL-17A expression through Th17 apoptosis, and show these cis elements including CNS-6 of IFN-γ and CNS2 of IL-17A can be a tool to predict the time for EAE mice therapeutic treatment at early disease stage.
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