Wang YaoYao, Shu Ye, Xiao YangFan, Wang Qing, Kanekura Takuro, Li YaPing, Wang JiuCun, Zhao Ming, Lu QianJin, Xiao Rong
Department of Dermatology, Second Xiangya Hospital, Central South University, 139 Ren-Min Road, Changsha, 410011 China ; Department of Dermatology, Sir Run Run Shaw Hospital, Zhejiang University, 3 East Qingchun Road, Hangzhou, 310016 China.
Department of Dermatology, Hunan Children's Hospital, 86 Zi-Yuan Road, Changsha, 410007 China.
Clin Epigenetics. 2014 Nov 11;6(1):25. doi: 10.1186/1868-7083-6-25. eCollection 2014.
The pathogenesis and etiology of systemic sclerosis (SSc) are complex and poorly understood. To date, several studies have demonstrated that the activation of the immune system undoubtedly plays a pivotal role in SSc pathogenesis. Activated immune effector T cells contribute to the release of various pro-inflammatory cytokines and drive the SSc-specific autoantibody responses. This, and a profibrotic environment, are all-important components of abnormal active immune responses that can lead to pathological disorders of SSc. CD11a is essential to inflammatory and immune responses, regulating adhesive and co-stimulatory interactions between CD4(+) T cells and other cells. Although CD11a is overexpressed in SSc patients, the mechanisms leading to this overexpression and its consequences remain unclear. DNA methylation, a main epigenetic modification, plays an important role in the regulation of gene expression and is involved in the pathogenesis of autoimmune diseases. This work aims to investigate the effect of DNA demethylation on CD11a expression in SSc CD4(+) T cells and to determine its functional significance. CD11a expression was measured using RT-PCR and flow cytometry. Bisulfite sequencing was used to determine the methylation status of the CD11a regulatory region. CD4(+) T cells were co-cultured with antigen-presenting cells, B cells, or fibroblasts with and without anti-CD11a, and proliferation of CD4(+) T cells, IgG production by B cells, and expression levels of COL1A2 mRNA by fibroblasts were evaluated.
Elevated CD11a expression levels were observed in CD4(+) T cells from SSc patients; these levels were found to be positively correlated with disease activity. The methylation levels of the CD11a regulatory sequences were lower in SSc patients than in controls and inversely correlated with CD11a mRNA expression. Treatment of CD4(+) T cells with 5-azacytidine (5-azaC) decreased CD11a promoter methylation and caused CD11a overexpression. SSc CD4(+) T cells and 5-azaC-treated CD4(+) T cells showed increased proliferation of CD4(+) T cells, increased production of IgG by co-cultured B cells, and induced expression of COL1A2 mRNA by co-cultured fibroblasts. These stimulatory effects were abrogated by anti-CD11a.
Demethylation of CD11a regulatory elements and subsequent CD11a overexpression in CD4(+) T cells may mediate immunological abnormalities and fibrotic processes in SSc.
系统性硬化症（SSc）的发病机制和病因复杂，目前尚不清楚。迄今为止，多项研究表明，免疫系统的激活无疑在SSc发病机制中起关键作用。活化的免疫效应T细胞有助于释放各种促炎细胞因子，并驱动SSc特异性自身抗体反应。这以及促纤维化环境，都是异常活跃免疫反应的重要组成部分，可导致SSc的病理紊乱。CD11a对炎症和免疫反应至关重要，它调节CD4(+) T细胞与其他细胞之间的黏附及共刺激相互作用。虽然CD11a在SSc患者中过度表达，但其过度表达的机制及其后果仍不清楚。DNA甲基化作为一种主要的表观遗传修饰，在基因表达调控中起重要作用，并参与自身免疫性疾病的发病机制。本研究旨在探讨DNA去甲基化对SSc患者CD4(+) T细胞中CD11a表达的影响，并确定其功能意义。采用逆转录聚合酶链反应（RT-PCR）和流式细胞术检测CD11a表达。亚硫酸氢盐测序用于确定CD11a调控区的甲基化状态。将CD4(+) T细胞与抗原呈递细胞、B细胞或成纤维细胞共培养，添加或不添加抗CD11a抗体，评估CD4(+) T细胞的增殖、B细胞产生IgG的情况以及成纤维细胞中COL1A2 mRNA的表达水平。
在SSc患者的CD4(+) T细胞中观察到CD11a表达水平升高；这些水平与疾病活动度呈正相关。SSc患者中CD11a调控序列的甲基化水平低于对照组，且与CD11a mRNA表达呈负相关。用5-氮杂胞苷（5-azaC）处理CD4(+) T细胞可降低CD11a启动子甲基化并导致CD11a过度表达。SSc患者的CD4(+) T细胞和经5-azaC处理的CD4(+) T细胞表现出CD4(+) T细胞增殖增加、共培养的B细胞产生IgG增加以及共培养的成纤维细胞中COL1A2 mRNA表达诱导增加。这些刺激作用被抗CD11a抗体消除。
CD11a调控元件的去甲基化以及随后CD4(+) T细胞中CD11a的过度表达可能介导了SSc中的免疫异常和纤维化过程。
