Perp在类风湿性关节炎外周Th17细胞存活中的作用研究

T helper type 17 cells (Th17) play a critical role in the pathogenesis of rheumatoid arthritis (RA), their secretion of IL-17A, IL-17F and IL-6 have been identified as important factors in articular cartilage and bone destruction via RANK-RANKL-OPG system. Peripheral Th17 cells from RA patients are significantly increased and abnormal survival, but the reason for its occurrence is unknown. Our preliminary gene chip screening found the reduced expression of Perp in collagen induced arthritis (CIA) rat model, and we validated in RA patients, Perp expression was also significantly lower in PBMCs of patients, and the Perp abnormal expression levels were significantly negative correlated with IL-17 mRNA and disease activity. Perp encoded by the PERP gene, belongs to the tetraspan membrane proteins, and is widely expressed in immune tissues and organs. Recent studies have shown the regulation function of Perp in lymphocyte survival and secretion of inflammatory cytokines. Taking together, we speculate that Perp might be involved in pathogenesis of RA through regulation of circulating Th17 cells survival. We will isolate peripheral CD4+ T cells of RA patients, and ensure that the abnormal Perp expression are related to apoptosis resistance of Th17 cells under the death signal stimulation; and establish the collagen-induced arthritis (CIA) mouse model on the background of CD4-cre Perp conditional knockout mice to study in-depth the function of Perp in peripheral Th17 cells survive, and the impact of the pathogenesis of RA. Furthermore, we will explore the molecular mechanisms between the decreased Perp expression and peripheral Th17 cells frequency imbalance, and this research will provide a new theoretical basis and experimental foundation for basic research and immunotherapy of RA.

Th17细胞在类风湿性关节炎（RA）的发病中发挥重要作用，其分泌的IL-17等细胞因子直接参与滑膜炎性改变、关节软骨以及骨的破坏。RA病人外周循环Th17细胞明显增加且存活异常，但原因不明。我们通过前期基因芯片筛选发现大鼠胶原诱导关节炎（CIA）模型中Perp表达降低，并进一步在RA病人PBMCs中证实Perp表达也明显降低，且Perp的异常表达还与IL-17以及疾病活动度评分等呈显著负相关。Perp是tetraspan膜蛋白家族成员，近期研究发现Perp与T淋巴细胞存活以及细胞因子分泌密切相关。由此推测，Perp很可能通过调控外周Th17细胞存活影响RA发病。我们将利用CD4-Cre Perp条件敲除鼠建立小鼠关节炎模型，从患者、动物、细胞及分子水平深入研究Perp调控外周Th17细胞存活影响RA发病的作用及机制，为RA的基础研究和免疫治疗提供新的理论依据和实验基础。

Th17细胞是近年来被发现的一类新型的分泌IL-17的CD4+T辅助细胞，大量临床前和临床数据均表明Th17细胞与自身免疫性疾病密切相关。如Th17细胞在RA的发病中存在重要作用，其分泌的IL-17直接参与滑膜炎性改变、关节软骨以及骨的破坏，临床研究亦发现RA病人外周Th17细胞显著增加且存活异常，但原因不明。前期我们通过基因芯片筛选发现大鼠CIA模型中Perp表达显著下调。Perp是tetraspan膜蛋白家族成员，近期研究发现Perp与T细胞存活以及细胞因子分泌密切相关。在本项目中，我们在预实验研究基础上扩大RA病人及健康对照样本，证实RA病人PBMCs中PerpmRNA表达水平较健康对照明显降低，且Perp mRNA的异常表达还与PBMCs中IL-17 mRNA的表达以及DAS28、RF、anti-CCP、ANA、CRP和ESR等临床指标亦呈显著相关。以上结果本人做为通讯作者已发表在CLIN DEV IMMUNOL（IF：3.064）。利用Perp-cKO小鼠，在体外研究中发现Perp显著调控外周Th17细胞的存活（对分化无影响），对Th1、Th2和Treg细胞的存活无显著影响。Perp-/- Th17细胞表现出显著的凋亡抵抗，其Smac以及活化的Caspase-3的表达水平显著降低。由于C57BL/6小鼠的CIA的发病率不稳定，我们采用另一种Th17细胞介导的自身免疫性疾病，MS的动物模型EAE。在体内研究中发现，Perp-cKO小鼠EAE的疾病活动度与WT小鼠相比显著增加，发病时间提前，脊髓的淋巴细胞浸润及脱髓鞘程度亦显著增加，且外周血中IL-17的水平显著提高，脾脏Th17细胞比例亦显著增加。以上结果已整理成文“Loss of Perp leads to exacerbation of EAE by modulating the survival, but not differentiation of peripheral Th17 Cells”，拟发表在较高IF的SCI杂志。总之，本项目首次从病人、动物模型、细胞及分子水平研究并发现Perp可以调控外周Th17细胞存活，进而影响自身免疫性疾病的发病，为自身免疫性疾病的基础研究和免疫治疗提供新的理论依据和实验基础。