IL-34促进类风湿关节炎滑膜细胞表达OX40L上调Tfh的机制研究

It has been confirmed that follicular helper T cells (Tfh) are involved in B cell activation and the autoantibodies production, which are closely associated with the pathogenesis of rheumatoid arthritis (RA). The generation of Tfh is regulated by the costimulator OX40-OX40L signal pathway, but the distinct mechanism is still not clarified. IL-34 is a newly discovered cytokine related with the course of RA. Our preliminary results showed that IL-34 had no direct effect on the CD4+T cells, but could induce fibroblast-like synoviocytes (FLS) to express OX40L, and FLS activated by IL-34 could upregulate the formation of Tfh in the coculture system, and the expression of mammalian target of rapamycin (mTOR) and glucose transporter protein 1 (Glut1) in CD4+T cells was also increased. So it would be speculated that the OX40L on FLS induced by IL-34 could interact with the OX40 on the surface of CD4+T cells, and then activate mTOR signaling pathway. Thereby the series of results would enhance the intracellular glucose metabolism, which might contribute to the differentiation of Tfh. Consequently our research will further focus on the signal transduction pathways of OX40L expression on FLS induced by IL-34, meanwhile explore the main pathway of glucose metabolism in the cells induced by the activation of mTOR through OX40-OX40L interaction, and thereby clarify how the expression of Glut1 would be promoted by mTOR and how the glucose metabolism might promote the Tfh differentiation. Finally it will provide a new target for the early intervention and the theoretical basis of RA treatment.

滤泡辅助性T细胞(Tfh)促进B细胞活化及自身抗体产生，与类风湿关节炎(RA)发病密切相关。IL-34是近年发现的细胞因子，已被我们证实参与RA发病，但具体作用尚不清楚。预实验显示，IL-34对CD4+T细胞无直接作用，但其活化的RA滑膜成纤维细胞(FLS)却促进CD4+T细胞中Tfh产生；IL-34也增加FLS表达OX40L。推测OX40L与T细胞膜OX40作用上调Tfh产生。此外，IL-34活化的FLS上调CD4+T细胞哺乳动物雷帕霉素靶蛋白（mTOR）及葡萄糖转运蛋白（Glut1）表达。推测OX40L-OX40通路激活CD4+T细胞内mTOR，影响胞内糖代谢促进Tfh分化。本课题将进一步研究IL-34上调FLS表达OX40L的信号通路，深入探讨OX40-OX40L轴激活mTOR的分子机制，进而阐明mTOR上调Glut1影响胞内糖代谢同时促进Tfh分化的具体机制，为RA治疗提供新靶点。

滤泡辅助性T细胞(Tfh)促进B细胞活化及自身抗体产生，与类风湿关节炎(RA)发病密切相关。炎性微环境对于免疫细胞的活化至关重要，IL-34是近年发现的细胞因子，在本课题中，我们发现：RA患者血清IL-34水平升高，且在IL-34增高的患者中其含量与RA疾病活动度呈正相关关系。IL-34可通过结合滑膜成纤维细胞(FLS)表面IL-34受体而影响其功能，但是IL-34本身对CD4+T细胞无直接作用，同时发现IL-34刺激后的FLS与健康人CD4+T细胞共培养后，FLS协同刺激分子OX40L表达上调，且体系中Tfh数量增加，CD4+T细胞中葡萄糖转运蛋白1(Glut1)和哺乳动物雷帕霉素靶蛋白(mTOR)表达增高。IL-34可通过促进FLS表达OX40L与CD4+T细胞OX40结合，继而激活Glut1/mTOR通路，影响胞内葡萄糖代谢水平而引起Tfh的数量增加。IL-34可通过激活P38、AKT1/2、ERK、NF-κB等信号分子调控FLS增殖、迁移、细胞周期及分泌炎性介质等特性，其中PI3K-Akt信号通路在IL-34上调FLS表达OX40L分子中发挥非常重要的作用。IL-34上调FLS表面表达OX40L，与CD4+T细胞表面OX40结合，作为协同刺激信号促进CD4+T细胞的活化，在FLS分泌的相应细胞因子作用下促进Tfh细胞的分化。同时发现OX40/OX40L轴可激活mTOR信号通路，进而引起Tfh细胞内糖酵解的增加，拮抗mTOR及糖酵解关键酶HK-2，可使Tfh细胞数量减少，因此认为mTOR及糖酵解在促进Tfh细胞数量增加上发挥非常重要的作用。RA微环境中除了IL-34，TL1A可以介导FLS线粒体功能障碍，并通过上调ROS参与RA发病。此外，单细胞分析技术发现FLS并非是单一群体，它们也跟CD4+T细胞一样具有众多亚群，并且在疾病的进展中发挥不同的作用。我们进一步研究证实不同亚群FLS对Tfh细胞数量的维系发挥不同的作用，CD90+FLS使Tfh细胞数量更多，且IL-34对其具有明显的作用，相反，CD90-FLS对Tfh细胞增殖作用不大，对IL-34的反应性不明显。在本项目执行中，已发表相关SCI文章4篇，参加会议交流2次。综上所述，该课题为揭示RA内环境中细胞因子影响FLS和Tfh在RA发病中的相互作用提供重要理论和实验依据。