Jagged1/Notch1信号途径调控Tc17细胞功能参与类风湿关节炎的实验研究

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different CD8+T cell subsets, especially the newly found subset Tc17 cells and the putative role of the Notch pathway on Tc17 cell effector function in rheumatoid arthritis (RA). Jagged1 is able to deliver an indirect negative signal into CD8+T cells in vivo, which suggests its therapeutic potential in the treatment of CD8+T cell-mediated diseases, including RA. Notch signalling is engaged in collagen II (CII)-specific Th1- and Th17-type expansion in which Notch3 receptor and Delta-like 1 ligand were involved. Selective inhibition of Notch signalling mediated by Notch3 may offer a new strategy for the treatment of RA. In our previous study, we found another CD8+T cell subset also producing interleukin-17 (IL-17), defined as Tc17, was elevated in RA patients and correlated positively with disease activity. Notch1 and Notch3, especially Notch1, were elevated on Tc17 cells in RA patients, and correlated positively with the levels of IL-17. Jagged1/Notch1 pathway may be involved in development and differentiation of Tc17 cells. Tc17 play an important role in RA, so it is necessary to explore the role of Jagged1/Notch1 pathway in Tc17 cells and the pathogenesis of RA. In the present study, we will study further the role of Jagged1/Notch1 pathway in the differentiation, expression and IL-17 production of Tc17 cells including collagen induced arthritis (CIA) mouse models and RA patients, by in vitro and in vivo experiments and Jagged1-Fc fusion protein, Notch1 monoclonal antibody and the γ-secretase inhibitor DAPT were used to observe the effect of blockade the Notch pathway on Tc17 function. The frequency of Tc17, its IL-17 and Notch1-4 receptor expression were detected by using flow cytometry; IL-17, Notch1-4 and NUMB mRNA were examined by RT-PCR; the levels of IL-17 in sera and supernatant of cell culture were detected by ELISA. Notch1 expression was elevated by gene transduction and reduced by RNA interference, in order to provide a new strategy for the target treatment of RA by Notch.

保守进化的Notch信号途径在CD8+T细胞的效应功能中发挥关键作用，为研究类风湿关节炎（RA）的发病机制提供了新的突破口和治疗靶点，但是否通过调节CD8+IL-17+T（Tc17）细胞亚群起作用尚不清楚。本项目在率先证实“Tc17细胞比例与功能异常是RA的重要发病机制之一，Tc17表达Notch1水平升高”的基础上，拟借助RA动物模型（胶原诱导的关节炎，CIA）和病人标本，采用受体的单抗或γ-分泌酶抑制剂阻断Notch信号途径，观察Notch配体Jagged1的融合蛋白对CIA关节炎病情、Tc17细胞分化的影响，明确Notch途径通过调控Tc17细胞参与RA发病；再通过基因转染及RNA干扰技术分别增强或减弱Notch1的表达，明确Jagged1/Notch1是参与Tc17细胞功能和分化的主要信号通路。从而为深入阐明RA发病机制和发展基于干预Notch信号途径的免疫治疗提供新的科学依据。

Notch同类分子在类风湿关节炎（RA）局部炎症组织和培养的滑膜细胞以及外周血辅助性T淋巴细胞中的表达均已有报道，但Notch 相关分子在RA外周血CD8+IL-17+（Tc17）细胞中的表达方式还不清楚。在本研究中，我们首先检测了Notch 受体及其下游分子在RA患者Tc17细胞中的表达方式。结果显示，活动期RA患者外周血中Tc17 细胞表达Notch1、3（主要是Notch1）受体水平升高，而Notch2、4 表达水平与健康对照组（HC）无显著差异；RA患者外周血Tc17细胞中Notch1、3（主要是Notch1） mRNA表达上调；RA患者Tc17细胞中Notch信号的负调节基因 NUMB表达下降；与对照组相比，RA患者血清、关节液中的IL-17水平均升高；RA患者关节液中IL-17水平高于外周血清。本研究结果表明，RA患者Tc17 细胞上Notch 受体的表达方式发生了显著改变，与健康对照相比，Notch1信号活性增强且与病情活动期有关。. 另外，不同的Notch配体在功能性T细胞分化过程中各具特征。然而，每种Notch 配体在自身免疫性疾病中的确切作用或用于治疗的可能性还远未明了。在本研究中，我们进一步检测了Jagged1是否能通过改变Tc17细胞反应调控胶原诱导的类风湿关节炎（CIA）模型。结果显示，在II型胶原（CII）初次免疫前甚至在免疫后注射可溶性Jagged1编码质粒sJag1-P可抑制CIA的病情严重性，降低促炎细胞因子（IL-17、TNF-α、IFN-γ、IL-6）水平而增加抑炎细胞因子（IL-10）水平。本研究结果表明，Jagged1 能够在体内向Tc17细胞传递一个间接的负性信号，对CIA小鼠发挥保护作用。. 综上所述，Jagged1可能对Tc17细胞介导的疾病，包括RA具有治疗作用，为RA靶向治疗提供了新的实验依据。