TLR4-MyD88信号途径在天然免疫与自身免疫性葡萄膜炎发病机制中的作用研究

The imbalance of immune microenvironment caused by microbial infection is critical to the pathogenesis of autoimmune diseases such as uveitis. The proposal is to study the role of lipopolysaccharides (LPS), the major component of a common infectious Gram-negative bacteria, in the pathogenesis of human autoimmune uveitis using a mouse model of experimental autoimmune uveitis (EAU). We will determine (1) the production of pro-inflammatory cytokines by TLR4-MyD88 signal-activated dendritic cell (DC)s with microarray and real-time PCR assays; (2) the role of the critical cytokines in the regulation of the Th1/Th17 effector T cell development and function in EAU with specific silencing siRNA and blocking antibodies; (3) the effect of the activated DCs on chemokine receptor expression and migration of T cells using flow cytometric and chemotaxis analysis; (4) the immunregulation role of regulatory T cells by LPS-activated DCs using the in vitro cell proliferation assay and the in vivo EAU mouse model.The goal of this proposal is to further clarify the direct relation between the infection-caused immunologic disturbance and the etiology of infection-related immune diseases including uveitis, and thereby to explore a novel therapeutic approach for these diseases.

病原微生物的持续感染导致机体内环境免疫应答紊乱是自身免疫性疾病发生的重要原因。本项目以实验性葡萄膜炎（EAU）为模型，观察常见感染菌菌壁成份脂多糖（LPS）在自身免疫性葡萄膜炎的致病作用：（1）用Microarray和real-time PCR方法，观察树突状细胞(DC)其TLR4-MyD88信号途径激活后的炎症因子表达；（2）用RNA干扰和/或中和抗体等手段，剖析DC活化后所产生的关键因子在调控Th1/Th17效应T细胞的产生及其在葡萄膜炎致病中的作用；（3）应用流式细胞仪及趋化试验，观察活化的DC对T细胞趋化因子受体表达、迁移的影响；（4）用增殖抑制试验及EAU模型，观察DC的MyD88途径对调节性T细胞功能的调节作用。本项目的完成将深入阐明感染导致机体的免疫损伤与葡萄膜炎发病及其与感染相关免疫性疾病的直接关系，为该类疾病的防治提供理论依据。

葡萄膜炎是一种常见的致盲疾病，其发病机制并未完全阐明。我们在小鼠实验性葡萄膜炎模型（EAU）中发现，MyD88基因敲除小鼠的发病程度减轻。本项目这一研究基础上，进一步研究了HMGB1-TLR4-MyD88信号通路在葡萄膜炎中的发病机制。我们在研究中发现，分离WT和MyD88-/- 小鼠的DC进行体外刺激后，MyD88-/- DC分泌的细胞因子和趋化介素的水平都下降，而且MyD88-/- DC的抗原呈递能力下降，同时在体内和体外促进T细胞分化为效应T细胞的能力也下降，最终导致了小鼠EAU发病减轻。进一步研究发现，在MyD88-/-过表达TNF-α能够拯救MyD88-/-DC带来的抗原呈递和促进T细胞分化能力的下降。我们还发现，TLR4-/-小鼠EAU发病情况同对照组相比无显著性差异，而HMGB1作为TLR4的重要配体，能够显著促进小鼠EAU的发病。这些结果说明HMGB1-TLR4-MyD88信号通路在EAU发病中起重要作用。本研究结果进一步阐明了HMGB1-TLR4-MyD88信号通路调节葡萄膜炎的发病机理，为该疾病的防治提供了新的靶点。