A20（TNFAIP3）在葡萄膜炎发病机制中的作用研究

Behcet's disease(BD) and Vogt-Koyanagi-Harada(VKH) syndrome are the two most common uveitis entities that lead to blindness in china. The imbalance of the number and function between effectory and Treg cells play a critical role in the pathogenesis of the two diseases. DCs play critical role in the differention and fucntion of different Th cells. Our preliminary studies showed that A20 was significantly decreased in the PBMCs from active BD patients as compared with that from inactive BD patients and normal controls, and there was a strong association between the gene polymorphisms of A20 with Behcet's disease and VKH disease. The goal of this project was to investigate the role of A20 in the pathogenesis of the aforementioned two uveitis entities. First, we will study the expression of A20 in the Mo-DC from BD patients and VKH patients to explore whether A20 was associated with the activity of the two uveitis. Secondly, we will investigate the effect of A20 on the differentiation, maturation and function of DCs and the subsequent different Th1, Th17 and Treg cell differentiation. Thirdly, we are going to study the effect of A20 on the phosphorylation of P38, ERK1/2 and JNK and the expression of NLRP3 inflammasome to clarify the mechanism involved in A20-mediated effect on DCs. Fourth, we will investigate the effect of A20 on the severity of experimental antoimmune uveitis(EAU) to validate the regulatory role of A20 in uveitis. Finally, we will assess the possible role of associated genetic polymorphisms in the expression and structure of A20, the activation and function of DCs. These studies are expected to clarify the pathogenesis of the two uveitis and provide a novel strategy in the prevention and treatment for these diseases.

Behcet病、VKH综合征是我国致盲率最高的葡萄膜炎类型，Th1/Th17/Treg细胞功能和数量异常是其发病基础，上述三种细胞失衡可上溯至树突状细胞（DC）活化及功能异常。我们最近预实验发现A20在活动期Behcet患者中表达显著降低，而且A20基因多态与Behcet病和VKH综合征显著相关。本项目以这两种疾病为研究对象，探讨A20在这两种患者DC中的表达水平、A20对DC活化、功能状态以及对Th1、Th17和Treg细胞分化的影响；探讨TLR信号通路中MAPK（P38、ERK1/2、JNK）的磷酸化水平和NLRP3炎症小体的表达等来确定A20对DC影响的机制；通过对A20的干预来探讨A20对动物模型葡萄膜炎的严重程度的作用；探讨A20基因变异对其本身基因翻译和结构、DC活化和功能、动物模型葡萄膜炎的影响。本研究旨在以A20为出发点阐明这两种不同性质疾病的发病机制，为其防治提供新策略。

A20，又名肿瘤坏死因子诱导蛋白3（TNFAIP3）,A20 是天然免疫受体如TNF受体，TOLL样受体和细胞内模式识别分子NOD2所介导的NF-κB活化终末信号所必需的分子。A20通过对调节TRAF6的去泛素化功能减弱NF-kB信号系统的活性，从而发挥对天然免疫反应的负性调节作用。葡萄膜炎是我国常见致盲性眼病之一，其中Behcet病和VKH为两种重要的葡萄膜炎类型。本课题通过检测Behcet病和VKH综合征患者PBMCs中A20 mRNA的表达水平，异常低表达的A20与Behcet病的发病有相关性。DC细胞作为抗原提呈细胞在免疫调节中起到重要作用，我们进一步发现BD患者外周血单核细胞诱导的DC细胞中A20的表达也低于正常人。随后，我们通过沉默DC细胞中A20 的表达，发现沉默A20后DC细胞分泌的IL-10和IL-27显著降低，而IL-1β和IL-6的表达显著上升。通过混合淋巴实验，我们进一步验证了A20对DC细胞的成熟以及抗原提呈作用有显著影响，并且这一作用与MAPK信号通路的激活有关。进一步我们发现在EAU的发病过程中，小鼠视网膜组织中A20的表达水平在炎症高峰期时显著降低，所以我们通过构建腺相关病毒过表达载体，局部调高眼局部组织中A20 的表达水平后诱导EAU。我们发现A20多表达能显著抑制EAU小鼠在炎症高峰期时的临床变现，并且抑制视网膜结构破坏。通过检测相关的炎症因子，我们发现A20过表达能显著抑制炎症因子MCP-1、TNF-α、IL-6、IL-10、IL-17和IFN-γ表达，并且抑制CD4+T细胞相关的转录因子T-bet、RORγT、GATA3和FOXP3的表达。综上所述，A20在葡萄膜炎的发病过程中起到重要的负性调控作用，这为今后葡萄膜炎的治疗提供新的靶点。在该项目资助下，共发表SCI论文5篇，待发表论文1篇。培养硕士研究生9人。项目投入73万元，支出43.82万元，各项支出与预算相符。剩余经费24.68万元，剩余经费计划用于本项目后续研究以及研究生的培养。