探讨PGRN在葡萄膜炎发病中的作用及机制

Behcet’s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome are the two most common uveitis entities and the leading cause of blindness in China. The disorder of Th1, Th17 and Treg cells in quantity and function are critical to the development of both diseases. Recent studies found that progranulin (PGRN) had an important negative regulatory effect on immune response and our recent preliminary experiment results revealed there was a decreased expression of PGRN in active BD and VKH patients. The project is therefore designed to investigate the role PGRN in uveits and the mechanism involved in. Administration of recombinant protein of PGRN or gene deficiency are preformed in animal model to confirm the role of PGRN in the occurrence, development and prognosis of uveitis and effect of PGRN on differentiation of Th17, Th1 and Treg cells. We also use retinal pigment epithelial cells as carrier to detect the role and the possible pathway of PGRN on the local immunoregulatory cells. Collectively, the aims of these studies are to disclose the role of PGRN in the pathogenesis of uveitis and to search for new strategies for the prevention and treatment of uveitis.

Behcet病、Vogt-小柳原田综合征是我国致盲率最高的葡萄膜炎类型，Th1、Th17和Treg细胞数量及功能紊乱是其发病基础。最近研究发现颗粒素蛋白前体（progranulin，PGRN)具有免疫调节作用，我们预实验发现活动期Behcet病和VKH综合征患者PGRN表达显著降低。因此本项目以PGRN为研究切入点，以葡萄膜炎动物模型和视网膜色素上皮细胞为研究对象，通过给予外源性重组PGRN或PGRN基因缺陷正反两方面验证PGRN对葡萄膜炎动物模型发生、发展及转归的影响及对Th17、Th1和Treg细胞分化和功能的影响；探讨PGRN对眼局部视网膜色素上皮细胞的作用及其具体通路。通过上述研究，以期阐明PGRN对葡萄膜炎的影响及机制，为葡萄膜炎防治研究寻找到重要靶分子。

目的背景:颗粒素蛋白前体(PGRN)在一些自身免疫性疾病中被认为具有重要的免疫调节作用，此项目主要研究PGRN在葡萄膜炎及其对应的实验性自身免疫性葡萄膜炎(EAU)和实验性自身免疫性脑脊髓炎(EAE)中的作用及机制。.方法:采用ELISA法测定BD、VKH患者及正常对照组血清PGRN水平，用B10RIII、WT和PGRN-/-小鼠诱导EAE和EAU，评价PGRN对这两种免疫介导疾病模型发生和发展的影响。采用ELISA、流式细胞仪和Real-time PCR检测局部和全身免疫改变。RNA测序检测PGRN发挥作用的关键基因和关键信号通路。.结果:活动性BD和VKH患者PGRN表达显著降低;重组PGRN显著降低EAU严重程度和体内免疫系统中Th17和Th1细胞频率。PGRN基因缺陷小鼠EAU和EAE疾病炎症严重程度严重上调，并伴有体内Th1和Th17细胞频率显著增加和调节性T (Treg)细胞频率减少;体外研究发现，rPGRN可抑制IRBP161-180特异性Th1和Th17细胞分化，促进Treg细胞的分化。但PGRN可以促进naïve CD4+T细胞向非抗原特异性Treg细胞极化，并伴随信号通路中STAT5磷酸化水平上调。通过RAN测序，我们发现了5个关键基因，包括Tnf、Il6、Il1b、Cxcl2和Ccl2，以及在PGRN-/- EAU小鼠中最显著富集的MAPK和Tnf信号通路。同时我们发现PGRN-/-小鼠EAE炎症严重程度的增加与体内M2向M1巨噬细胞转化有关。.结论:我们的研究结果表明PGRN在EAU和EAE中具有重要的保护作用，这些研究提示PGRN在防治Th1/Th17介导的自身免疫性疾病可作为免疫调节治疗靶点。