The Dynamic Research Of Th17/Treg Cells And Relative Inflammatory Cytokines Expression During Autoimmune Uveitis

Purpuse: 1. To study the expression of Th17 cells, Treg cells and related cytokines in Behcet's disease, VKH syndrome-- two kinds of representative uveitis, and their different expression in disease different periods. 2. To observe the dynamic changes of Th17 cells,Treg cells and related cytokines expression during the experientmal autoimmune uveitis(EAU). We also analyzed whether the balance of Th17/Treg was involved in EAU models.Methods: 1. Collecting 4 ml peripheral venous blood of Behcet's disease, syndrome of VKH patients in activity and resting stage and healthy people. Using flow cytometry to analyze the frequency of Th17 and Treg cells. The levels of cytokines(IL-17, IL-6, IL-10,TGF-?) in serums were detected by ELISA. 2. EAU was induced in Lewis rats(6-8 weeks)by injection of complete Freund adjuvant(CFA) containing interphotoreceptor retinoid-binding protein(IRBP1177-1191) and tuberculin(TB) into footpads, napes and back of the body. Clinical assessment by Genesis-D camera examination of ocular inflammation was conducted daily after immunization. EAU models were evaluated pathologically on days 9, 13, 18 and 23 post immunization. The rats were put to death on days 9, 13, 18, 23,28, 35 and 48 post immunization. Lymphocytes isolated from lymph nodes and spleens were colleced by Ficoll-Hypaque density gradient centrifugation. Then the Lymphocytes were subjected to flow cytometry to analyze the frequency of Th17 and Treg cells. The levels of cytokines(IL-17, IL-6, IL-10, TGF-?) in serums were detected by ELISA at different time points post immunization. Real-time quantitative PCR(RT-PCR) was used for measuring the m RNA expression level of IL-17, IL-6, TGF-? and Foxp3 in lymph nodes and spleens at different time points post immunization.Results:1. By flow cytometry, we observed that the percentage of Th17 cells in peripheral blood of Behcet's disease and VKH syndrome patients in activity period with higher proportion, and the differences are statistically significant compare to stationary phase and healthy controls. The percentage of Th17 cells Behcet disease and VKH syndrome patients and the health control group no statistical difference. The percentage of Treg cells in peripheral blood of Behcet's disease and VKH syndrome patients in activity period with lower proportion, and the differences are statistically significant compare to stationary phase and healthy controls. The percentage of Treg cells Behcet disease and VKH syndrome patients and the health control group no statistical difference. By ELISA, we observed that the changes of IL-17, IL-6 and TGF-? in serums and the changes of the percentage of Th17 cells had consistent trend. While the changes of IL-10 and the changes of the percentage of Treg cells were consistent. 2. The ocular inflammation of EAU models was observed on day 7-8, peaked on day 13, then ocular inflammation was gradually resolved and no inflammation was observed on day 21. The results of histopathologic examination were in conformity with the camera observations. By flow cytometry, we observed that the percentage of Th17 cells from EAU models lymph nodes and spleens were all signaficantly higher on day 9(P<0.01 compared to normal rats). And they peaked on day 13, then gradually decreased. There were statistically difference on day 23(P<0.05),and there was no statistically significant difference between rats on day 28 and normal rats.The frequency of Treg cells in lymph nodes was observed a signifacatnly higher on day13(P<0.01),in contrast, the frequency of Treg cells in spleens was observed a signifacatnly higher on day 9(P<0.01), and they all reached peak on day 18. The percentage of Treg cells in lymph nodes and spleens still maintained a high level on day 48(P < 0.01 compared to the normal rats). The ratio of Treg/Th17 in lymph nodes was observed significantly higher on day 18(P<0.05), in contrast, the ratio of Treg/Th17 in spleens was observed significantly higher on day 23(P<0.01), and they all reached peak on day 28. And this ratio still maintained a high level on day 48(P < 0.01 compared to the normal rats). By ELISA,we observed that the changes of IL-17 and IL-6 in serums, the changes of the percentage of Th17 cells and ocular inflammatory changes during EAU course had consistent trend.While the changes of IL-10 and TGF-? in serums and the changes of the percentage of Treg cells were consistent. Th17 and Treg cells expression in gene level were confirmed by real-time quantitative PCR.Conclusions: 1. Th17 cells proportion and related cytokine levels of uveitis patients in activity period were elevated, and proportion of Treg cells and related cytokines levels drop, so the imbalance of Th17 / Treg cells exists in patients with uveitis. 2. The percentage of Th17 cells and Th17 related cytokines were all observed a signifacatnly higher at initiation phase of uveitis, a peak at effector phase, a normal level at recovery phase of uveitis, which were consistent with ocular inflammatory changes of EAU rats.While the percentage of Treg cells and Treg related cytokines and transcription factors all peaked at inactive phase of uveitis, and they maintained a high level until the recovery phase of uveitis. So the balance of Th17/Treg was involved in EAU models and this balance play crucial role in EAU progress.