DC及小胶质细胞经Th17/Treg介导参与糖尿病视网膜病变的作用机制

There is increasing interest in recent years to understand the immune-inflammatory mechanism in the pathogenesis of diabetic vascular complications. The dendritic cell (DC) is a key component of immunity and is critical in modulating CD4+T cell differentiation. The range of identified effectors CD4+T cell lineages has recently expanded with the description of an IL17-producing subset, called Th17, which is distinct from Th1 and Th2 lineages; while Treg cells play an indispensable role in maintaining immunological unresponsiveness. Regarding to the immune response, there is a new paradigm that the progression of immunity-related pathogenesis may be associated with skewed balance between Th17 and Treg toward Th17. The retinal microglia, a key component of retinal immunity, might be activated by specific pathway and served as local DC. Our previous study has demonstrated that the retinal glia was involved in the pathogenesis of retinal vascular damage. Furthermore, in a pilot study, we found that there was an increased ratio of Th17/Treg in patients with diabetic retinopathy (DR) compared with healthy controls. Based on these findings, we hypothesize that the DC and the resident retinal microglia might be activated by Toll-like receptors (TLRs) signaling, inducing excess of IL-6 and IL-23 production, which might drive the differentiation of Th17 cells and suppress the generation of Treg cells; we further postulate that this imbalance between Th17 and Treg cells exacerbates retinal vascular and neural injuries in diabetic retina. The goal of this study is to investigate the effect of DC and microglia on Th17/Treg polarization in DR. The four specific aims included: 1) To demonstrate whether the imbalance of Th17/Treg cells in patients is correlated with the pathogenesis progress of DR. 2) To determine the mechanisms of the activation of DC/microglia through the TLR signaling pathway; DC and microglia isolated from TLR2-/- and TLR4-/- knockout mice will be utilized to determine how TLR2/4 influence their activation. 3) To determine the mechanisms of DC/microglia in altering the balance of Th17 and Treg cells. The requirement of IL-6 and/or IL-23 produced by the activated DCs and microglia for shifting the balance of Th17 and Treg cells will be determined by establishing a cell co-culture assay and measuring some related cytokines. 4) To demonstrate the above hypothesis in diabetic retinopathy model and oxygen-induced retinopathy model in vivo. The proposed study may provide new evidence for immune response involved DC/ microglia activation and the imbalance of Th17/Treg in the earlier retinal pathologic changes in DR. It will expand our understanding on the immune-inflammatory mechanism in diabetic retinopathy and may be relevant for the development of new therapeutic strategy for vascular and neural injuries in DR.

免疫炎症在糖尿病并发症中的作用倍受关注。树突状细胞(DC)调控CD4+T细胞各亚群的增殖分化是免疫应答的核心环节; Th17细胞是新发现的致炎性CD4+T细胞，但其功能受Treg细胞抑制。视网膜小胶质细胞具有特殊的免疫调节能力, 经活化后也可发挥类似DC的功能。本课题组既往已证实视网膜神经胶质细胞参与糖尿病视网膜病变(DR)血管损伤；而我们近期发现DR患者可能存在Th17/Treg比例失衡。由此提出新假说：糖尿病性损害可能通过Toll 样受体通路, 活化DC和视网膜小胶质细胞，经IL-6,IL-23等细胞因子激活CD4+T 细胞特定的转录通路，引发Th17/Treg比例失衡，导致视网膜炎症损伤。本项目以DC及小胶质细胞为切入点，在细胞、动物及临床研究中, 探索循环DC及视网膜小胶质细胞的活化机制及其促发Th17/Treg免疫失衡参与DR的作用机制，为临床防治DR提供新的介入靶点和理论基础。

糖尿病性视网膜病变(DR)是最常见的糖尿病并发症之一，增殖期DR以视网膜新生血管形成为主要病理特征，但其机制目前仍不清楚。越来越多的研究发现DR是视网膜慢性免疫炎症介导的神经组织及微血管的损伤。本课题组前期研究也发现免疫细胞及其反应与视网膜神经、血管损伤密切相关，本项目即以致炎性Th17细胞和免疫调节性Treg 细胞的调节为切入点，创新性提出小胶质细胞及DC细胞可经TLR2/4受体活化后释放细胞因子IL-6、IL-23等，从而调控Th17/Treg 细胞比例，介导糖尿病视网膜神经血管损伤。我们的研究结果也证实了在糖尿病性视网膜病变患者的外周血中致炎性Th17细胞增加，免疫调节性Treg细胞减少，二者比例明显失衡。在体外细胞实验中发现TLR2、TLR4信号通路介导小胶质细胞活化，并可促进CD4+T细胞向Th17分化；在视网膜新生血管病变的小鼠模型中，发现视网膜小胶质细胞明显活化并呈M1极化表现，Th17/Treg细胞比例失衡，并利用TLR2/TLR4基因敲除鼠证实TLR2和TLR4介导小胶质细胞调控Th17/Treg失衡过程。从而证实病理刺激下，TLR2和TLR4信号通路活化后激活视网膜小胶质细胞，促进CD4细胞向Th17细胞分化，减少Treg细胞形成，介导Th17/Treg细胞比例失衡，导致视网膜神经和血管损伤的科学假说。该项目的研究成果至今已发表SCI论文4篇。本研究从免疫炎症的角度，探索了视网膜新生血管形成的新机制，拓展了我们对视网膜新生血管性疾病中免疫炎症因素的认识，为视网膜新生血管疾病的治疗提供了新思路和新方向。