TREM-2/β-catenin介导的角膜保护作用及分子调控机制研究

Triggering receptors expressed on myeloid cells 2(TREM-2) is a new pattern recognition receptor, however, the role of TREM-2 in ocular immunity remains unclear. Our preliminary data showed that, bacterial infection induced TREM-2 expression in the mouse cornea, indicating that TREM-2 may participate in the ocular immune response. Moreover, we found that silencing of TREM-2 accelerated the disease progression and enhanced the viable bacterial number as well as pro-inflammaoty cytokine expression in the mouse cornea, suggesting that TREM-2 plays a protective role at the ocular surface. Next question is to explore the molecular mechanism of TREM-2 mediated corneal protection. Our preliminary data further demonstrated that the anti-inflammatory activity of TREM-2 is β-catenin-dependent. Although little is known regarding the specific ligand and downstream signaling of TREM-2, based on the literature and our preliminary data, we hypothesize that TREM-2 activates β-catenin signaling pathway by binding to its endogenous ligand heat shock protein 60 (HSP60), and protects the cornea by promoting bacterial clearance and by suppressing inflammtory response. This project will use different murine models of Pseudomonas aeruginosa keratitis to test the role of TREM-2 in bacterial keratitis and to explore the mechanism of TREM-2 mediated immune regulation. Furthermore, we will identify the molecular mechanism by which TREM-2 induced the activation of β-catenin. This project will provide substantive information for prevention and clinical treatment of bacterial keratitis.

TREM-2是新近发现的模式识别受体，然而其在眼免疫中的功能仍不清楚。预实验结果显示，细菌感染小鼠角膜诱导TREM-2表达，提示TREM-2可能与角膜免疫相关。沉默了TREM-2的小鼠角膜的细菌量和炎症因子表达上调，疾病加重，提示TREM-2参与角膜保护。那么，TREM-2介导角膜保护的分子机制如何？进一步实验发现，TREM-2的抑炎作用依赖于β-catenin。目前TREM-2的配体及下游信号通路仍不明确，结合文献报道和预实验结果，我们首次提出"TREM-2通过结合内源性配体HSP60激活β-catenin通路，进而促进细菌清除、抑制过度炎症，保护角膜"的假说。本项目拟通过抗体交联活化和RNA干扰沉默等动物模型及体外实验，从正、反两方面揭示TREM-2介导的角膜保护作用及其机制，确定在角膜炎中，TREM-2的关键配体及其激活β-catenin的分子机制，为细菌性角膜炎的防治提供理论依据。

本课题通过体内外实验，研究了铜绿假单胞菌（ Pseudomas aeruginosa,PA）感染条件下，髓样细胞抑制受体TREM2通过活化beta-catenin，抑制过度炎症，促进细菌清除，从而减轻角膜炎症损伤的作用和分子机制，取得的主要成果包括：.1, 体外实验揭示了 PA感染后通过激活NLRP3炎症小体诱导巨噬细胞自噬，从而抑制巨噬细胞对PA的胞内杀伤 (Infection and Immunity, 2015) 。.2, 体外实验揭示了TREM2通过激活beta-catenin抑制巨噬细胞焦亡，从而促进巨噬细胞对于PA的吞噬杀伤的作用机制(Cell Death & Diseases, in revision) .3, 体内实验揭示了TREM2在PA角膜炎中通过抑制Caspase-1介导的炎症小体活化，抑制角膜炎症，促进细菌清除，从而发挥角膜保护作用的机制(Journal of Infectious Diseases, in revision).4, 体外实验揭示了TREM-2通过PI3K/Akt通路促进巨噬细胞对PA清除的分子机制(Scandinavian Journal of Immunology, 2014).5, 体内实验揭示了TREM-2表达和PA角膜炎病程进展密切相关；并在小鼠模型中应用TREM-2 siRNA或活化抗体干预，揭示了TREM-2通过PI3K/Akt通路抑制过度炎症，促进细菌清除，发挥角膜保护作用 (Investigative Ophthalmology & Visual Science, 2013)。.6,通过体内和体外实验探明了beta-catenin在PA感染后的角膜组织和炎症细胞中的动态表达，并揭示了beta-catenin通过抑制角膜炎症、促进细菌清除，发挥角膜保护作用　(Journal of Infection, 2013)。.7,Co-IP实验证明HSP60不是TREM2内源性配体，而DAP12是介导TREM2和beta-catenin相互作用的关键分子。.上述工作揭示了TREM2和beta-catenin通过抑制炎症小体活化和巨噬细胞焦亡，从而抑制角膜过度炎症，促进细菌清除的分子机制，并发现PA感染诱导炎症小体活化和巨噬细胞焦亡可能是一种新的细菌免疫逃逸机制，为PA角膜炎及其他感染性疾病治疗提供了新的靶点和理论依据。