防御素抑制巨噬细胞自噬对角膜抗感染免疫的影响及其机制

As a family of antimicrobial peptide, defensins play an important role in the anti-infectious immunity.We previously reported that beta-defensin (BD) 2 and 3 play a protective role in Pseudomonas aeruginosa (PA) keratitis by enhancing bacterial clearance. However, the regulatory mechanism remains unknown. Our preliminary data showed that BD2 and BD3 inhibited the PA-induced autophagy in macrophages and that autophagy resulted in impaired phagocytosis and bacterial clearance.This observation suggests that BD2 and BD3 may promote macrophage-mediated bacterial killing by decreasing the autophagy. To further explore the signaling pathway involved, we examined the totoal and phosphorylated protein levels of PI3K, p38,ERK and JNK.Our results showed that BD2 and BD3 induced activation of PI3K, but not p38,ERK and JNK MAPK. Therefore, we hypothesize that BD2 and BD3 will enhance bacterial clearance by inhibiting macrophage autophagy via a PI3K/Akt pathway. We will explore the immune regulatory mechanisms of BD2 and BD3 in bacterial clearance by using both in vitro and in vivo infection models as well as various experimental methods including RNA interference, immunofluorescence, immunoblot, phagocytosis and killing assay. Therefore, our research may provide potential targets for the treatment of PA keratitis as well as other ocular infectious diseases.

防御素是一类阳离子抗菌肽，在机体抗感染免疫中发挥着重要作用。我们先前报道了Beta-防御素(BD)2和3在铜绿假单胞菌(Pseudomonas aeruginosa,PA)角膜炎中促进细菌清除、保护角膜的作用，然而其促进细菌清除的免疫调控机制仍不清楚。预实验结果显示，BD2和BD3可以抑制PA诱导的巨噬细胞自噬，而自噬可以抑制巨噬细胞对PA的吞噬和清除，提示BD2和BD3可能通过抑制自噬来促进细菌清除。那么，BD2和BD3抑制自噬的信号机制如何？进一步研究发现，BD2和BD3可诱导Akt的活化，而对MAPK通路无影响。由此，我们首次提出"BD2和BD3通过PI3K/Akt途径抑制巨噬细胞自噬，促进细菌清除"的假说，并拟通过细胞培养和动物模型，采用RNA干扰、免疫荧光、免疫印迹、吞噬杀伤等实验技术，深入研究BD2和BD3介导的免疫杀伤机制，为PA角膜炎等感染性眼病的防治提供理论依据。

本课题围绕巨噬细胞自噬和吞噬杀伤的关系展开，研究了PA感染条件下，防御素对巨噬细胞自噬和吞噬杀伤的调控作用及分子机制，并在此基础上对影响巨噬细胞自噬、吞噬杀伤和炎症反应的一系列关键分子和信号通路进行了研究，取得的主要成果包括：.1, 揭示了绿脓杆菌(Pseudomonas aeruginosa, PA)可以诱导巨噬细胞自噬，而自噬可以下调吞噬受体表达以及ROS和NO的产生，从而抑制巨噬细胞对PA的吞噬和胞内杀伤 (PLOS ONE, in revision).2, 揭示了防御素BD2和BD3通过抑制巨噬细胞自噬，促进巨噬细胞对PA的吞噬和胞内杀伤。.3, 揭示了防御素BD2和BD3调控巨噬细胞自噬和吞噬杀伤的关键分子机制：BD2和BD3可以下调EGR1和c-FOS的表达，上调PI3K/Akt/Rheb/mTOR通路，从而抑制巨噬细胞自噬，促进其对PA的清除。.4,首次揭示了PA感染人巨噬细胞后激活NLRP3炎症小体，而活化的NLRP3炎症小体通过分泌IL-1促进自噬，进而抑制巨噬细胞对PA的胞内杀伤 (Infection and Immunity, 2015) 。.5,首次揭示了髓样细胞抑制受体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, 2013a)。.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, 揭示了MRP8/14表达水平和PA角膜炎疾病进程的相关性，并通过体内外实验揭示，尽管MRP8/14可以促进细菌清除，但最终由于放大炎症反应导致角膜免疫病理损伤 (Investigative Ophthalmology & Visual Science, 2013b)。.上述工作揭示了一系列天然抗感染免疫的关键分子调控机制，并首次发现PA诱导的炎症小体和自噬可能是一种新的免疫逃逸机制，为PA角膜炎的治疗的提供了新的靶点和理论依据.