NLRP6介导的角膜保护作用及分子调控机制

NLRP6 is a newly identified cytosolic pattern recognition receptor, whereas its role in anti-infectious immunity and inflammatory response remains unclear. Our preliminary data showed that Pseudomonas aeruginonsa (PA) infection induced NLRP6 expression in mouse corneas. In vivo knockdown of NLRP6 exacerbated corneal ulceration after PA infection, with increased pro-inflammatory cytokine expression and bacterial load, indicating that NLRP6 may play a protective role in bacterial keratitis (corneal infection) by suppressing corneal inflammation and enhancing bacterial elimination. Next question is, what is the underlying mechanism of NLRP6-mediated corneal resistance? In vitro experiments suggested that NLRP6 specifically inhibited activation of NLRP3 inflammasome. Our previous study reported that activation of NLRP3 inflammasome amplified the inflammatory response, and suppressed macrophage-mediated bacterial clearance of PA. Based on the literature and our preliminary data, we proposed a new hypothesis that NLRP6 negatively regulates NLRP3 inflammasome activation by suppressing canonical NF-kB pathway and competitively interacting with the adaptor molecules MAVS and ASC, therefore enhances bacterial elimination and reduces corneal inflammation, which finally leads to corneal resistance to PA infection. This project is designed to explore the role and molecular regulatory mechanism of NLRP6 in corneal anti-infectious immunity and NLRP3 inflammasome activation. Our findings may provide new targets and strategy for prevention and clinical treatment of bacterial keratitis.

NLRP6是新近发现的胞内模式识别受体，然而其在抗感染免疫与炎症中的功能仍不清楚。预实验结果显示，铜绿假单胞菌感染小鼠角膜可诱导NLRP6表达，而沉默NLRP6使角膜促炎因子表达和荷菌量上调，角膜溃疡加重，提示NLRP6可能通过抑制炎症、促进细菌清除，介导角膜保护作用。那么，NLRP6介导免疫调控的分子机制是什么？体外实验发现，NLRP6能特异性抑制NLRP3炎症小体活化。我们先前报道，NLRP3炎症小体可以放大炎症，抑制巨噬细胞对铜绿假单胞菌的清除。结合文献报道和预实验结果，我们提出“NLRP6通过抑制经典NF-kB通路和竞争结合接头分子MAVS/ASC来负调控NLRP3炎症小体，从而抑制过度炎症、促进细菌清除，保护角膜”的假说，并拟通过动物模型和体外实验，从正、反两方面揭示NLRP6介导角膜保护的免疫调控机制及其负调控NLRP3炎症小体的分子机制，为角膜炎防治提供新的靶点和理论依据。

本课题主要通过体内外实验，研究了模式识别受体NLRP6在铜绿假单胞菌（Pseudomas aeruginosa, PA）角膜炎中的作用及分子机制， 并进一步探究了NLRP6在不同免疫细胞介导的炎症免疫损伤中的作用及其调控机制。.取得的主要成果包括：.① 体内实验揭示了Caspase-1炎症小体和巨噬细胞焦亡介导PA角膜炎症损伤 (Qu et al., Front Immunol, 2018) 。.② 体外实验揭示了PA感染诱导巨噬细胞焦亡和自噬，从而逃逸巨噬细胞吞噬杀伤的分子机制（Wu et al. Int Immunopharmacol. 2016；Wu et al., Front Immunol. 2018)。 .③ 体内外实验揭示了NLRP6可以抑制Caspase11非经典炎症小体活化和巨噬细胞焦亡以及M1极化，减轻角膜炎症损伤 (J Infect Dis., in revision）。.④ 体外实验揭示了NLRP6在活化的CD4+T细胞中高表达，且通过抑制NLRP3炎症小体活化来诱导Th17分化，加重结肠炎症损伤。.⑤ 体内外实验揭示了胞内DNA受体STING（NLRP3炎症小体的重要调控分子之一）在 PA 角膜炎中的作用机制(Chen et al. Front Immunol. 2018)。 .⑥ 体内外实验揭示了SLAMF7诱导巨噬细胞M2极化，减轻角膜炎症损伤的作用机制(Zhu et al. J Infect Dis., 2020)。.上述工作表明，NLRP6在巨噬细胞和CD4+T细胞介导的免疫炎症损伤中发挥了不同作用，并揭示了其分子调控机制，为细菌性角膜炎及其他感染或炎症性疾病的防治提供了理论依据。