STING介导的角膜保护作用及其免疫调节机制研究

Stimulator of interferon genes (STING) is a newly identified pattern recognition receptor, which can directly bind cyclic diguanylate nucleotide (c-di-NMP) and is also a critical downstream adaptor of intracelluar DNA sensors. During infection, the activation of STING and its downstream molecules leads to production of cytokines (such as type I interferons) participating in immuno-regulation and host defense response. The involvement and the role of STING in acute suppurative keratitis caused by Pseudomonas aeruginosa are unknown. Our previous study indicated that cyclic diguanylate monophosphate (c-di-GMP) from Pseudomonas aeruginosa, a bacterial second messenger, resulted in phosphorylation of interferon regulatory factor 3 (p-IRF3) and activated STING signaling pathway in vitro and in animal model of kiratitis. Further results showed that STING activation promoted host resistance to Pseudomonas aeruginosa kiratitis, and the next problem is what the mechanism of STING in the disease. Preliminary experiment indicated that STING inhibited inflammation and promoted bacterial killing. According to all previous results, we propose the hypothesis that “STING ameliorates disease progress of Pseudomonas aeruginosa kiratitis, by regulating inflammation and bacterial elimination”. However, the protective role of STING in Pseudomonas aeruginosa kiratitis depends on type I interferon remains unknown. The project is to explore the role and the regulatory mechanism of STING in Pseudomonas aeruginosa keratitis, which may hold promise for alternative clinical treatment of the disease, decrease the occurrence and blindness of Pseudomonas aeruginosa keratitis, and have critical clinical significance.

STING新近发现的模式识别受体识别病原体第二信使（环化二核苷酸），并是胞浆DNA受体下游必需通路分子，其激活后导致细胞因子的释放（如I型干扰素），调节炎症反应和免疫防御。那么STING是否参与到铜绿假单胞菌角膜炎中，并在其中发挥什么作用？预实验显示，铜绿假单胞菌的第二信使环化二鸟苷酸在体内外均能激活STING通路，其下游分子磷酸化干扰素调节因子3表达显上调；且发现，激活STING能够延缓角膜炎的疾病进程。那么其发挥角膜保护的机制是什么？进一步结果显示，STING可以抑制炎症反应并促进细菌清除。由此，我们首次提出“STING通过调节炎症和细菌清除，延缓角膜溃疡穿孔”的假说。然而STING发挥的角膜保护作用是否依赖于I型干扰素呢？本项目拟通过体内外实验深入研究STING抑制角膜溃疡穿孔的免疫调节机制，即该机制是否依赖I型干扰素。本研究为开发预防和治疗化脓性角膜炎的新措施，提供可靠的理论依据。

铜绿假单胞菌（Pseudomonas aeruginosa，PA）是导致细菌性角膜炎的首要致病菌之一，尤其是在自身免疫系统被抑制的患者和佩戴角膜接触镜的人群中。天然免疫是抵抗PA感染的第一道防线。当病原体感染机体时，病原体被初始免疫应答细胞上的模式识别受体（pattern recognition receptors，PRRs）所识别，触发机体的天然免疫应答。据报道，多个PRRs如toll-样受体（Toll-like receptors，TLR）4/5/9、NOD样受体家族NLRP3和NLRC4均参与铜绿假单胞菌角膜炎的致病过程。干扰素基因刺激因子（stimulator of interferon genes，STING）是胞浆DNA受体通路中的桥联分子，可参与多种病原体-宿主相关性疾病。然而，STING在对PA诱导的角膜炎症的调节和对PA清除方面的作用尚不清楚。我们的研究发现，STING参与到PA角膜炎的动物模型和细胞模型中。为了探讨STING在PA角膜炎中发挥的作用，我们在体内外应用siRNA沉默STING或者用2’3’-cGAMP激活STING在观察STING的调节作用。动物实验结果发现，沉默STING 加速疾病的进程，然而激活STING延缓疾病的进展。这些结果说明，STING在铜绿假单胞菌角膜炎中发挥宿主保护作用。为了探讨STING发挥调节作用的机制，我们采用荧光定量PCR和细菌平板计数的方法检测炎性细胞因子的表达和荷菌量的变化。我们体内外结果发现STING抑制炎性因子的分泌并促进细菌的清除。动物实验同时发现STING抑制感染角膜中炎性细胞的浸润。总结，我们的研究发现STING通过抑制角膜炎症反应并促进细菌清除在角膜炎中发挥角膜保护作用。