Mechanisms of flagellin-induced protection against microbial keratitis

描述（由申请人提供）：微生物性角膜炎是视力丧失的常见原因。角膜对炎症介导的损伤非常敏感，因此具有强大的先天防御能力。然而，当上皮屏障功能被破坏时，机会性细菌和真菌病原体可以进入上皮细胞层并在角膜中定植，导致感染。在最初的授权期间，鞭毛蛋白，Toll样受体5（TLR 5）的配体，被用来加强角膜先天免疫，并提供对广谱角膜炎病原体的抗性，包括铜绿假单胞菌，白色念珠菌和烟曲霉。这种高度可诱导和强大的先天性粘膜保护可归因于上皮中鞭毛蛋白诱导的基因组重编程，这反过来又与专业免疫细胞相互作用以控制炎症并根除入侵病原体。本申请中的假设是鞭毛蛋白通过TLR 5刺激保护性角膜先天粘膜免疫，其涉及上皮细胞、浸润的PMN和活化的树突状细胞，共同赋予对微生物角膜炎的强大抗性。提出了三个具体的目标来验证这一假设：1）确定鞭毛蛋白诱导的重编程在角膜上皮细胞和角膜中是如何调节的。这可以通过使用染色质免疫沉淀试验和组蛋白去乙酰化酶抑制剂进行基因特异性染色质修饰，并通过使用siRNA和敲除小鼠来描绘激活转录因子-3（一种由fg诱导的转录因子）在调节角膜先天免疫应答中的作用来评估。2)确定上皮细胞如何参与和协调鞭毛蛋白诱导的针对微生物性角膜炎的粘膜天然免疫防御。上皮细胞的作用及其与树突状细胞在先天防御中的相互作用将通过使用各种转基因和基因敲除小鼠、骨髓重建和细胞耗竭来确定。3)确定鞭毛蛋白如何诱导角膜对非鞭毛病原体的保护作用。鞭毛蛋白的治疗潜力及其作用机制将通过使用真菌性角膜炎的小鼠模型（念珠菌属和曲霉属作为病原体以及感染后局部应用鞭毛蛋白）和双TLR敲除小鼠来表征。拟议的研究结果应阐明角膜先天免疫及其诱导，并可能导致新的预防/治疗模式的发展，预防和治疗微生物角膜炎。 公共卫生相关性：本研究旨在探讨鞭毛蛋白诱导的保护性角膜粘膜免疫的机制，并探索其治疗微生物性角膜炎（一种常见的视力丧失原因）的潜力。所获得的知识对于开发基于机制的、有效的预防/治疗方式来预防和治疗感染性角膜炎的长期目标将是至关重要的。

DESCRIPTION (provided by applicant): Microbial keratitis is a common cause of vision loss. The cornea is exquisitely sensitive to inflammation- mediated damage and therefore has strong innate defenses. However, when the epithelial barrier function is breached, opportunistic bacterial and fungal pathogens can gain access to the epithelial cell layers and colonize in the cornea, leading to infection. During the initial grant period, flagellin, the ligand of Toll-like receptor 5 (TLR5), was used to fortify corneal innate immunity and render resistance to a broad spectrum of keratitis causing pathogens, including Pseudomonas aeruginosa, Candida albicans and Aspergillus fumigates. This highly inducible and robust innate mucosal protection can be attributed to flagellin-induced genomic reprogramming in the epithelium which, in turn, interacts with professional immune cells to control inflammation and to eradicate invading pathogens. The hypothesis in this application is that flagellin, through TLR5, stimulates protective corneal innate mucosal immunity that involves epithelial cells, infiltrated PMNs, and activated dendritic cells, collectively conferring robust resistance to microbial keratitis. Three specific aims are proposed to test this hypothesis: 1) To determine how flagellin-induced reprogramming is regulated in corneal epithelial cells and in the cornea. This can be assessed by using the chromatin immunoprecipitation assay and histone deacetylase inhibitors for gene-specific chromatin modification and by using siRNA and knockout mice for delineating the role of activating transcription factor-3, a transcription factor induced by fg, in regulating the innate immune response in the cornea. 2) To determine how the epithelium participates in and coordinates flagellin-induced mucosal innate immune defense against microbial keratitis. The role of the epithelium and its interaction with dendritic cells in innate defense will be determined by using various transgenic and knockout mice, bone marrow reconstitution, and cell depletion. 3) To determine how flagellin induces protection against non-flagellated pathogens in the cornea. The therapeutic potential of flagellin and its mechanisms of action will be characterized by using mouse models of fungal keratitis (Candida and Aspergillus as pathogens and post-infection topical flagellin application) and double TLR knockout mice. The results of the proposed study should shed light on corneal innate immunity and its induction, and may lead to the development of new prophylactic/therapeutic modalities for preventing and treating microbial keratitis. PUBLIC HEALTH RELEVANCE: This proposal is to determine the mechanisms underlying flagellin-induced protective corneal mucosal immunity and to explore its therapeutic potential to treat microbial keratitis, a common cause of vision loss. The knowledge gained will be critical for the long-term goal of developing mechanism-based, efficacious prophylactic/therapeutic modalities for preventing and treating infectious keratitis.