Mechanisms of flagellin-induced protection against microbial keratitis

微生物角膜炎是导致视力丧失的常见原因。角膜对炎症非常敏感-- 中介伤害，因此具有强大的天生防御能力。然而，当上皮屏障功能是 被破坏的、有机会的细菌和真菌病原体可以进入上皮细胞层并 定植于角膜，导致感染。在最初的授权期内，Toll-like的配体鞭毛蛋白 受体5(TLR5)被用来加强角膜的天然免疫，并对广谱的 引起角膜炎的病原体包括铜绿假单胞菌、白色念珠菌和曲霉菌 熏蒸剂。这种高度可诱导和强大的天然粘膜保护可以归因于鞭毛蛋白诱导的 上皮细胞的基因组重编程，进而与专业免疫细胞相互作用来控制 发炎和根除入侵的病原体。这个应用中的假设是鞭毛蛋白，通过 TLR5，刺激保护性角膜黏膜免疫，包括上皮细胞，浸润性PMN， 和激活的树突状细胞，共同赋予对微生物角膜炎的强大抵抗力。三个具体的 目的是为了验证这一假说：1)确定鞭毛蛋白诱导的重编程是如何调节的 在角膜上皮细胞和角膜中。这可以通过使用染色质来评估 免疫沉淀法和组蛋白脱乙酰酶抑制剂用于基因特异性染色质修饰和 利用siRNA和基因敲除小鼠描述激活转录因子-3的作用 由纤维蛋白原诱导的调节角膜天然免疫反应的因子。2)确定如何 上皮细胞参与和协调鞭毛蛋白诱导的黏膜天然免疫防御 角膜炎。上皮的作用及其与树突状细胞在天然防御中的相互作用将被确定。 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。3)至 确定鞭毛蛋白如何诱导对角膜中非鞭毛病原体的保护。治疗性 鞭毛蛋白的潜力及其作用机制将通过建立小鼠真菌模型来表征 角膜炎(作为病原体的念珠菌和曲霉菌以及感染后局部使用鞭毛)和 TLR基因敲除小鼠。这项拟议的研究结果应该有助于阐明角膜的先天免疫及其 诱导，并可能导致新的预防/治疗方式的发展，以预防和 治疗微生物角膜炎。

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.