Mechanisms of flagellin-induced protection against microbial keratitis

描述（由申请人提供）：细菌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，因此具有很强的先天防御能力。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。在最初的资助期间，鞭毛蛋白，toll样受体5 （TLR5）的配体，被用来加强角膜先天免疫，并对广泛的角膜炎病原体，包括铜绿假单胞菌、白色念珠菌和烟熏曲霉产生抗性。这种高度诱导和强大的先天粘膜保护可归因于鞭毛蛋白诱导的上皮基因组重编程，反过来，鞭毛蛋白与专业免疫细胞相互作用以控制炎症并根除入侵的病原体。本应用的假设是鞭毛蛋白通过TLR5刺激保护性角膜先天黏膜免疫，包括上皮细胞、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抵抗力。为了验证这一假设，我们提出了三个具体的目标：1)确定鞭毛蛋白诱导的重编程是如何在角膜上皮细胞和角膜中被调节的。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，以及使用siRNA和敲除小鼠来描述激活转录因子-3（一种由fg诱导的转录因子）在调节角膜先天免疫反应中的作用来评估。2)确定上皮如何参与和协调鞭毛蛋白诱导的黏膜对微生物角膜炎的先天免疫防御。上皮及其与树突状细胞的相互作用在先天防御中的作用将通过使用各种转基因和敲除小鼠、骨髓重建和细胞耗尽来确定。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。利用真菌性角膜炎小鼠模型（假丝酵母和曲霉菌作为病原体，感染后局部应用鞭毛蛋白）和双TLR敲除小鼠来表征鞭毛蛋白的治疗潜力及其作用机制。这项研究的结果将揭示角膜先天免疫及其诱导，并可能导致开发新的预防/治疗方式来预防和治疗微生物角膜炎。

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.