Role of Toll-Like Receptors in Bacterial Keratitis

描述（由申请方提供）：铜绿假单胞菌（P. aeruginosa）是一种常见的与细菌性角膜炎相关的机会性微生物，尤其是在长期配戴接触透镜的使用者中。我们的目标是确定细菌性角膜炎发展的分子机制，特别是Toll样受体（TLR）4的作用，目前在眼睛中的作用仍有待研究。我们假设TLR 4在角膜中对铜绿假单胞菌的先天免疫应答中起关键作用，并且通过这种TLR的激活和信号传导将调节疾病结果。提出了三个具体的目的：1）测试TLR 4调节感染角膜中的炎性血管生成的假设。2)为了验证TLR 4调节感染角膜细胞凋亡的假设。3)为了验证TLR 4调节感染角膜中抗菌肽的假设。实验将包括使用的技术，如真实的时间RT-PCR，短干扰RNA（siRNA），以及平板计数，酶联免疫吸附试验（ELISA），髓过氧化物酶测定（MPO）的中性粒细胞（PMN）定量，双抗体免疫染色，组织病理学，Tunel测定，DNA梯状，NF？B功能测定和Western印迹。我们的长期目标是了解小鼠角膜中细菌和免疫反应之间的相互作用，以便为人类患者开发针对角膜炎的非常规疗法。由于仅在美国，每年微生物角膜炎的发病率为25，000 - 30，000例，治疗费用估计为1500 - 3000万美元，并且出现了细菌耐药性，因此这些研究与人类健康相关，并具有相当大的医学和经济影响。公共卫生相关性铜绿假单胞菌是一种与细菌性角膜炎相关的机会性革兰氏阴性病原体，尤其是在长期配戴接触透镜的使用者中。阐明TLR 4在微生物性角膜炎中的确切作用，包括调节先天性和适应性免疫反应，调节炎性血管生成，细胞凋亡和抗菌肽的产生，将为细菌性角膜炎的发病机制提供实质性的见解。最终，这项建议的发现将在临床上用于开发更好的治疗方法，以减少细菌性角膜炎和预防失明。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic organism associated with bacterial keratitis, especially in extended wear contact lens users. Our goal is to determine the molecular mechanisms for development of bacterial keratitis, specifically, the role of Toll-like receptor (TLR) 4, which at present remains underexplored in the eye. We hypothesize that TLR4 plays a critical role in the innate immune response to P. aeruginosa in the cornea and that the activation and signaling through this TLR will regulate disease outcome. Three specific aims are proposed: 1) To test the hypothesis that TLR4 regulates inflammatory angiogenesis in the infected cornea. 2) To test the hypothesis that TLR4 regulates apoptosis in the infected cornea. 3) To test the hypothesis that TLR4 regulates antimicrobial peptides in the infected cornea. Experiments will include use of techniques such as real time RT-PCR, short interfering RNA (siRNA), as well as plate counts, enyme linked immunosorbant assay (ELISA), myeloperoxidase assay (MPO) for neutrophil (PMN) quantitation, dual antibody immunostaining, histopathology, Tunel assay, DNA laddering, NF?B functional assays and Western blotting. Our long-term objective is to understand the interactions between bacteria and the immune response in the mouse cornea so that non-conventional therapies against keratitis can be developed for human patients. Since in the United States alone the incidence of microbial keratitis is 25,000-30,000 cases annually, with cost of treatment estimated at $15-30 million, and with emerging bacterial resistance, the studies are of relevance to human health and have considerable medical and economic impact. PUBLIC HEALTH RELEVANCE P. aeruginosa is an opportunistic, Gram-negative pathogen associated with bacterial keratitis, especially in extended wear contact lens users. Elucidating the precise role of TLR4 in microbial keratitis, including regulation of innate and adaptive immune responses, modulation of inflammatory angiogenesis, apoptosis and antimicrobial peptide production, will provide substantive insight into the pathogenesis of bacterial keratitis. Ultimately, the findings from this proposal will be useful clinically in development of better treatments to reduce bacterial keratitis and prevent blindness.