Mechanisms of flagellin induced protection against bacterial keratitis

鞭毛蛋白诱导预防细菌性角膜炎的机制

基本信息

批准号：
7461874
负责人：
Fu-Shin X Yu
金额：
$ 33.86万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7461874
关键词：
AKT Signaling Pathway Adjuvant Therapy Adrenal Cortex Hormones Anti-Inflammatory Agents Anti-inflammatory Antibiotics Antimicrobial Resistance Attenuated Bacterial Infections Biological Assay Blindness C57BL/6 Mouse Cell surface Cells Clinical Trials Coculture Techniques Contact Lenses Cornea Corneal Diseases Data Development Disease Outbreaks Down-Regulation Epidermal Growth Factor Receptor Epithelial Epithelial Cells Exposure to Family member Feedback Flagella Flagellin Genes Gram-Negative Bacteria Host Defense Human IRAK1 gene IRAK3 gene IRAK4 gene In Vitro Infection Infection prevention Infectious Agent Infiltration Inflammation Inflammatory Inflammatory Response Invaded Keratitis Knockout Mice Laboratory Study Lead Light MAPK14 gene MAPK8 gene Maintenance Measures Mediating Modeling Mus Neutrophil Infiltration Outcome PI3K/AKT Pathway interactions Patients Perforation Phenotype Prevention Production Prophylactic treatment Protein Family Pseudomonas Pseudomonas Infections Pseudomonas aeruginosa Public Health Research Resolution Role Role playing therapy Signal Pathway Signal Transduction Small Interfering RNA Solutions Stromal Cells Structural Protein TNF receptor-associated factor 6 TRAF6 gene Testing Therapeutic Tissues Toll-Like Receptor 5 Trauma Treatment Protocols Vision antimicrobial base chemokine corneal epithelium corneal scar cytokine design exposed human population human IRAK4 protein improved in vivo inhibitor/antagonist interleukin-1 receptor-associated kinase killings knockout gene lipoxin A4 microbial migration mouse model neutrophil novel novel strategies pathogen prevent prophylactic response

项目摘要

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa keratitis is a sight-threatening corneal disease associated with trauma and contact-lens wearing. Although an antibiotic regimen can eliminate the infectious organisms, the host inflammation that occurs, if not suppressed, can cause corneal scarring that can potentially lead to permanent vision loss in some patients. Previous studies from the laboratory revealed that human corneal epithelial cells (HCECs) sense Pseudomonas via recognition of flagellin, the structural protein of bacterial flagella, by Toll-like receptor-5 (TLR5) and respond to Pseudomonas-challenge by producing pro-inflammatory cytokines. To date, how the corneal inflammatory response is modulated after TLR stimulation is largely unresolved. We recently discovered that a prior exposure to flagellin resulted in cell reprogramming, as manifested by decreased production of pro-inflammatory cytokines and augmented expression of antimicrobial molecules in HCECs and in development of protective mechanisms, including enhanced bacterial clearance and downregulated expression of cytokines/chemokines, and accelerated inflammation resolution in the cornea of Pseudomonas-infected C57BL/6 (B6) mice. We hypothesize that TLR-mediated epithelial reprogramming is a key determinant of corneal innate defense and the underlying mechanisms can be exploited as novel approaches for anti-inflammatory and/or anti-infection therapies. To understand flagellin-induced cell reprogramming and ocular protective mechanisms, we propose the following Specific Aims: 1) To assess how the TLR5-mediated signaling pathways are modulated by the IRAK family of proteins and PI3K/AKT and what roles they play in acquiring tolerance phenotype in HCECs. This will be achieved by using siRNA and specific inhibitors to define the role of signal transducing molecules of the TLR-pathway in HCEC reprogramming and its consequences in vitro. 2) To determine how flagellin-induced epithelial reprogramming differentially regulates the expression of antimicrobial genes and modulates neutrophil infiltration and stromal cell activation in the cornea in response to Pseudomonas infection. This will be done using in vitro bacterial killing assay and co-culture of epithelial-neutrophil and epithelial-stromal cells. 3) To exploit the underlying mechanisms of tolerance/reprogramming as novel approaches for anti-inflammatory and anti-infection therapies in murine model of Pseudomonas keratitis. C57BL/6 mouse model of Pseudomonas keratitis, along with gene knockout mice, will be used to test this hypothesis and to determine therapeutic potential of flagellin in preventing infection- and inflammation-caused corneal tissue damage. The results of this study will provide a logical basis for the design of novel anti-inflammatory and anti-infection therapies for prophylaxis and/or treatment of bacterial keratitis. PUBLIC HEALTH RELEVANCE. This study explores the possibility of using flagellin as a prophylactic measure to prevent microbial keratitis and as an adjuvant therapy to traditional antibiotic regimen to suppress the ongoing inflammation after bacterial infection of the cornea. In the light of emerging antimicrobial resistance, the controversial role of corticosteroids, and an increased outbreak of contact solution-associated microbial keratitis, this study is of paramount importance and may lead to clinical trials for the use of flagellin in preventing and/or treating infectious keratitis.

描述（由申请人提供）：铜绿假单胞菌角膜炎是一种与外伤和佩戴隐形眼镜相关的威胁视力的角膜疾病。尽管抗生素疗法可以消除传染性微生物，但发生的宿主炎症如果不加以抑制，可能会导致角膜疤痕，从而可能导致某些患者永久性视力丧失。实验室之前的研究表明，人角膜上皮细胞 (HCEC) 通过 Toll 样受体 5 (TLR5) 识别鞭毛蛋白（细菌鞭毛的结构蛋白）来感知假单胞菌，并通过产生促炎细胞因子来应对假单胞菌的攻击。。迄今为止，TLR 刺激后如何调节角膜炎症反应在很大程度上尚未解决。我们最近发现，先前接触鞭毛蛋白会导致细胞重编程，表现为促炎细胞因子的产生减少和 HCEC 中抗菌分子的表达增加，以及保护机制的发展，包括增强细菌清除和下调细胞因子/趋化因子的表达，并加速假单胞菌感染的 C57BL/6 (B6) 小鼠角膜的炎症消退。我们假设 TLR 介导的上皮重编程是角膜先天防御的关键决定因素，其潜在机制可作为抗炎和/或抗感染治疗的新方法。为了了解鞭毛蛋白诱导的细胞重编程和眼部保护机制，我们提出以下具体目标：1) 评估 IRAK 蛋白家族和 PI3K/AKT 如何调节 TLR5 介导的信号通路以及它们在获得耐受性中发挥的作用HCEC 中的表型。这将通过使用 siRNA 和特异性抑制剂来定义 TLR 通路信号转导分子在 HCEC 重编程及其体外后果中的作用来实现。 2)确定鞭毛蛋白诱导的上皮重编程如何差异调节抗菌基因的表达并调节角膜中中性粒细胞浸润和基质细胞活化以响应假单胞菌感染。这将通过体外细菌杀灭测定以及上皮中性粒细胞和上皮基质细胞的共培养来完成。 3）探索耐受/重编程的潜在机制作为假单胞菌角膜炎小鼠模型抗炎和抗感染治疗的新方法。假单胞菌角膜炎的 C57BL/6 小鼠模型以及基因敲除小鼠将用于测试这一假设，并确定鞭毛蛋白在预防感染和炎症引起的角膜组织损伤方面的治疗潜力。这项研究的结果将为设计用于预防和/或治疗细菌性角膜炎的新型抗炎和抗感染疗法提供逻辑基础。公共卫生相关性。这项研究探讨了使用鞭毛蛋白作为预防微生物性角膜炎的预防措施以及作为传统抗生素疗法的辅助疗法以抑制角膜细菌感染后持续炎症的可能性。鉴于新出现的抗菌药物耐药性、皮质类固醇的争议性作用以及接触溶液相关微生物角膜炎爆发的增加，这项研究至关重要，可能会导致使用鞭毛蛋白预防和/或治疗感染性角膜炎的临床试验。角膜炎。