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

• 批准号: 7615662
• 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
• 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/7615662
• 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; Play; Prevention; Production; Prophylactic treatment; Protein Family; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; Research; Resolution; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Solutions; Stromal Cells; Structural Protein; 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; improved; in vivo; inhibitor/antagonist; interleukin-1 receptor-associated kinase; killings; lipoxin A4; microbial; migration; mouse model; neutrophil; novel; novel strategies; pathogen; prevent; prophylactic; public health relevance; response

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创伤和佩戴隐形眼镜有关的威胁视力的角膜疾病。虽然抗生素疗法可以消除感染性微生物，但宿主炎症的发生，如果不加以抑制，可能会导致角膜疤痕，这可能会导致一些患者永久失明。先前的实验室研究表明，人角膜上皮细胞（HCECs）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响应假单胞菌的攻击。迄今为止，在TLR刺激后，角膜炎症反应是如何被调节的，在很大程度上尚未解决。我们最近发现，先前暴露于鞭毛蛋白导致细胞重编程，表现为HCECs中促炎细胞因子的产生减少和抗菌分子的表达增加，以及保护机制的发展，包括细菌清除增强和细胞因子/趋化因子表达下调，以及假单胞菌感染的C57BL/6 （B6）小鼠角膜炎症消退加速。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，其潜在机制可以作为抗炎和/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HCECs耐受表型的获得中发挥的作用。这将通过使用siRNA和特异性抑制剂来确定tlr途径的信号转导分子在HCEC重编程中的作用及其在体外的后果来实现。2)确定鞭毛蛋白诱导的上皮重编程在假单胞菌感染下如何调控抗菌基因的表达、调节角膜中性粒细胞浸润和基质细胞活化。这将通过体外细菌杀灭试验和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，为小鼠假单胞菌角膜炎模型的抗炎和抗感染治疗提供新途径。C57BL/6假单胞菌角膜炎小鼠模型，以及基因敲除小鼠，将用于验证这一假设，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。本研究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。本研究探讨了使用鞭毛蛋白作为预防微生物角膜炎的预防性措施，以及作为传统抗生素治疗方案的辅助治疗，以抑制角膜细菌感染后持续炎症的可能性。鉴于新出现的抗菌素耐药性，皮质类固醇的争议作用，以及接触液相关的微生物角膜炎爆发的增加，这项研究具有至关重要的意义，并可能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。