The role of galectin-8 in the regulation of corneal infection and inflammation

项目摘要 铜绿假单胞菌（PA）是角膜微生物感染的主要原因。之一 PA角膜感染的严重后果是由持续的角膜炎症导致的失明。 在美国，每年发生大约30，000例微生物角膜炎。先天免疫系统 是抵抗病原体的第一道防线，由模式识别受体启动， 入侵的微生物目前已知IL-1β在PA诱导免疫应答中起重要作用 角膜炎。在对细菌感染的经典免疫应答中，成熟IL-1β的产生是两个步骤， 过程第一步是诱导pro-IL-1β表达，这通常通过TLR介导的IL-1β表达来实现。 激活NF-κ B B通路，导致IL-1β前体的诱导。然后第二个信号触发装配 导致半胱天冬酶-1裂解的炎性小体。活性（裂解）半胱天冬酶-1裂解pro-IL-1β， 产生活性IL-1β，其从细胞分泌以介导下游炎症作用， 感染最近，我们有一个令人兴奋的观察，半乳糖凝集素-8敲除（Gal-8 KO）小鼠耐药 PA感染。额外的初步研究表明，Gal-8具有调节这两种激活的潜力。 TLR和炎性体途径。该项目的目标是描述Gal-8在调节中的作用， 的PA诱导的免疫病理学，并制定有效的策略，预防和消退， 不受控制的炎症反应，导致角膜广泛损伤和视力损害。在 目的1，为了了解Gal-8 KO小鼠对PA角膜炎的抵抗机制，我们将测试 假设Gal-8具有抑制炎性小体介导的成熟IL-1β产生的能力， 影响先天免疫反应。具体地，在该目的中，使用Gal-8 KO小鼠和Gal-8缺陷小鼠， 巨噬细胞和中性粒细胞，我们将描述Gal-8在炎症小体调节中的作用。 PA角膜炎的途径。在目标2中，我们将通过以下方式确定Gal-8是否影响PA角膜炎的结果： 调节嗜中性粒细胞弹性蛋白酶和/或TLR途径。为了更好地表征Gal-8在发病机制中的作用， 在Aim 3A中，我们建议对PA角膜炎的转录组和细胞因子/趋化因子蛋白质组进行分析， WT和Gal-8 KO小鼠以及WT和Gal-8 KO巨噬细胞和中性粒细胞的PA感染角膜。在Aim中 在图3B中，将进行研究以确定Gal-8是否可以被靶向以控制过度活跃的免疫应答 和PA角膜炎小鼠模型中的角膜炎症。我们预计，拟议的研究将提供 突破性的数据，不仅为了解PA角膜炎的分子机制，而且为 开发一种新的基于半乳糖凝集素的治疗方法，可以治疗由以下两种原因引起的致盲免疫病理学： 细菌性角膜炎以及其它眼部疾病，如角膜移植排斥和干眼病。

Project Summary The bacterium Pseudomonas aeruginosa (PA) is a leading cause of microbial infection of the cornea. One of the serious consequences of PA corneal infection is blindness resulting from persistent corneal inflammation. Approximately 30,000 cases of microbial keratitis occur annually in the United States. The innate immune system is the first line of defense against pathogens and is initiated by pattern recognition receptors which respond to invading microbes. It is known that IL-1β plays an important role in the induction of immune response in PA keratitis. In the classical immune response to bacterial infection, generation of mature IL-1β is a two-step process. The first step is the induction of pro-IL-1β expression, which is generally achieved by TLR-mediated activation of NF-B pathway that results in the induction of pro-IL-1β. A second signal then triggers the assembly of inflammasomes leading to the cleavage of caspase-1. Active (cleaved) caspase-1 cleaves pro-IL-1β to generate active IL-1β, which is secreted from the cell to mediate downstream inflammatory effects that clear the infection. Recently, we have made an exciting observation that galectin-8 knockout (Gal-8 KO) mice are resistant to PA infection. Additional pilot studies revealed that Gal-8 has the potential to regulate the activation of both TLR and inflammasome pathways. The goal of this project is to characterize the role of Gal-8 in the regulation of PA-induced immunopathology, and to develop effective strategies for prevention and regression of uncontrolled inflammatory response that results in extensive damage to the cornea and visual impairment. In Aim 1, in an effort to understand the mechanisms that render Gal-8 KO mice resistant to PA keratitis, we will test the hypothesis that Gal-8 has the capacity to dampen inflammasome-mediated generation of mature IL-1β to influence innate immune response. Specifically, in this Aim, using Gal-8 KO mice and Gal-8 deficient macrophages and neutrophils, we will characterize the role of Gal-8 in the regulation of the inflammasome pathway in PA keratitis. In Aim 2, we will determine whether Gal-8 influences the outcome of PA keratitis by modulating neutrophil elastase and/or TLR pathway. To better characterize the role of Gal-8 in the pathogenesis of PA keratitis, in Aim 3A, we propose to perform transcriptome and cytokine/chemokine proteome analyses of PA-infected corneas of WT and Gal-8 KO mice, and WT and Gal-8 KO macrophages and neutrophils. In Aim 3B, studies will be performed to determine whether Gal-8 can be targeted to control overactive immune response and corneal inflammation in the mouse model of PA keratitis. We expect that the proposed study will provide ground-breaking data, not only for understanding the molecular mechanism of PA keratitis, but also for the development of novel, galectin-based therapy that can treat blinding immunopathology resulting both from bacterial keratitis, as well as from other ocular disorders, such as corneal graft rejection and dry eye disease.