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

项目摘要 铜绿假单胞菌(PA)是引起角膜微生物感染的主要原因。其中一个 PA角膜感染的严重后果是持续性角膜炎症导致的失明。 在美国，每年大约有30,000例微生物角膜炎。先天免疫系统 是抵御病原体的第一道防线，由模式识别受体启动，模式识别受体对 入侵的微生物。已知IL-1β在诱导PA免疫应答中起重要作用 角膜炎。在对细菌感染的经典免疫反应中，成熟的IL-1β的产生分两步进行 进程。第一步是诱导前IL-1β的表达，这通常是通过TLR介导实现的 激活核因子-B通路，诱导前-IL-1β。然后，第二个信号触发组件 导致caspase-1裂解的炎性小体。活性(切割)caspase-1将前-IL-1β切割成 产生活性的IL-1β，它由细胞分泌，介导下游的炎症效应，清除 感染。最近，我们做了一个令人兴奋的观察，Galectin-8基因敲除(Galectin-8 KO)小鼠具有抵抗力 与PA感染有关。更多的初步研究表明，Gal-8有可能调节这两种受体的激活 TLR和炎症体通路。这个项目的目标是描述Gal-8在调节中的作用 对PA诱导的免疫病理进行研究，并制定有效的策略预防和消退 不受控制的炎症反应，导致对角膜的广泛损害和视力损害。在……里面 目的1，为了了解Gal-8KO小鼠对PA角膜炎产生抗药性的机制，我们将测试 Gal-8具有抑制炎症小体介导的成熟IL-1β产生的假说 影响先天免疫反应。具体地说，在这个目的中，使用Gal-8 KO小鼠和Gal-8缺陷 巨噬细胞和中性粒细胞，我们将描述Gal-8在调节炎症小体中的作用 PA角膜炎的发病途径。在目标2中，我们将确定Gal-8是否通过以下方式影响PA角膜炎的结局 调节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 对于PA角膜炎，在Aim 3A中，我们建议进行转录组和细胞因子/趋化因子蛋白质组分析 感染PA的WT和Gal-8KO小鼠角膜，WT和Gal-8KO巨噬细胞和中性粒细胞。在AIM 3B，将进行研究以确定Gal-8是否可以作为靶点来控制过度活跃的免疫反应 以及PA角膜炎小鼠模型中的角膜炎症。我们预期拟议的研究将会提供 开创性的数据，不仅对于了解PA角膜炎的分子机制，而且对于 以半乳糖凝集素为基础的新型疗法的开发，可治疗由两种疾病引起的致盲免疫病理 细菌性角膜炎以及其他眼病，如角膜移植排斥反应和干眼病。

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.