Conjunctival Goblet Cell NLRP3 Inflammasome in Ocular Surface Bacterial Infection

描述（由申请人提供）：细菌性结膜炎和角膜炎发生在大约25,000美国人每年与金黄色葡萄球菌（S。金黄色葡萄球菌）是感染的主要原因。如果不治疗，角膜感染尤其会威胁视力。金黄色葡萄球菌使得控制眼表面感染的新方法的开发成为直接目标。角膜对感染的反应受到限制，但结膜可以反应旺盛。特别地，分泌MUC 5AC的结膜杯状细胞是眼表面的第一道先天免疫防御，其分泌的粘蛋白（MUC 5AC）捕获和去除细菌。然而，尚不清楚细菌是否与杯状细胞相互作用。本项目的长期目标是确定：a）细菌是否直接与结膜杯状细胞相互作用，B）杯状细胞中触发了什么样的细胞信号机制和功能，c）这些功能的激活是否预防细菌性角膜炎和结膜炎，以及d）是否可以开发激活这些功能的药物来治疗眼表感染。一个创新的假设是，细菌与结膜杯状细胞相互作用，刺激两种不同的反应。首先，杯状细胞分泌粘蛋白来捕获和清除细菌。其次，杯状细胞激活新发现的Nod样受体（NLRP）3，这是一种对细菌的细胞内反应。NLRP 3的激活导致多组分复合物的形成，炎性小体导致成熟IL-1b的分泌，其启动先天介导的炎症。这一假说的第二个组成部分是致病性而非致病性细菌与杯状细胞的相互作用不同，因此杯状细胞对致病性而非致病性细菌产生免疫反应。本研究的具体目的是：1）致病性产毒S.金黄色葡萄球菌，而不是由葡萄球菌，非产芽孢杆菌。表皮葡萄球菌，通过刺激杯状细胞粘蛋白分泌引起保护性反应，并且分泌反应是不同的，因为这两种类型的细菌激活不同的Toll样受体（TLR）二聚体（TLR 2/1与TLR 2/6）和不同的信号传导途径（[Ca 2 +]/细胞外调节激酶（ERK）1/2与磷脂酰肌醇-3激酶（PI-3 K）.AKT）？（2）致病性S.金黄色葡萄球菌，而非金黄色葡萄球菌。表皮，激活杯状细胞NLRP 3炎性体以产生成熟的IL-1 B，并且激活的机制是：a）通道形成或B）活性氧物质（ROS）的产生，？培养的大鼠和人结膜杯状细胞将与细菌或病原体相关分子模式（PAMP）的脂蛋白一起孵育。将通过荧光显微镜测量细胞内[Ca 2 +]，通过蛋白质印迹法测量ERK 1/2，并通过生化测定法测量粘蛋白分泌。将通过免疫沉淀和蛋白质印迹法研究NLRP 3形成，通过FLICA测定研究NLRP 3活化，通过ELISA研究成熟IL-1b的分泌。激动剂、拮抗剂、化学抑制剂和siRNA将用于表征应答。

DESCRIPTION (provided by applicant): Bacterial conjunctivitis and keratitis occur in approximately 25,000 Americans annually with Staphylococcus aureus (S. aureus) being the leading cause of infection. If untreated, corneal infections in particular can be sight threatening In addition, the emergence of methicillin-resistant S. aureus makes development of new approaches to control ocular surface infections an immediate goal. Corneal response to infection is constrained, but conjunctiva can respond exuberantly. In particular the MUC5AC-secreting conjunctival goblet cells are the first line of innate immune defense of the ocular surface with their secreted mucin (MUC5AC) trapping and removing the bacteria. It is not known, however, if bacteria interact with goblet cells. The long-term object of this project is to determine: a) if bacteria interact directly with conjunctival goblet cells, b) what cellular signalng mechanisms and functions are triggered in the goblet cells, c) if activation of these functions prevents bacterial keratitis and conjunctivitis, and d) if drugs that activate these functions can e developed to treat ocular surface infections. An innovative hypothesis is that bacteria interact with conjunctival goblet cells and stimulate two distinct responses. First, goblet cells secrete mucin to trap and remove bacteria. Second, goblet cells activate the newly discovered Nod-like receptor (NLRP) 3 an intracellular responder to bacteria. Activation of NLRP3 causes formation of a multi-component complex, the inflammasome that result in the secretion of mature IL-1b that initiates innate mediated inflammation. A second component of this hypothesis is that pathogenic, but not commensal, bacteria interact differently with the goblet cells so that goblet cells mount an immune response against pathogenic, but not commensal, bacteria. The following specific aims will be investigated: 1) Does interaction with goblet cells by pathogenic toxin-forming S. aureus, but not by commensal, non-toxigenic S. epidermidis, cause a protective response by stimulating goblet cell mucin secretion and are the secretory responses distinct because the two types of bacteria activate diverse Toll-like receptor (TLR) dimers (TLR2/1 versus TLR2/6) and different signaling pathways ([Ca2+]/extracellular regulated-kinase (ERK)1/2 versus phosphatidylinositol-3 kinase (PI-3K).AKT)? and 2) Does interaction of pathogenic S. aureus, but not of commensal S. epidermidis, activate the goblet cell NLRP3 inflammasome to produce mature IL-1b and is the mechanism activated: a) channel formation or b) production of reactive oxygen species (ROS),? Cultured rat and human conjunctival goblet cells will be incubated with bacteria or lipoproteins that are pathogen-associated molecular patterns (PAMPs). Intracellular [Ca2+] will be measured by fluorescence microscopy, ERK1/2 by western blotting, and mucin secretion by biochemical assay. NLRP3 formation will be investigated by immunoprecipitation and western blotting, NLRP3 activation by FLICA assay, and secretion of mature IL-1b by ELISA. Agonists, antagonists, chemical inhibitors, and siRNA will be used to characterize responses.