Conjunctival Goblet Cell NLRP3 Inflammasome in Ocular Surface Bacterial Infection

描述(申请人提供)：细菌性结膜炎和角膜炎每年在美国约25,000人中发生，金黄色葡萄球菌(金黄色葡萄球菌)是感染的主要原因。如果不治疗，特别是角膜感染可能会威胁视力此外，耐甲氧西林金黄色葡萄球菌的出现使开发新的方法来控制眼表感染成为一个迫在眉睫的目标。角膜对感染的反应是有限的，但结膜可以做出强烈的反应。特别是，分泌MUC5AC的结膜杯状细胞是眼表天然免疫防御的第一道防线，其分泌的粘蛋白(MUC5AC)捕获和清除细菌。然而，细菌是否与杯状细胞相互作用尚不清楚。该项目的长期目标是确定：a)细菌是否与结膜杯状细胞直接相互作用，b)杯状细胞中触发了什么细胞信号机制和功能，c)这些功能的激活是否可以防止细菌性角膜炎和结膜炎，以及d)是否可以开发出激活这些功能的药物来治疗眼表感染。一种创新的假设是细菌与结膜杯状细胞相互作用，并激发两种不同的反应。首先，杯状细胞分泌粘蛋白来捕获和清除细菌。其次，杯状细胞激活了新发现的NOD样受体(NLRP)3，这是一种细胞内对细菌的反应。NLRP3的激活导致多组分复合体的形成，炎症体导致成熟的IL-1b的分泌，从而启动先天介导的炎症。这一假设的第二个组成部分是，致病细菌而不是共生细菌与杯状细胞相互作用不同，因此杯状细胞对致病细菌而不是共生细菌产生免疫反应。将研究以下特定目标：1)致病毒素形成金黄色葡萄球菌与杯状细胞的相互作用，但不是通过共生的非产毒表皮葡萄球菌，是否通过刺激杯状细胞粘蛋白分泌引起保护性反应，以及这种分泌反应是否因为两种类型的细菌激活不同的Toll样受体二聚体(TLR2/1对TLR2/6)和不同的信号通路([Ca2+]/细胞外调节激酶(ERK)1/2对磷脂酰肌醇-3激酶(PI-3K).AKT)而不同？2)致病金黄色葡萄球菌的相互作用，而不是共生表皮葡萄球菌的相互作用，是否激活了杯状细胞NLRP3炎症体以产生成熟的IL-1b，其机制是激活的：a)通道形成或b)产生活性氧(ROS)，？培养的大鼠和人结膜杯状细胞将与细菌或脂蛋白孵育，这些细菌或脂蛋白是病原体相关分子模式(PAMP)。细胞内[Ca~(2+)]用荧光显微镜测定，ERK1/2用免疫印迹法测定，粘蛋白分泌用生化法测定。免疫沉淀法和免疫印迹法检测NLRP3的形成，FLICA法检测NLRP3的活化，ELISA法检测成熟IL-1b的分泌。激动剂、拮抗剂、化学抑制剂和siRNA将用于表征反应。 公共卫生相关性：细菌性结膜炎和角膜炎每年在大约25,000名美国人中发生。如果不治疗，他们可能会威胁视力。此外，出现了抗药性细菌株，使这些感染对一线治疗产生抗药性。因此，开发新的方法来控制眼表感染是一个重要的目标。

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. PUBLIC HEALTH RELEVANCE: Bacterial conjunctivitis and keratitis occur in approximately 25,000 Americans annually. If untreated, they can be sight threatening. Furthermore, antibiotic resistant strains of bacteria have emerged, making these infections resistant to first line treatments. Therefore, developing new approaches to control ocular surface infection is an important goal.