Conjunctival Goblet Cell NLRP3 Inflammasome in Ocular Surface Bacterial Infection

描述（由申请人提供）：每年约有25,000名美国人发生细菌性结膜炎和角膜炎，金黄色葡萄球菌（S. aureus）是感染的主要原因。如果不及时治疗，尤其是角膜感染会对视力造成威胁。此外，耐甲氧西林金黄色葡萄球菌的出现使得开发控制眼表感染的新方法成为当务之急。角膜对感染的反应是受限的，但结膜反应旺盛。特别是分泌MUC5AC的结膜杯状细胞是眼表先天免疫防御的第一道防线，它们分泌的粘蛋白（MUC5AC）可以捕获和清除细菌。然而，尚不清楚细菌是否与杯状细胞相互作用。该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)杯状细胞中触发了哪些细胞信号机制和功能；c)这些功能的激活是否可以预防细菌性角膜炎和结膜炎；d)是否可以开发出激活这些功能的药物来治疗眼表感染。一个创新的假设是，细菌与结膜杯状细胞相互作用，并刺激两种不同的反应。首先，杯状细胞分泌黏液来捕获和清除细菌。其次，杯状细胞激活新发现的nod样受体（NLRP） 3，这是细胞内对细菌的反应。NLRP3的激活导致多组分复合物的形成，炎症小体导致成熟IL-1b的分泌，引发先天介导的炎症。该假说的第二个组成部分是致病细菌而非共生细菌与杯状细胞的相互作用不同，因此杯状细胞对致病细菌而非共生细菌产生免疫反应。将调查以下具体目标：1)致病性产毒金黄色葡萄球菌与杯状细胞相互作用，而非共生的非产毒性表皮葡萄球菌与杯状细胞相互作用，是否通过刺激杯状细胞粘蛋白分泌而引起保护反应？分泌反应是否不同，因为两种细菌激活不同的toll样受体（TLR）二聚体（TLR2/1 vs TLR2/6）和不同的信号通路（[Ca2+]/细胞外调节激酶（ERK）1/2 vs磷脂酰肌醇-3激酶（PI-3K）.AKT）？2)致病金黄色葡萄球菌而非共生表皮葡萄球菌的相互作用是否激活杯状细胞NLRP3炎性体产生成熟的IL-1b，其激活机制是a)通道形成还是b)活性氧（ROS）的产生？培养的大鼠和人结膜杯状细胞将与病原体相关分子模式（PAMPs）的细菌或脂蛋白一起培养。细胞内[Ca2+]将通过荧光显微镜测量，ERK1/2通过western blotting，粘蛋白分泌通过生化测定。NLRP3的形成将通过免疫沉淀和western blotting进行研究，NLRP3的激活将通过FLICA试验进行研究，成熟IL-1b的分泌将通过ELISA进行研究。激动剂、拮抗剂、化学抑制剂和siRNA将用于描述反应。

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.