Conjunctival Goblet Cell NLRP3 Inflammasome in Ocular Surface Bacterial Infection

眼表细菌感染中的结膜杯状细胞NLRP3炎症小体

基本信息

批准号：
8461558
负责人：
Darlene A Dartt
金额：
$ 41.47万
依托单位：
SCHEPENS EYE RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8461558
关键词：
1-Phosphatidylinositol 3-Kinase Agonist American Antibiotic Resistance Bacteria Bacterial Infections Bacterial conjunctivitis Binding Biochemical Biological Assay Caspase-1 Cell Death Cell secretion Cells Chemicals Cleaved cell Complex Conjunctivitis Cornea Development Enzyme-Linked Immunosorbent Assay Epidermal Growth Factor Receptor Exocytosis Figs - dietary Film Fluorescence Microscopy Gel Goals Goblet Cells Hemolysin Human Immune Immune response Immunoprecipitation Incubated Infection Infection prevention Inflammation Inflammatory Response Keratitis Life Lipoproteins MAPK3 gene MUC5AC gene Measures Mediating Molecular Mucins Neutrophil Infiltration Organism P2X-receptor Pattern Pharmaceutical Preparations Phosphotransferases Production Proto-Oncogene Proteins c-akt Rattus Reactive Oxygen Species Resistance Signal Pathway Signal Transduction Small Interfering RNA Squamous Cell Staphylococcus aureus TLR1 gene TLR2 gene TLR6 gene Toll-Like Receptor 2 Toll-like receptors Toxin Vision Western Blotting cell type commensal microbes conjunctiva cytokine dimer extracellular fluoroquinolone resistance inhibitor/antagonist innovation macrophage methicillin resistant Staphylococcus aureus novel strategies ocular surface pathogen pathogenic bacteria prevent receptor resistant strain response

项目摘要

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.

描述（由申请人提供）：每年大约有 25,000 名美国人患有细菌性结膜炎和角膜炎，其中金黄色葡萄球菌 (S. aureus) 是感染的主要原因。如果不及时治疗，角膜感染尤其可能威胁视力。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前的目标。角膜对感染的反应受到限制，但结膜反应强烈。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，其分泌的粘蛋白 (MUC5AC) 可以捕获并清除细菌。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和 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，它是细菌的细胞内反应物。 NLRP3 的激活导致多组分复合物的形成，即炎症小体，导致成熟 IL-1b 的分泌，从而引发先天介导的炎症。该假设的第二个组成部分是致病菌（而非共生菌）与杯状细胞的相互作用不同，因此杯状细胞对致病菌（而非共生菌）产生免疫反应。将研究以下具体目标： 1) 致病性毒素形成金黄色葡萄球菌（而非共生的非产毒表皮葡萄球菌）与杯状细胞的相互作用是否通过刺激杯状细胞粘蛋白分泌而引起保护性反应，并且是分泌性的反应不同，因为两种类型的细菌激活不同的 Toll 样受体 (TLR) 二聚体（TLR2/1 与 TLR2/6）和不同的信号通路（[Ca2+]/细胞外调节激酶 (ERK)1/2 与磷脂酰肌醇 3 激酶 (PI-3K).AKT）？ 2) 致病性金黄色葡萄球菌（而非共生表皮葡萄球菌）的相互作用是否会激活杯状细胞 NLRP3 炎性体以产生成熟的 IL-1b，并且激活的机制是：a) 通道形成或 b) 活性氧的产生（ ROS），？培养的大鼠和人类结膜杯状细胞将与病原体相关分子模式 (PAMP) 的细菌或脂蛋白一起孵育。细胞内 [Ca2+] 将通过荧光显微镜检测，ERK1/2 通过蛋白质印迹检测，粘蛋白分泌通过生化检测检测。将通过免疫沉淀和蛋白质印迹研究NLRP3的形成，通过FLICA测定研究NLRP3的激活，并通过ELISA研究成熟IL-1b的分泌。激动剂、拮抗剂、化学抑制剂和 siRNA 将用于表征反应。