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：
8274619
负责人：
Darlene A Dartt
金额：
$ 43.65万
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/8274619
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. 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.

描述（由申请人提供）：每年大约25,000名美国人发生细菌结膜炎和角膜炎，金黄色葡萄球菌（金黄色葡萄球菌）是感染的主要原因。如果未经治疗，尤其是角膜感染也可能是危险的威胁，那么耐甲氧西林的金黄色葡萄球菌的出现就会使控制眼表感染的新方法发展成为直接的目标。角膜对感染的反应受到限制，但结膜可以反应良好。尤其是分泌粘液量的结膜杯状细胞是眼表的先天免疫防御的第一线，其分泌的粘蛋白（MUC5AC）诱捕并去除细菌。但是，如果细菌与杯状细胞相互作用，则尚不清楚。该项目的长期目的是确定：a）如果细菌与结膜杯状细胞直接相互作用，b）在杯状细胞中触发了哪些细胞信号机制和功能，c）如果这些功能的激活会阻止细菌性角膜炎和结膜炎，并且可以激活这些功能，则可以使这些功能发挥作用。创新的假设是细菌与结膜杯状细胞相互作用并刺激两个不同的反应。首先，杯状细胞分泌粘蛋白捕获并去除细菌。其次，杯状细胞激活了新发现的点头样受体（NLRP）3对细菌的细胞内响应者。 NLRP3的激活导致多组分复合体的形成，多组分复合体导致成熟的IL-1B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，细菌与杯状细胞的相互作用不同，因此与杯状细胞相互作用不同，因此杯状细胞对致病性，但不含共生细菌的免疫反应。 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）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）具有致病性金黄色葡萄球菌的相互作用，但没有共生链球菌的相互作用，激活了杯状细胞NLRP3炎性体以产生成熟的IL-1B，并且是被激活的机制：a）通道形成或b）产生活性氧（ROS）（ROS），？？？培养的大鼠和人结膜杯状细胞将与与病原体相关的分子模式（PAMP）的细菌或脂蛋白一起孵育。细胞内[Ca2+]将通过荧光显微镜，ERK1/2通过生化测定法测量。 NLRP3形成将通过免疫沉淀和蛋白质印迹，FLICA测定激活以及ELISA对成熟IL-1B的分泌进行研究。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：每年约25,000名美国人发生细菌结膜炎和角膜炎。如果未经治疗，它们可能会威胁到视力。此外，已经出现了细菌的抗生素抗性菌株，使这些感染对第一线治疗具有抗药性。因此，开发控制眼表感染的新方法是一个重要目标。