TLR3 SIGNALING IN PULMONARY MUCOSAL EPITHELIAL CELLS

肺粘膜上皮细胞中的 TLR3 信号传导

• 批准号: 8360020
• 负责人: THERESE M MCGINN
• 金额: $ 3.54万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360020
• 关键词: Agonist; Animals; Antiviral Agents; Cell Line; Cell Maturation; Cells; Communication; Dendritic Cells; Double-Stranded RNA; Environment; Epithelial Cells; Epithelium; Funding; Goals; Grant; Immune response; Immunophenotyping; In Vitro; Infection; Inflammatory; Inflammatory Response; Influenza; Lead; Ligands; Ligation; Link; Lung; Measurement; Mediating; Mediator of activation protein; Modeling; Monitor; Mucous Membrane; NF-kappa B; National Center for Research Resources; Natural Immunity; Nebraska; Paracrine Communication; Principal Investigator; Production; Publishing; RNA Viruses; Reporting; Research; Research Infrastructure; Resources; Respiratory System; Respiratory syncytial virus; Respiratory tract structure; Role; Signal Pathway; Signal Transduction; Source; Stimulus; Surface; System; Testing; United States National Institutes of Health; Viral; Viral Pathogenesis; Virus Diseases; Virus Replication; Work; acquired immunity; adaptive immunity; chemokine; cost; cytokine; functional genomics; human TLR3 protein; pathogen; protein expression; respiratory; response; treatment strategy; virus development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Pulmonary mucosal epithelia serve as barriers from the external environment and as targets for infection with RNA viruses such as respiratory syncytial virus (RSV) and influenza. Virus infection of pulmonary epithelial cells triggers inflammatory responses that culminate in production of antiviral cytokines and chemokines. While often a crucial component of a successful immune response to airway pathogens, these responses, if not appropriately controlled, can lead to pathological complications. Production of inflammatory cytokines by airway epithelial cells is initiated upon engagement of Toll-like receptor 3 (TLR3) by double stranded RNA (dsRNA) produced during RNA virus replication. Numerous published reports have indicated that TLR3 ligation in dendritic cells (DCs) promotes DC maturation and may contribute to development of virus-specific acquired immunity. However, the role of TLR3 signaling in the interaction between RNA virus-infected airway epithelial cells and underlying DCs has not been defined. Current models for investigating DC maturation in the context of mucosal tissues rely on the use of animals. An in vitro system that facilitates discrete analysis of epithelial cells or DCs would promote efforts to elucidate mechanisms of communication between these cells. The long-term goal of the proposed work is to define the paracrine signals between airway epithelial cells and submucosal DCs that occur in response to external stimuli, including TLR agonists, and to understand how such signaling pathways participate in viral pathogenesis. We anticipate that such expanded understanding will potentiate identification of treatment strategies for viral and other pulmonary conditions. We hypothesize that: a) TLR3 signaling in pulmonary epithelial cells promotes maturation of DC and provides a link between innate and adaptive immunity in the respiratory tract; b) interaction between TLR3-activated respiratory epithelial cells and submucosal DCs is mediated through soluble factors that promote promote DC maturation. In order to test these hypotheses, the following aims are proposed: 1. We will establish in vitro cultures of respiratory epithelial cell line BEAS-2B. a. Verify TLR3 protein expression and identify cellular localization of TLR3 in cultured epithelial cell lines. b. Assess activation status of epithelial cell lines in vitro after stimulation with the synthetic TLR3 ligand by measurement of NF-kB activation in cells and cytokine production in culture supernatants. 2. We will determine whether TLR3 stimulation in pulmonary epithelia promotes maturation of dendritic cells in a two-component system. a. An in vitro Transwell system will be used to test the requirement for soluble mediators, or cell-cell contact, in communication between pulmonary epithelial cells and DCs. b. The maturation state of the DCs will be monitored by surface immunophenotyping.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 肺粘膜上皮细胞是抵御外部环境的屏障，也是呼吸道合胞病毒（RSV）和流感等RNA病毒感染的目标。肺上皮细胞的病毒感染引发炎症反应，最终产生抗病毒细胞因子和趋化因子。虽然这些反应通常是对气道病原体成功免疫反应的关键组成部分，但如果不适当控制，可能会导致病理并发症。 当 RNA 病毒复制过程中产生的双链 RNA (dsRNA) 与 Toll 样受体 3 (TLR3) 结合时，气道上皮细胞就会开始产生炎症细胞因子。 许多已发表的报告表明，树突状细胞 (DC) 中的 TLR3 连接可促进 DC 成熟，并可能有助于病毒特异性获得性免疫的发展。 然而，TLR3 信号传导在 RNA 病毒感染的气道上皮细胞和潜在 DC 之间相互作用中的作用尚未明确。 目前研究粘膜组织中 DC 成熟的模型依赖于动物的使用。有助于对上皮细胞或树突状细胞进行离散分析的体外系统将促进阐明这些细胞之间的通讯机制的努力。 这项工作的长期目标是定义气道上皮细胞和粘膜下 DC 之间响应外部刺激（包括 TLR 激动剂）而发生的旁分泌信号，并了解此类信号通路如何参与病毒发病机制。我们预计，这种扩大的了解将有助于确定病毒和其他肺部疾病的治疗策略。我们假设： a) 肺上皮细胞中的 TLR3 信号传导促进 DC 的成熟，并在呼吸道的先天免疫和适应性免疫之间提供联系； b) TLR3激活的呼吸道上皮细胞和粘膜下DC之间的相互作用是通过促进DC成熟的可溶性因子介导的。 为了检验这些假设，提出以下目标： 1. 我们将建立呼吸道上皮细胞系BEAS-2B的体外培养。 一个。 验证 TLR3 蛋白表达并确定培养的上皮细胞系中 TLR3 的细胞定位。 b. 通过测量细胞中 NF-kB 的激活和培养物上清液中细胞因子的产生，评估合成 TLR3 配体刺激后体外上皮细胞系的激活状态。 2.我们将确定肺上皮细胞中的TLR3刺激是否促进二元系统中树突状细胞的成熟。 一个。 体外 Transwell 系统将用于测试肺上皮细胞和 DC 之间通讯时对可溶性介质或细胞间接触的需求。 b. DC 的成熟状态将通过表面免疫表型进行监测。