TLR3 SIGNALING IN PULMONARY MUCOSAL EPITHELIAL CELLS

肺粘膜上皮细胞中的 TLR3 信号传导

• 批准号: 7725206
• 负责人: THERESE M MCGINN
• 金额: $ 2.49万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7725206
• 关键词: Agonist; Animals; Antiviral Agents; Cell Line; Cell Maturation; Cells; Communication; Computer Retrieval of Information on Scientific Projects Database; Condition; Dendritic Cells; Double-Stranded RNA; Environment; Epithelial Cells; Epithelium; Funding; Goals; Grant; Immune response; Immunity; In Vitro; Infection; Inflammatory; Inflammatory Response; Influenza; Institution; Lead; Ligands; Ligation; Link; Lung; Measurement; Mediating; Mediator of activation protein; Modeling; Mucous Membrane; NF-kappa B; Paracrine Communication; Production; Publishing; RNA Viruses; Reporting; Research; Research Personnel; Resources; Respiratory System; Respiratory syncytial virus; Role; Signal Pathway; Signal Transduction; Source; Stimulus; System; Testing; United States National Institutes of Health; Viral; Viral Pathogenesis; Virus Diseases; Virus Replication; Work; acquired immunity; chemokine; cytokine; pathogen; protein expression; receptor; respiratory; response; virus development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 Tolllike 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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 肺粘膜上皮细胞作为来自外部环境的屏障，并作为RNA病毒如呼吸道合胞病毒（RSV）和流感病毒感染的靶标。病毒感染肺上皮细胞引发炎症反应 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的成功免疫应答的关键组成部分，但如果不是， 如果控制得当，可能会导致病理并发症。在RNA病毒复制期间产生的双链RNA（dsRNA）与Toll样受体3（TLR 3）接合后，气道上皮细胞产生炎性细胞因子。许多已发表的报告表明，TLR 3连接在树突状细胞（DC）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，TLR 3信号转导在RNA病毒感染的气道上皮细胞和底层DC之间的相互作用中的作用尚未确定。 目前研究粘膜组织中DC成熟的模型依赖于动物的使用。一个在体外系统，有利于离散分析上皮细胞或DC将促进努力阐明这些细胞之间的通信机制。这项工作的长期目标是确定气道上皮细胞和粘膜下树突状细胞之间的旁分泌信号，这些信号是对外部刺激的反应， 包括TLR激动剂，并了解这些信号通路如何参与病毒的发病机制。我们预计，这种扩展的理解将加强识别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TLR 3信号传导促进DC的成熟，并提供呼吸道中先天免疫和适应性免疫之间的联系; B）TLR 3活化的呼吸道上皮细胞和粘膜下DC之间的相互作用通过促进DC成熟的可溶性因子介导。为了检验这些假设，提出了以下目标： 1.我们将建立呼吸道上皮细胞系BEAS-2B的体外培养。 a.验证TLR 3蛋白表达并鉴定TLR 3在培养的上皮细胞系中的细胞定位。 B.通过测量细胞中的NF-kB活化和培养上清液中细胞因子的产生，评估合成TLR 3配体刺激后体外上皮细胞系的活化状态。 2.我们将确定肺上皮细胞中的TLR 3刺激是否促进双组分系统中树突状细胞的成熟。 a.将使用体外Transwell系统来测试肺上皮细胞和DC之间的通信中对可溶性介质或细胞-细胞接触的需求。 B. DC的成熟状态