TLR3 SIGNALING IN PULMONARY MUCOSAL EPITHELIAL CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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。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呼吸道病原体的成功免疫反应的关键组成部分，但如果不适当控制，这些反应可能导致病理并发症。 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DC之间的旁分泌信号，这些信号是响应于外部刺激（包括TLR激动剂）而发生的，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-κ B活化和培养上清液中的细胞因子产生，评估用合成的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. 通过表面免疫表型分析监测DC的成熟状态。

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.