TLR3 SIGNALING IN PULMONARY MUCOSAL EPITHELIAL CELLS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 肺粘膜上皮是外界环境的屏障，也是呼吸道合胞病毒(RSV)和流感等RNA病毒感染的靶点。病毒感染肺上皮细胞会触发炎症反应，最终导致抗病毒细胞因子和趋化因子的产生。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，但如果控制不当，可能会导致病理并发症。呼吸道上皮细胞产生的炎性细胞因子是由RNA病毒复制过程中产生的双链RNA(DsRNA)与Toll样受体3(TLR3)结合而启动的。大量报道表明，树突状细胞(DC)中的TLR3连接促进了DC的成熟，可能有助于病毒特异性获得性免疫的形成。然而，TLR3信号在RNA病毒感染的呼吸道上皮细胞和潜在的DC之间的相互作用中的作用尚未确定。目前在粘膜组织背景下研究DC成熟的模型依赖于动物的使用。一个便于对上皮细胞或树突状细胞进行离散分析的体外系统将有助于阐明这些细胞之间的通讯机制。这项工作的长期目标是确定呼吸道上皮细胞和粘膜下树突状细胞之间对外界刺激(包括TLR激动剂)的旁分泌信号，并了解这些信号通路如何参与病毒的致病过程。我们预计，这种扩展的理解将加强对病毒和其他肺部疾病的治疗策略的识别。我们假设：a)肺上皮细胞中的TLR3信号促进DC的成熟，并在呼吸道天然免疫和获得性免疫之间提供联系；b)TLR3激活的呼吸上皮细胞和粘膜下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，我们提出了以下目标： 1.建立呼吸道上皮细胞系BEAS-2B的体外培养模型。 A.验证TLR3蛋白在培养上皮细胞系中的表达并鉴定其在细胞中的定位。 B.通过测定细胞中核因子-kB的活性和培养上清液中细胞因子的产生，评估合成的TLR3配体刺激后的上皮细胞株的激活状态。 2.我们将在双组分系统中确定TLR3刺激肺上皮细胞是否促进树突状细胞的成熟。 A.体外Transwell系统将用于测试肺上皮细胞和DC之间通信中对可溶性介质或细胞-细胞接触的需求。 B.树突状细胞的成熟状态将通过表面免疫表型进行监测。

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.