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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Exploratory Grants (P20)
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Special Emphasis Panel (ZRR1-RI-4 (01))
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University of Nebraska Medical Center
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