The long-term goal of this proposal is to define the immune function of dendritic cells (DC) in the lung. That tight junction (TJ) integrity is maintained as DC traverse pulmonary epithelium to sample inhaled air for antigens and the discovery that tricellulin, a newly discovered TJ protein, appears to regulate trans- epithelial DC migration, prompts the following questions central to lung DC biology. 1. What are the mechanisms by which tricellulin regulates DC migration across airway and alveolar epithelia while preserving the TJ barrier? Tricellulin's role in DC migration is evaluated by siRNA-mediated knockdown of tricellulin in MTE7b cells and/or DC. After determining the turnover rate and fate of tricellulin in the mouse tracheal epithelial MTE7b cell line and bone marrow derived DC, the effect of reduced tricellulin levels on DC migration and TJ integrity is evaluated using chemotaxis and permeability assays, respectively. 2. Which domains of tricellulin regulate the TJ seal as DC migrate through TJs of MTE7b monolayers? SiRNA induced tricellulin knockdown in MTE7b cells is used to minimize confounding effects of endogenous tricellulin. Introduction of defined siRNA-resistant deletional mutant tricellulin constructs containing deletions of either the C- or N-terminal domains or deletion of 16 or 12 amino acids from the 1st or 2nd extra-cellular loops, respectively, will identify those domains that regulate trans-junctional DC migration while maintaining the TJ barrier. Epitope tagged deletional constructs are localized by immunofluorescence microscopy. Permeability and chemotaxis assays assess the functional effect of each deletion. 3. Does DC migration through TJs depend on tricellulin regulated RhoA/ROCK1 signaling and is there a direct link between tricellulin and the actin cytoskeleton? Preliminary studies show that knockdown of tricellulin stimulates a marked increase in Rho-GTP levels that is associated with enhanced migration of DC across MTE7b monolayers. In these studies DC are stimulated to migrate across either control or tricellulin knockdown MTE7b cells expressing the four siRNA resistant tricellulin deletional mutants. Rho-GTP is localized at the site of DC migration using a novel in situ Rho-GTP assay. Rho-GTP levels are quantified by Rhotekin pulldown assays. Pull-down experiments, using GST fusion peptides of the N- and C-termini of tricellulin, identify those members of the actin cytoskeleton that are linked to tricellulin. 4. Is tricellulin required for transepithelial DC migration in vivo? In mice with Cre-lox-regulated conditional RNA interference of tricellulin expression, an ovalbumin- induced model of asthma will examine the role of tricellulin in regulating DC trans-epithelial trafficking in vivo. These studies will contribute important new insights into the role of tricellulin in pulmonary DC trafficking and will form the basis for devising new strategies to regulate DC interactions with inhaled pathogens.
. Results of the proposed studies will provide new knowledge regarding the regulation of dendritic cell trafficking at the interface of the lung epithelial cells and inhaled foreign proteins, bacteria and viruses. They provide basic information that will facilitate the devising of new strategies to control and regulate the immune response in the lung. With ever increasing populations and more frequent air transportation, the worldwide spread of inhaled pathogens is becoming an increasing threat. ? ? ?
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