The long-term objective of this project is to elucidate the molecular and cellular pathways activated by the binding of the cystic fibrosis transmembrane conductance regulator (CFTR) to Pseudomonas aeruginosa which initiates a cascade of effective host innate immune responses that fail to occur in individuals with CF. This underlies their hypersusceptibility to life shortening, chronic P. aeruginosa infection.
The aims are directed at analyzing how binding of P. aeruginosa to CFTR initiates release of interleukin-1 (IL-1), rapid activation and nuclear entry of NF-kB via signaling through the IL-1 receptor (IL-1 R), recruitment of CFTR and other proteins to membrane lipid rafts, and initiation of other epithelial cell responses including bacterial ingestion, cytokine secretion and apoptosis. Studies with human cells with WT or mutant CFTR infected with P. aeruginosa will determine the kinetics and magnitude of IL-1 release and IL-1 R signaling through the molecular adaptor MyD88 to define the contribution of CFTR to these responses. Transgenic mice lacking these mediators will be evaluated in a model of chronic P. aeruginosa oropharyngeal infection for loss of resistance to infection, which occurs in transgenic CF mice. Additional cellular studies will analyze the contribution to effective innate immunity of 5 proteins - major vault protein; Carmal; Pag/Cbp; Clathrin; and migration inhibition factor - identified as rapidly entering into membrane lipid rafts in a CFTR-dependent fashion following epithelial cell infection with P. aeruginosa. Readouts include release of IL-1, NF-kB activation, endocytosis of P. aeruginosa, cytokine secretion, and apoptosis. Transgenic mice lacking one of these factors, or mice treated with short-interfering RNA (siRNA) to knock down the production of the factor in the lung, will be evaluated for their susceptibility to P. aeruginosa infection. siRNA to CFTR will also be used in vitro to determine if a CF-phenotype can be produced in a cell with endogenous WT-CFTR. siRNA to CFTR will be inoculated into the lungs of mice to transiently knock down protein expression, the animals then chronically infected with P. aeruginosa, and the suppression of CFTR production relieved at various time points to evaluate the effect of restoration of wild-type (WT)-CFTR function on the pathogenesis of P. aeruginosa infection. These studies will help define strategies for use in clinical trials of drugs or therapies designed to induce WT-CFTR function in CF patients. Overall, an enhanced understanding of the normal resistance to P. aeruginosa infection and the role of CFTR in this process will be obtained from the proposed aims. ? ?
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