Infection with Pseudomonas aeruginosa (Pa) is a hallmark of lung disease in cystic fibrosis (CF). Alveolar macrophages (AM) play an important part in the pulmonary inflammatory response. The analysis of transcript profiles in AM infected with Pa identified a variety of expected and novel factors related to inflammation and apoptosis. Although AM express the cystic fibrosis transmembrane regulator (CFTR), its function in AM is not known and there is evidence of a dysregulated inflammatory response of AM in CF. This proposal focuses on the role of CFTR expressed in AM in their response to Pa. Based on the difficulty to obtain unstimulated or uninfected AM from individuals with CF, CFTR-expression in AM will be silenced using genetic modification with small interfering RNA (siRNA) delivered by an adenovirus (Ad) vector. The overall goal is to evaluate the role of CFTR in the response of AM to Pa.
Two specific aims outline the studies to achieve this goal.
Aim 1. To assess if silencing of CFTR in AM results in a proinflammatory phenotype. SiRNA constructs specific for the CFTR mRNA will be screened in epithelial cell lines. An Ad vector expressing the most effective siRNA-CFTR (AdsiRNA-CFTR) construct will be used to silence CFTR expression in AM. The expression of CFTR mRNA and protein will be monitored and the secretion of IL-8 following stimulation will be evaluated to assess if silencing of CFTR in AM results in a proinflammatory phenotype as known for epithelial cells. Two alternate controls will be evaluated to achieve decreased CFTR function: (1) An Ad vector expressing the CFTR R-domain and (2) the thiazilodine CFTR inhibitor CFTRinh-172.constructed to silence CFTR expression in human AM.
Aim 2. To evaluate if AM with silenced CFTR expression display an altered response to Pa in vitro. To assess the hypothesis that CFTR expression in AM affects the response to Pa, human AM , genetically modified with AdsiRNA-CFTR will be infected with Pa or other control bacteria and evaluated for (1) Pa phagocytosis, cell death/apoptosis, and chemokine/cytokine release; and (2) the expression of genes related to inflammation and apoptosis, previously identified by transcript profiles of human AM exposed to Pa.
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