WS2.3 Pseudomonas aeruginosa enhanced gap junctional communication by a TLR5 and MAPK-dependent mechanism in airway epithelial cells

Objectives: Inflammation, infection and the accumulation of misfolded CFTR have all been reported to instigate an ER stress response in cystic fibrosis. Emerging evidence now implicates microRNA (miRNA) in providing an additional layer of control over the Unfolded Protein Response (UPR). Here the role of miRNA in regulating basal and stimulus-induced ER stress in bronchial epithelial cells in vitro and in vivo was investigated. Methods and Results: The miRNA expression profile of bronchial brushings taken from CF and non-CF individuals (n = 5 each) was quantified via in situ qRT-PCR. Using in silico analysis groups of miRNA predicted to target components of the UPR were identified that were collectively up regulated in CF. Expression of a selection of UPR genes that can be induced via ER stress in non-CF cells was observed to be decreased in CF in vivo (ATF6, Grp78, PERK, Erp57, ATF3, Derlin-1, XBP-1) and in vitro (ATF6, Grp78, Erp57, XBP-1) under basal conditions. ATF6 was experimentally validated as a direct molecular target of miR-145, -221 and -494 via pre-miR over expression and anti-miR inhibition experiments, and through the use of a luciferase reporter vector containing the full length 3’UTR of ATF6. Reduction of these miRNA and reciprocal ATF6 expression was observed in the presence of LPS. Conclusions: These results indicate a lack of an active UPR in CF bronchial epithelial cells in vivo or in vitro under basal conditions which may be in part due to suppression of genes involved by increased miRNA expression in CF.