055 - Modulation of the pro-inflammatory phenotype of CFTR mutated epithelial cells - 05/12/08

Background: Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). dF508 CFTR deficient targeting to the cell membrane may activate the cellular stress response and induce a pro-inflammatory phenotype by activating potent transcription factors, including NF-κB In the lung, elafin and SLPI are important anti-proteases known for their anti-microbial and anti-inflammatory (anti-NFκB) properties. The aim of this study is to assess whether correction of dF508-CFTR pro-inflammatory phenotype is possible using elafin or SLPI.

Methods: Human epithelial cells and CFTR over-expression: a: IB3 (dF508-CFTR over-expressers) and S9 control bronchial epithelial cells; b: over-expression of WT-CFTR and dF508-CFTR in A549 alveolar cells and BEAS-2B bronchial cells using recombinant adenovirus (Ad) vectors.

Measurement of ‘CF cells’ pro-inflammatory phenotype: Sub-confluent epithelial cells were either mock-infected or infected with Ad-NF-κB-luciferase. 24 hrs later, cells were either mock-stimulated or stimulated with increased concentrations of TNF-. Secretion of IL-8 and/or luciferase activity were then measured.

Modulation of CF phenotype with antiproteases: Whenever possible, the modulation of the pro-inflammatory phenotype of ‘CF cells’ was attempted by using Ad derived elafin or SLPI. Cells were infected at subconfluence with 50 MOI of Ad. After 24 hrs, cells were either further mock-infected or infected with Ad-NF-kB-luciferase and/or stimulated with TNF-. As above, secretion of IL-8 and/or luciferase activity were measured.

Results: IB3 dF508-CFTR cells secreted 4- to 6-fold more IL-8 than S9 corrected cells when stimulated with 1, 10 or 50ng/ml of TNF-. BEAS-2B- or A549-Ad-dF508-CFTR cells also secreted high levels of IL-8, when stimulated with low levels of TNF-, when compared to BEAS-2B- or A549-Ad-WT-CFTR controls, respectively. The IL-8 differential output in ‘CF cells’ seems mediated by the NF-kB pathway in mutated IB3 cells (2- to 4-fold increase in luciferase activity, when compared to S9-corrected cells).

Conclusion: We confirm that the dF508-CFTR mutation confers a pro-inflammatory phenotype to bronchial and alveolar epithelial cells which may be modulated by anti-proteases.