Abstract
BackgroundMutation of the cystic fibrosis transmembrane-conductance regulator (CFTR) causes cystic fibrosis (CF) but not all CF aspects can easily be explained by deficient ion transport. CF-inflammation provides one example but its pathogenesis remains controversial. Here, we tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-κB activity.Methodology/Principal FindingsIn lung epithelial (H441) and engineered (H57) cell lines; we report that inflammatory markers are significantly suppressed by wild-type CFTR. Transient-transfection of wild-type CFTR into CFTR-naïve H441 cells, dose-dependently down-regulates both basal and Tumour Necrosis Factor-α evoked NF-κB activity when compared to transfection with empty vector alone (p<0.01, n>5). This effect was also observed in CFTR-naïve H57-HeLa cells which stably express a reporter of NF-κB activity, confirming that the CFTR-mediated repression of inflammation was not due to variable reporter gene transfection efficiency. In contrast, H57 cells transfected with a control cyano-fluorescent protein show a significantly elevated basal level of NF-κB activity above control. Initial cell seeding density may be a critical factor in mediating the suppressive effects of CFTR on inflammation as only at a certain density (1×105 cells/well) did we observe the reduction in NF-κB activity. CFTR channel activity may be necessary for this suppression because the CFTR specific inhibitor CFTRinh172 significantly stimulates NF-κB activity by ∼30% in CFTR expressing 16HBE14o− cells whereas pharmacological elevation of cyclic-AMP depresses activity by ∼25% below baseline.Conclusions/SignificanceThese data indicate that CFTR has inherent anti-inflammatory properties. We propose that the hyper-inflammation found in CF may arise as a consequence of disrupted repression of NF-κB signalling which is normally mediated by CFTR. Our data therefore concur with in vivo and in vitro data from Vij and colleagues which highlights CFTR as a suppressor of basal inflammation acting through NF-κB, a central hub in inflammatory signalling.
Highlights
Cystic fibrosis (CF) is widely regarded solely as a defect in ion transport arising from a heritable mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) [1]
Wild type CFTR suppresses inflammation in a number of cell lines To test the proposal that wild-type CFTR could itself suppress inflammation, a wt-CFTR expressing vector was transfected into the pulmonary epithelial cell line, H411 as detailed in the methods to measure its effects on nuclear factor kB (NF-kB) activity
Our studies arose in part from the finding that when CFTR is expressed for the first time in the developing fetal lung, the rapidly rising level of CFTR expression inversely correlated with endogenous NF-kB activity/interleukin 8 (IL-8) release coupled to our interest in lung development [21,22,23,24,25]
Summary
Cystic fibrosis (CF) is widely regarded solely as a defect in ion transport arising from a heritable mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) [1]. Verhaeghe et al showed that there is increased activation of a number of NF-kB driven genes in a 24 week old CF fetus compared to non-CF fetuses of similar age (22 weeks and 23 weeks) [9], whilst others report that in CF and non-CF human fetal tracheal grafts explanted under the skin of immuno-deficient ‘SCID’ mice, there was a significantly increased intraluminal IL-8 content and consistently elevated accumulation of murine leukocytes in the sub-epithelial region in the CF grafts compared to controls [10] These data are consistent with reports showing increased activation of NF-kB in a variety of CF cell lines [11,12] where infection is not an issue. We tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-kB activity
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