Respiratory virus infection is a major cause of exacerbation of cystic fibrosis (CF) lung disease and CF patients suffer more severe consequences of virus infection. Our goals are to understand the impact of respiratory viruses on the pathogenesis of CF lung disease. To study the effects of virus infection on the human CF ciliated airway epithelium we used an in vitro model of human ciliated airway epithelium (HAE) and respiratory syncytial virus (RSV) as a virus that commonly infects CF patients. Using non-CF HAE, we show that RSV infects only ciliated cells and causes an acute increase in airway surface liquid height (ASL) height as measured by Texas red-dextran staining of ASL followed by confocal microscopy. This effect was maximum at ~3 days post-inoculation (pi) in non-CF HAE (ctrl,7.71?0.5;RSV,14.8?0.9?m, n=4). Parallel experiments with CF HAE revealed that RSV infection of ciliated cells failed to induce an increase in ASL (CF-ctrl,5.9?0.1;CF-RSV,5.9?0.1?m; n=4). These data suggest that non-CF HAE, but not CF HAE, respond to RSV infection by stimulation of fluid secretion into the airway lumen and is likely mediated by epithelial cell ion transport mechanisms. To determine whether the increase in ASL height after RSV was due to active Cl- secretion, we used bumetanide (100?M) which significantly inhibited RSV-induced ASL height (ctrl,7.3?0.6; RSV,14.4?1.3; RSV+bumetanide,9?0.6; ctrl+bumetanide,5.6?0.1?m; n=4). To determine the specific contribution of CFTR-mediated Cl- secretion to this response we used CFTR172 that also significantly inhibited RSV-induced ASL height (ctrl,6.6?0.3;RSV,11.4?1;RSV+CFTR172, 8 ?0.3; ctrl+CFTR172, 6.8?0.5?m; n=4). Since adenosine triphosphate (ATP) and adenosine (ADO) have been previously shown to regulate Cl- secretion and ASL height in non-CF HAE, we determined the ASL concentration of ATP and ADO 3 days after RSV infection in non-CF HAE and found that both were increased by RSV infection (ATP, ctrl,16?5; RSV,26?3nM; ADO, ctrl,151?25; RSV,245?15nM, n=3) suggesting that the increased ASL after RSV infection was due to the release of nucleotides known to regulate ion transport in airway epithelium. We conclude that RSV infection results in a CFTR-dependent increase in ASL height which is mediated by increased nucleotide levels in the ASL and that this cell response to infection is defective in CF airway epithelial cells. We speculate that increased fluid secretion is a host defense mechanism that attempts to clear virus from the airways and this clearance is diminished in CF airways possibly resulting in prolonged RSV infection of the CF airways.
Author(s): Erin N Worthington, Lucy Clunes, Robert Tarran, Raymond J Pickles