Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 Spike protein
BackgroundSARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper- proinflammatory state involves activation of signaling by NF{kappa}B and ENaC, and expression of high levels of cytokines and chemokines. Post-infection inflammation may contribute to "Long COVID", and there are long term consequences for acute severe COVID-19, which double or triple the chances of dying from any cause within a year. Enhanced signaling by NF{kappa}B and ENaC also marks the airway of patients suffering from cystic fibrosis, a lethal proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might be due to inhibition of CFTR signaling by SARS- CoV-2 Spike protein. MethodsThis hypothesis was tested using the hTERT-transformed BCi-NS1.1 basal stem cell, previously derived from small airway epithelia, which were differentiated into a model of small airway epithelia on an air-liquid-interface (ALI). CyclicAMP-activated CFTR chloride channel activity was measured using an Ussing Chamber. Cell surface-CFTR was labeled with the impermeant biotin method. ResultsExposure of differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of CFTR protein expression. As hypothesized, TNF/NF{kappa}B signaling was activated, based on increased protein expression of TNFR1, the TNF receptor; TRADD, the first intracellular adaptor for the TNF/TNFR1 complex; phosphorylated I{kappa}B, and the chemokine IL8. ENaC activity was also activated, based on specific changes in molecular weights for and {gamma} ENaC. Exposure of the epithelia to viral Spike protein suppressed cAMP-activated CFTR chloride channel activity. However, 30 nM concentrations of cardiac glycoside drugs ouabain, digitoxin and digoxin, prevented loss of channel activity. ACE2 and CFTR were found to co- immunoprecipitate (co-IP) in both basal cells and epithelia, suggesting that the mechanism for Spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, Spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane, suggesting that failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. ConclusionBased on experiments with this model of small airway epithelia, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by SARS-CoV-2 Spike protein, thus inducing a CFTR-null, cystic fibrosis-like clinical phenotype.