COVID-19 airway inflammation is due to Spike inhibition of CFTR signaling

Abstract Over 860,000 COVID-19 deaths have occurred in the U.S., and more than 5.5 million deaths have occurred world-wide. Consequently, development of effective antiviral drugs that block infectivity and airway inflammation continue to be a global health priority. Inflammation in the COVID-19 airway is due to increased NFκB and Epithelial Sodium channel (ENaC) signaling. Cystic fibrosis (CF), a genetic disease caused by inactivating mutations in the CFTR gene, also has the same proinflammatory NFκB and ENaC signaling phenotype in the airway. Using a differentiated human epithelial "lung-on-a-chip" platform, we find that ACE2, the receptor protein for the SARS-CoV-2 Spike protein, co-immunoprecipitates with CFTR. Furthermore, exposure of differentiated epithelia to the SARS-COV-2 Spike protein dose-dependently suppresses cyclic AMP-dependent CFTR chloride channel activity and CFTR protein expression. Spike-dependent loss of CFTR also activates TRADD-dependent NFκB signaling and proteolytically activates ENaC. We have also found that Spike-dependent loss of CFTR may be due to failure of endosomal recycling to return apical CFTR to the plasma membrane. Finally, we find that nanoMolar concentrations of cardiac glycoside drugs such as ouabain, digitoxin and digoxin, which competitively inhibit Spike:ACE2 binding, rescue Spike-dependent reduction in CFTR activities. We have therefore hypothesized that binding of SARS-CoV-2 Spike protein to ACE2 in the lung causes loss of CFTR signaling and activation of proinflammatory NFκB and ENaC signaling. To further test this hypothesis we propose the following Specific Aims: SA #1: To define the mechanism by which SARS-CoV-2 Spike protein reduces CFTR channel activity and CFTR protein levels. We will determine the mechanism by which ACE2 interacts with CFTR. We will determine the mechanism by which Spike protein interaction with ACE2 leads to loss of CFTR. SA#2: To define the mechanism by which SARS-CoV-2 Spike protein drives activation of ENaC and NFκB signaling. We will determine the mechanism by which Spike-induced loss of CFTR protein results in activation of TRADD- dependent NFκB ENaC signaling. SA#3: To identify protective mechanisms of cardiac glycosides on cell and animal models of COVID-19 disease. We will test whether cardiac glycoside drugs block native SARS- CoV-2 infection of epithelia and rescue hamster models of COVID-19. . Novelty and Significance: To our knowledge this is the first time COVID-19 airway inflammation has been traced to inhibition of CFTR signaling. Consistently, COVID-19 patients who are also CF carriers, with only one wildtype CFTR gene and only 50% of CFTR function, were recently reported to suffer more severe COVID-19 symptoms and earlier death than normal subjects with COVID-19.