Cystic fibrosis airway cells are less susceptible to sars-cov-2 infection and have altered ph regulatory protein expression

Introduction: In the airways of people with Cystic Fibrosis, reduction in HCO3- secondary to loss of CFTR protein function contributes to low extracellular pH and impaired mucus clearance making them more susceptible to infection.1 It may also contribute to the higher intracellular pH seen in CF primary human airway epithelial cells (HAECs). Respiratory viruses are a leading cause of pulmonary exacerbations in people with CF. While it was expected that SARS-CoV-2 would lead to severe infection in this population, only 500 confirmed cases have been identified with most being categorized as mild. We have recently shown that increasing intracellular pH with a novel inhaled alkaline treatment, Optate, inhibits the replication of SARS-CoV-2 in normal HAECs. We hypothesize that the baseline increased intracellular pH in the airways of people with CF is protective against SARS-CoV-2 infection. We further hypothesized that pH regulatory proteins may contribute to this increased intracellular pH in CF beyond decreased secretion of bicarbonate. Methods: CF and non-CF primary HAECs were infected with SARS-CoV-2 at a multiplicity of infection of 1. SARS-CoV-2 viral titers in cell media were subsequently quantified by plaque assay. Cytopathic effects (CPE) were evaluated by visual microscopy. Results were compared between CF and non-CF HAECs. Levels of pH regulatory proteins NHERF1, LDHd, LDHb, LDHa, glutaminase, S-nitrosoglutathione reductase (GSNOR), DUOX1, CCNO, carbonic anhydrase 2 (CA2), carbonic anhydrase 12 (CA12), and ATP12a S-were measured in cell lysates from the same CF and non-CF HAECs by JESS automated western blot (ProteinSimple, San Jose, CA, USA). Finally, pH was compared between the CF and non-CF HAECs using a pH-sensitive fluorescent dye, pHrodo Red (Thermo Fisher Scientific, Waltham MA, USA) imaged during fluorescent microscopy. Results: Intracellular pH of CF HAECs was significantly higher than non-CF HAECs (n = 6, p < 0.001). CF HAECs had significantly less SARS-CoV-2 viral titers than non-CF HAECs (n = 6, p = 0.0192). No difference in CPE was observed between groups. CA2 and CA12 protein levels were significantly increased in CF HAECs compared to non-CF HAECs (n = 6, p = 0.0094 and 0.0113, respectively). Conclusions: CF HAECs are less susceptible to SARS-CoV-2 viral infection that non-CF HAECs. This may be due to the increased intracellular pH in CF HAECs compared to non-CF HAECs. Since carbonic anhydrases produce bicarbonate ions, CA2 and CA12 upregulation may contribute to increased intracellular pH in CF along with decreased bicarbonate extrusion due to CFTR function loss.