Cigarette Smoke Exposure Increases ACE-2 Expression and SARS-CoV-2 Infection Severity in Human and Ferret Airways and Induces Apoptotic Cell Injury In Vitro

Abstract

RATIONALE: Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking could alter expression of angiotensin converting enzyme-2 (ACE2), which functions as the cellular receptor and point of entry. Here we investigated the severity of SARS CoV-2 infection ex vivo and in vitro and using tissue samples and primary ferret and human airway epithelial cells with and without antecedent exposure to cigarette smoke. Methods: ACE2 expression measured by Quantitative PCR (Q-PCR) of ferret lungs exposed to 6 months of cigarette smoke, and findings validated by immunofluorescence (IF). Primary airway cells isolated from airways of ferrets or human non-smokers or COPD subjects were grown until terminally differentiated at air liquid interface. Cells were then exposed to cigarette smoke extract (CSE) or vehicle control, then infected with SARS CoV-2 (3 MOI) or mock control. Viral copy was measured by Q-PCR. Viral infection was quantified by foci forming assay (FFU/mL) using VeroE6 cells. Results: Ferret lungs following 6 months of smoke exposure had increased ACE2 as compared to air controls by Q-PCR (>1.5 fold, P<0.05, N=6), and IF staining. Higher ACE2 expression was also observed in normal ferret airway cells exposed to CSE (>1.5 fold, P<0.05, N=3), normal Human Bronchial Epithelial (HBE) cells exposed to CSE (>2 fold, P<0.001, N=4), and HBE cells from COPD donors as compared to healthy controls (>2 fold, P<0.001, N=4). When ferret airway cells were inoculated with SARS-CoV-2, intracellular viral load of SARS-CoV-2 was increased in CSE exposed cells as compared to vehicle controls (103-104 vehicle Vs CSE 105 106 copy/μL). Viral infection was also increased >2 fold (P<0.01, N=5). Likewise, CSE exposed (105-107 copy/μL, P<0.0001, N=4) and COPD (106-108 copy/μL, P<0.0001, N=4) HBE had increased viral load as compared to controls (103-104 copy/μL, P <0.001, N=4), and 2 to 3-fold increase in viral infection, respectively. TUNEL staining was increased in infected cells, indicating apoptosis. Transcript analysis of HBE cells with and without SARS-CoV-2 by RNASeq to identify differentially expressed genes in CSE exposed cells as compared to controls is in progress. Conclusion: Cigarette smoke and CSE increased ACE2 expression in ferrets, and ferret and human cells respectively. CSE-induction increased viral replication and infection severity, resulting in increased apoptosis. Cigarette smoking likely influences the severity of SARS-CoV-2 infection by altering expression of ACE2, inducing airway cell apoptosis upon infection.