Cigarette Smoke Preferentially Induces Full Length ACE2 Expression in Differentiated Primary Human Airway Cultures But Does Not Alter Susceptibility to SARS-CoV-2 Infection

Cigarette smoking has many serious negative health consequences. The relationship between smoking and SARS-CoV-2 infection is controversial, specifically whether smokers are at increased risk of infection. We investigated the impact of cigarette smoke on ACE2 isoform expression and SARS-CoV-2 infection in differentiated primary human bronchial epithelial cells at the air-liquid-interface (ALI). We assessed the expression of ACE2 in response to CSE and therapeutics reported to modulate ACE2. We exposed ALI cultures to cigarette smoke extract (CSE) and then infected them with SARS-CoV-2. We measured cellular infection using flow cytometry and whole-transwell immunofluorescence. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated isoform (dACE2) that lacks the capacity to bind SARS-CoV-2. CSE did not have a significant impact on key mediators of the innate immune response. Importantly, we show that, despite the increase in flACE2, CSE did not alter airway cell infection after CSE exposure. We found that nicotine does not significantly alter flACE2 expression but that NRF2 agonists do lead to an increase in flACE2 expression. This increase was not associated with an increase in SARS-CoV-2 infection. Our results are consistent with the epidemiological data suggesting that current smokers do not have an excess of SARS-CoV-2 infection. but that those with chronic respiratory or cardiovascular disease are more vulnerable to severe COVID-19. They suggest that at physiological expression levels, flACE2 does not limit airway SARS-CoV-2 infection.Funding Information: We are grateful for the generous support of the UKRI COVID Immunology Consortium, Addenbrooke’s Charitable Trust (15/20A) and the NIHR Cambridge Biomedical Research Centre. This work was supported by a Wellcome Trust Principal Research Fellowship (084957/Z/08/Z) and MRC research grant MR/V011561/1 to P.J.L. This work was supported by the NC3Rs NC/S001204/1 project grant and the Roy Castle Lung Cancer Foundation grant (2015/10/McCaughan) to FM.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Cambridge University Hospitals NHS Trust (Research Ethics Committee Reference 19/SW/0152; Donor 2).