Paradoxical effects of cigarette smoke and COPD on SARS-CoV-2 infection and disease.

BACKGROUND: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro. METHODS: We quantified: (1) pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and/or TMPRSS2 mRNA levels by RT-qPCR in two independent human cohorts; and (2) pulmonary ACE2 protein levels by immunostaining and ELISA in C57BL/6 WT mice exposed to air or CS for up to 6 months. The effects of CS exposure on SARS-CoV-2 infection were evaluated after in vitro infection of Calu-3 cells and differentiated human bronchial epithelial cells (HBECs), respectively. RESULTS: ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus controls but similar in central airways. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice. CS treatment decreased viral replication in Calu-3 cells, as determined by immunofluorescence staining for replicative double-stranded RNA (dsRNA) and western blot for viral N protein. Acute CS exposure decreased in vitro SARS-CoV-2 replication in HBECs, as determined by plaque assay and RT-qPCR. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-exposure potently inhibited SARS-CoV-2 replication in vitro. These findings urge to investigate further the controversial effects of CS and COPD on SARS-CoV-2 infection.

Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease.

INTRODUCTION: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. METHODS: In Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. RESULTS: In both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.