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

Abstract

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.

Fig. 1

ACE2 expression in bronchial and alveolar epithelium from COPD patients, smoker and never-smoker (NS) controls. The number of ACE2+ cells in the central airway bronchial epithelium was similar between patients with chronic obstructive pulmonary disease (COPD), smokers without COPD and NS controls. In A, representative IHC for ACE2 images of central airways of a COPD patient (upper panel) and a smoker without COPD. The insets show details of the ciliated bronchial epithelium. The number of ACE2+ cells in the alveolar epithelium (B) and peripheral airway epithelium (C), normalized for length of the alveolar wall or basement membrane, respectively, was lower in patients with COPD versus smokers without COPD and NS controls. In D, triple immunofluorescence representative images of alveolar (upper panels) and bronchiolar epithelium (lower panels) from a COPD patient, a smoker without COPD, and a never-smoker (NS) where ACE2 staining is identified by green fluorochrome, the epithelium is identified by red fluorochrome, and the color yellow is obtained by merging the two fluorochromes. In E, the levels of ACE2 mRNA from peripheral lung samples were decreased between patients with chronic obstructive pulmonary disease (COPD) versus both smoker without COPD and NS controls. The red circles indicate the current smokers among the smoker controls and COPD patients

Fig. 2

ACE2 expression in bronchial and alveolar epithelium from mice exposed to room air and acutely or chronically to cigarette smoke (CS). WT C57BL6 mice were exposed to air or cigarette smoke (CS) for up to 6 months (n = 3/4 group). In A, the number of ACE2+ cells in the bronchial epithelium were decreased in 6-month CS-exposed mice versus 1-, 3-, and 6- month air-exposed mice. The number of ACE2+ cells in the bronchial epithelium was decreased in 3-month CS-exposed mice versus 3- and 6-month air-exposed mice. The number of ACE2+ cells in the bronchial epithelium was decreased in 6-month CS-exposed mice versus 1-month CS-exposed mice. Also, the number of ACE2+ cells in the alveolar epithelium was decreased in 6- and 3-month CS-exposed mice versus 1- and 6- month air-exposed mice. The number of ACE2+ cells in the alveolar epithelium was decreased in 6-month CS-exposed mice versus 1-month CS-exposed mice. In B, representative images of air-exposed (upper 3 panels) and CS-exposed (lower 3 panels) murine small airways where bronchial epithelial cells are identified by staining with a red fluorophore, mucin-producing cells by a cyan fluorochrome, and ACE2+ cells by a green fluorophore. In C, representative images of air-exposed (upper three panels) and CS-exposed (lower three panels) murine alveolar cells where the epithelial cells are identified by staining with a red fluorophore, and ACE2+ cells by a green fluorophore. The staining isotype control for each staining is also shown. In D, the ACE2 protein levels measured by ELISA in total lung homogenates were decreased in mice exposed to CS for one month versus air (n = 10–15/group). * = P < 0.05; ** = P < 0.01; *** = P < 0.001. CS cigarette smoke

Fig. 3

Cigarette Smoke (CS) extract blocks in vitro SARS-CoV-2 replication in Calu-3 cells. To investigate the effects of cigarette smoke (CS) exposure on SARS-CoV-2 infection, we performed a 72 h in vitro infection of Calu-3 cells, a line permissive to SARS-CoV2 infection and replication. Cells were sham- or CSE-treated for 24 h. Supernatants (SN) and cytoplasmic lysates were obtained from a cell aliquot to measure ACE2 levels by ELISA. Then, cells were infected with SARS-CoV-2 (2 h viral infection in normal media, then remove inoculum). Every 24 h cells were fixed for IF staining of infection, and cell lysates were harvested for SDS-PAGE and WB of viral nucleocapsid (N) protein. dsRNA intermediates arise during the replication of viral RNA (vRNA), and IF staining with dsRNA-specific J2 monoclonal Ab is a good marker for SARS-CoV-2 replication. A Nikon Ti2 automated microscopy was used to quantitatively measure infection, as seen by dsRNA signal. Whereas replication of vRNA peaked at 48 h (A, B) in sham-treated cells, CSE-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 72 h (C). In C, immunoblots show two bands used for densitometry and separated with a horizontal white line, one for the N protein, and one for GADPH. The two bands were cropped from original gels that are available in a Supplementary repository. ACE2 protein levels were undetectable in the SN, but were unchanged in CSE-treated versus sham cell lysates (not shown). In summary, CSE-pre-exposure increased ACE2 levels but potently abrogated SARS-CoV-2 replication in this in vitro model. The figure is representative of three independent experiments

Fig. 4

Acute CS treatment blocks in vitro SARS-CoV-2 replication in differentiated primary airway epithelium. HBECs, fully differentiated into airway epithelium by culture at air–liquid interface, were subjected to acute sham- or CS-exposure prior to SARS-CoV-2 infection. Cells were infected via apical inoculation with SARS-CoV-2 at 0.05 PFU/cell MOI for 1 h at 37C, washed to remove unbound virus, and infections were incubated for 24 h, 48 h, and 72 h. A Samples are collected for RNA purification and RT-qPCR to detect the level of SARS-CoV-2 nucleocapsid gene in infected cells by following the procedure described in method session. The SARS-CoV-2 N gene level is lower in 48 h and 72 h post-infected CS-treated cells compare to Sham-treated cells (n = 3 technique replicates). * = P < 0.05. ** = P < 0.01. CS cigarette smoke. B The infected HBEC cells were sampled by addition of 200 uL fresh media to the apical side of the transwell to collect all the progeny virus that is released from the infected cells. The amount of the progeny viruses was tittered by plaque assay as described in method session. CS-treated cells generated less SARS-CoV-2 progeny compared to Sham-treated cells after 48 and 72 h of infection (n = 4). * = P < 0.05. CS cigarette smoke