Fig 1: 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 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: 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
Supplier Page from MyBioSource.com for Mouse ACE2 ELISA Kit