SARS-CoV-2 (COVID-19) adhesion site protein upregulation in small airways, type II pneumocytes, and alveolar macrophages of smokers and patients with COPD.

COPD is the third leading cause of death globally, a chronic lung disease characterised by partially reversible airflow obstruction, with increased exacerbations, dyspnoea, and decreased quality of life (1). Smoking primarily causes COPD, although air pollution through fossil fuel burning is a contributing factor too and is an increasingly likely comorbidity risk for severe COVID-19 (2, 3). COPD is partly characterised by the variable immune response, which sees increased macrophage recruitment in conjunction with other pathological inflammatory mediators (4, 5). COPD patients are highly susceptible to respiratory infections, with a study showing 30% of COPD exacerbations related to respiratory viruses (6). Therefore, both cigarette smoking and COPD are likely risk factors for an increased severity of COVID-19. Along with cigarette smoking and COPD being linked to dysfunctional immunity, they are also shown to relate to an increased expression of proteins linked to pathogen adhesion, driving smoking-induced pneumonia and other respiratory infections (7-12).
Since the emergence of the novel Coronavirus SARS-CoV-2 at the close of 2019, COVID-19 has spread rapidly around the globe. As of early 2023, there are over 763 million reported cases of COVID-19, nearly 7 million deaths across 223 countries worldwide (13). SARS-CoV-2, the virus behind COVID-19, is one member of the large coronavirus family (14). COVID-19 has a range of symptomatic presentations, from respiratory distress and airway damage to death (15-17). In approximately 80% of COVID-19 infections, patients present with mild respiratory illness (18). Risk factors for more severe infections include age, comorbidities, hypoxia, and severe immune response (19).
Human angiotensin-converting enzyme 2 (ACE2), Furin, and transmembrane serine protease 2 (TMPRSS2), are utilised by respiratory viruses as a receptor for cell adhesion and entry (20). SARS-CoV-2 utilises the ACE2 enzyme as the critical entry point into human cells (20, 21). This study hypothesised that smoking could upregulate ACE2 expression. If confirmed, smoking-induced upregulation of ACE2 in the lung would be a largely avoidable risk factor linked to an increased susceptibility of developing COVID-19. The cells targeted by SARS-CoV-2 are predominately type II pneumocytes and alveolar macrophages, with studies showing increases in these cell types attributed to smoking and COPD (22-27). New data is drawing attention to various host mechanisms by which SARS-CoV-2 enters the cell (28). Furin is a proprotein convertase that is believed to be an essential protein in the configuration of the SARS-CoV-2 envelope, processing essential membrane proteins (29). SARS-CoV-2 utilises the S protein, a granule?shaped structural protein, which aids in viral-cellular binding and is activated by Furin (30, 31). TMPRSS2, like Furin, is believed to cleave the viral S glycoprotein with a similar outcome. These processes are also seen in other coronaviruses, such as SARS-CoV, and viruses in the Orthomyxoviridae family, such as influenza and H1N1 (32). Current data suggests that smoking may upregulate Furin expression in lung tissue, however, the effect of smoking on TMPRSS2 is debated, with some reporting no effect while others show increased expression (33, 34).
Among the 42 patients, 16 patients were diagnosed with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or stage II COPD, of which 8 were current smokers and 8 were ex-smokers (>1 year smoking cessation); 9 patients had small airway disease only, 7 were normal lung function smokers; 10 were never-smoking normal controls. Surgical resections, taken during lung cancer surgery, were fixed in formalin within minutes of surgery. Never smoking normal control tissue was taken at autopsy from patients who were deceased due to non-respiratory related reasons. After processing, the resected small airway (<2 mm internal diameter) tissue blocks were separately embedded in paraffin wax for research analyses. The tissue was sectioned at 3.5µm and processed with standard immunohistochemical staining procedures. Immunostaining was completed for ACE2, Furin and TMPRSS2. Antibody binding was visualised by a substrate 3,3'-diaminobenzidine (DAB) reaction. This produces a brown colour, indicating positive staining. Nuclear counterstain was achieved with gills hematoxylin. All slides were coded and randomised to blind the analyst. Images were taken of the small airway epithelium (airways less than 2 mm in diameter and lacking cartilaginous support) at x40 magnification and of lung parenchyma at x20. Epithelial ACE2, Furin and TMPRSS2 staining were measured as a percentage of positive staining in the small airway epithelium. In addition, Type-2 pneumocytes, both positive and negative for ACE2, Furin and TMPRSS2 for type-2 pneumocytes, were counted per parenchymal tissue area in the alveolar epithelium. Similarly, alveolar macrophages staining positive and negative for ACE2, Furin and TMPRSS2 were counted per parenchymal tissue area.
Primary small airway epithelial cells were seeded in culture slides and incubated to attain at least 80 percent confluence. Cells were treated with cigarette smoke extract or appropriate vehicle control and further incubated. The cells were rinsed and after blocking were stained with anti-human ACE2, Furin or TMPRSS2. Following subsequent preparations, the cells were examined, and images were taken using an Olympus FV1200 confocal laser scanning microscope. Alexa Fluor?488 images were captured under excitation. With the aid of ImageJ software, averaged corrected total cell fluorescence was calculated by measuring the integrated density of individual cells minus the integrated density of the background. Co-localisation of immunofluorescent staining and Pearson’s coefficients were calculated with ImageJ software by splitting the colour channels of the images and running the JACoP plugin with conserved thresholds.
Analysis showed an increase in the expression of ACE2, Furin and TMPRSS2 in the small airway epithelium, type II pneumocytes and alveolar macrophages of smokers and COPD patients. This is the first report on the significant increase in type II pneumocytes in smokers and patients with COPD, suggesting active interstitial pathology. This increase indicates that smokers and patients with COPD could be at higher risk of developing post-COVID-19 interstitial pulmonary fibrosis. The analysis also provides links between increased expression of ACE2 because of smoking, SAD, and COPD in both type II pneumocytes and macrophages, correlating this with previous information linking possible pathogenic phenotypes between COPD and COVID-19. This study shows that along with increased ACE2 expression, the cofactors Furin and TMPRSS2 also increase in smokers, SAD patients, COPD patients. These findings were further supported by the cell culture analysis using cigarette smoke extract treatment. Overexpression of these proteins links smokers and COPD patients, increased susceptibility to the SARS-CoV-2 virus, and the potential for severe manifestations of COVID-19. Mounting evidence supports the hypothesis that smoking is an avoidable risk factor during the COVID-19 pandemic. This study also found novel changes in COPD respiratory cell populations that may offer links to idiopathic pulmonary fibrosis. These cell population changes were mirrored in the literature, in COVID-19 postmortems suggesting possible links between pathological mechanisms of COPD, and ‘long’ or chronic COVID that fits a more fibrotic phenotype.