key: cord-331895-3srslmgk
authors: Jacobs, M.; Van Eeckhoutte, H. P.; Wijnant, S. R.; Janssens, W.; Joos, G. F.; Brusselle, G. G.; Bracke, K. R.
title: Increased expression of ACE2, the SARS-CoV-2 entry receptor, in alveolar and bronchial epithelium of smokers and COPD subjects
date: 2020-06-02
journal: nan
DOI: 10.1101/2020.05.27.20114298
sha: 
doc_id: 331895
cord_uid: 3srslmgk

Rationale: Smokers and patients with chronic obstructive pulmonary disease (COPD) are at increased risk for severe Coronavirus Disease 2019 (COVID-19). Objectives: We investigated the expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptor ACE2 and the protease TMPRSS2 in lung tissue from never smokers and smokers with and without COPD. Methods: In a cross-sectional, observational study we measured mRNA expression of ACE2 and TMPRSS2 by RT-PCR in lung tissue samples from 120 well phenotyped subjects. Next, protein levels of ACE2 were visualized by immunohistochemistry on paraffin sections from 87 subjects and quantified in alveolar and bronchial epithelium. Finally, primary human bronchial epithelial cells (HBECs) were cultured at air liquid interface and exposed to air or cigarette smoke. Results: ACE2 mRNA expression was significantly higher in lung tissue from current smokers and subjects with moderate to very severe COPD and correlated with physiological parameters of airway obstruction and emphysema. Pulmonary expression levels of TMPRSS2 were significantly higher in patients with (very) severe COPD and correlated significantly with ACE2 expression. Importantly, protein levels of ACE2 were elevated in both alveolar and bronchial epithelium of current smokers and subjects with moderate to very severe COPD. Finally, TMPRSS2 mRNA expression increased in in vitro cultured HBECs upon acute exposure to cigarette smoke. Conclusions: We demonstrate increased expression of ACE2 in lungs of smokers and COPD subjects, which might facilitate host cell entry of SARS-CoV-2. These findings help identifying populations at risk for severe COVID-19.

Coronavirus Disease 19 (COVID-19) is a novel emerging respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is causing a vast pandemic with huge medical, social, personal and financial impact. The virus first appeared in Wuhan, China and rapidly spread across the rest of the world, making COVID-19 the third large-scale pandemic caused by coronaviruses after SARS in 2002 and Middle East respiratory syndrome (MERS) in 2012 [1, 2] . The clinical course of the disease ranges from an asymptomatic course to progressive respiratory failure with need for ventilatory support and even death [1] .

Angiotensin-converting enzyme 2 (ACE2) was identified as the cell entry receptor used by SARS-CoV-1 and SARS-CoV-2 [1, 3] . ACE2 is a metallopeptidase I and a homologue of the ACE-receptor. Although both receptors play important roles in the renin-angiotensinaldosterone system (RAAS), they have profoundly different roles. Whereas ACE converts angiotensin I (Ang I) to angiotensin II (Ang II), a potent vasoconstrictor, ACE2 converts both Ang I and Ang II to Ang-(1-9) and Ang-(1-7) respectively, thereby counteracting the effects of ACE and Ang II [4, 5] . Expression of ACE2 was shown in various cells and tissues, including alveolar type II cells and bronchial epithelial cells, as well as in the oral cavity, oesophagus, heart, kidney, and ileum [6] . In addition, transmembrane protease, serine 2 (TMPRSS2) was identified as the host cell protease used by SARS-CoV-2 for S protein priming, which is an essential part of the viral entry process [7] .

Although SARS-CoV-2 can infect any individual, most severe cases are reported in those with comorbidities such as hypertension, diabetes, and chronic obstructive pulmonary disease (COPD) [2, [8] [9] [10] . COPD is a highly prevalent chronic respiratory disease, characterized by an abnormal inflammatory response to cigarette smoke [11] . Importantly, both smokers and patients with COPD are at increased risk for severe complications and a higher mortality upon . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05. 27.20114298 doi: medRxiv preprint SARS-CoV-2 infection [12, 13] . Whether this is due to an increased expression of ACE2 in the lungs, and more specifically in the alveolar tissue where pneumonia occurs in severe COVID- 19 , needs thorough investigation. A recent paper reported increased ACE2 expression in bronchial brushings and airway epithelia of current smokers and patients with COPD, compared to healthy controls [14] . Previous work done by our group, showed upregulation of dipeptidyl peptidase 4 (DPP4), the viral entry receptor for the MERS coronavirus, in alveolar tissue of smokers and patients with COPD [15] .

We hypothesized that the increased risk for a more severe course of COVID-19 in smokers and patients with COPD is due to an increased expression of the SARS-CoV-2 entry receptor ACE2 in the lung. Therefore, we aimed to investigate the expression of ACE2, as well as the host cell protease TMPRSS2, in a large number of lung tissue specimens of well phenotyped subjects, including never smokers and smokers with and without airflow limitation. We quantified ACE2 and TMPRSS2 mRNA expression in the lung, as well as ACE2 protein levels in both alveolar and bronchial epithelium. Next, we investigated the independent determinants of pulmonary ACE2 expression by linear regression analysis. Finally, we determined mRNA expression of ACE2 and TMPRSS2 in cigarette smoke-exposed primary human bronchial epithelial cells.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. 

Methodological details are available in the online data supplement. Study design, hypothesis, methods, analyses, and findings are reported according to the STROBE checklist for crosssectional studies [16, 17] .

In this cross-sectional, observational study, we analyzed lung tissue specimens from a total of 134 subjects: 106 lung samples from our large lung tissue biobank at Ghent University Hospital (collected from lung resections for solitary pulmonary tumors and currently encompassing 360 subjects) and 28 samples from explant lungs from end-stage COPD patients collected at UZ Gasthuisberg Leuven, Belgium. All lung samples were collected between January 2002 and February 2020, before the first reported case of COVID-19 in Belgium. A flow-chart illustrating the selection of lung samples for RT-PCR (n=120) and immunohistochemical (n=87) analyses is shown in Figure E1 . Based on medical history, smoking history, questionnaires and preoperative spirometry, patients were categorized as never-smokers with normal lung function, smokers without airflow limitation or subjects with COPD. Subjects were considered exsmokers when they had quitted smoking for more than 1 year. COPD severity was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification.

None of the patients were treated with neo-adjuvant chemotherapy. Lung tissue of patients diagnosed with solitary pulmonary tumors was obtained at a maximum distance from the pulmonary lesions and without signs of retro-obstructive pneumonia or tumor invasion and collected by a pathologist. Written informed consent was obtained from all subjects. This study was approved by the medical ethical committees of the Ghent University Hospital (2011/0114; 2016/0132; 2019/0537) and the University Hospital Gasthuisberg Leuven (S51577).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. 

Primary human bronchial epithelial cells (HBECs) were isolated by enzymatic digestion from lung resection specimens derived from 6 donors (1 non-COPD and 5 COPD GOLD stage II or III) during surgery for lung cancer, as described previously [18] . After a 14-day ALI culture period, cells were exposed to mainstream cigarette smoke or air, as described previously, and harvested after 3, 6 or 24 hours [19] .

RNA extraction from lung tissue blocks of 120 subjects (including 28 never-smokers, 36 smokers without airflow limitation, 42 patients with COPD GOLD II and 14 patients with COPD GOLD III-IV, see Table 1 for patient characteristics), as well as from the ALI cultured HBECs, was performed with the miRNeasy Mini kit (Qiagen, Hilden, Germany), between 2015 and 2020. Next, cDNA was prepared with the EvoScript Universal cDNA Master Kit (Roche) in March 2020, followed by RT-PCR analysis for ACE2, TMPRSS2 and 3 reference genes as described previously [20, 21] .

Sections from formalin fixed paraffin embedded lung tissue blocks of 87 subjects (including 20 never-smokers, 24 smokers without COPD, 29 subjects with COPD GOLD II and 14 subjects with COPD GOLD III-IV, see supplementary Table E1 for patient characteristics) were stained for ACE2 in March and April 2020. After antigen retrieval with citrate buffer (Scytek), the slides were incubated with anti-ACE2 antibody (polyclonal rabbit-anti-human, Abcam ab15248). Next, slides were colored with diaminobenzidine (Dako, Carpinteria, CA, USA) and counterstained with Mayer's hematoxylin (Sigma-Aldrich, St-Louis, MO, USA). The isotype control was rabbit IgG (R&D systems, AB-150-C).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint

Quantitative measurements of the ACE2-positive signal in alveolar tissue and bronchial epithelium was performed on images of stained paraffin sections as described previously [15] .

A detailed description of the methods can be found in the online supplement. Briefly, to quantify the amount of ACE2-positive signal in alveolar tissue, 10 images of alveolar tissue (not containing airways or blood vessels) were recorded from an average of 3 tissue blocks per patient. The area of brown ACE2-positive staining was measured and normalized to the total area of alveolar tissue present in each image. Additionally, the number of ACE2-positive cells was manually counted in each image and again normalized to the total area of alveolar tissue.

To quantify the amount of ACE2-positive signal in airway epithelium, images of airways were recorded from an average of 3 tissue blocks per patient. The amount of ACE2-positive signal was measured only in the airway epithelial layer and normalized to the length of the basement membrane (Pbm). The number of airways per patient was between 3 and 20.

Statistical analysis was performed with Sigma Stat software (SPSS 26.0, Chicago, IL, USA) and R3.5.1, using Student's t-test, Kruskal-Wallis and Mann-Whitney U test, Fisher's exact test, Spearman's Rank correlations, and multivariate linear regression analyses. Additional details and covariates for the regression analyses are described in the online supplement.

Characteristics of the study population are presented as median (interquartile range).

Differences at p-values < 0.05 were considered to be significant (*P<0.05, **P<0.01 and *** P<0.001).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint

Using real time quantitative PCR, ACE2 mRNA levels were determined in lung tissue from 120 subjects. Lung tissue from 20 never smokers, 36 smokers without airflow limitation (including 22 current smokers and 14 ex-smokers) and 42 patients with COPD GOLD stage II (including 28 current smokers and 14 ex-smokers) was derived from lobectomy specimens, whereas tissue from 14 subjects with COPD GOLD stage III-IV was derived from explant lungs after lung transplantation. Demographic and clinical patient characteristics are described in Table 1 .

ACE2 mRNA expression was significantly higher in lung tissue of current smokers without airflow limitation, current smokers with COPD GOLD stage II, and smokers with COPD GOLD stage III-IV, compared to never smokers ( Figure 1A ). In addition, ex-smokers without airflow limitation showed significantly lower ACE2 mRNA levels, compared to current smokers. TMPRSS2 mRNA expression was significantly higher in lung tissue of patients with COPD GOLD stage III-IV, compared to never smokers, smokers without airflow limitation and patients with COPD GOLD stage II ( Figure 1B) . Moreover, there was a significant positive correlation between pulmonary TMRPSS2 mRNA expression and ACE2 mRNA expression, even after excluding the high TMPRSS2 expressing GOLD stage III-IV patients ( Figure 1G ).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint ACE2 protein levels are increased in alveolar tissue and bronchial epithelium of smokers and COPD subjects.

By immunohistochemical (IHC) staining, ACE2 protein levels were assessed in lung tissue from 87 subjects: 20 never smokers, 24 smokers without COPD (15 current smokers and 9 exsmokers), 29 patients with COPD GOLD stage II (20 current smokers and 9 ex-smokers) and

14 smokers with COPD GOLD stage III-IV. Patient characteristics are described in supplementary Table E1 . Quantification of ACE2 staining in bronchial epithelium revealed numerically higher levels in current smokers without airflow limitation and current smokers with COPD GOLD II, and significantly higher levels in patients with COPD GOLD III-IV, compared to never smokers ( Figure 4A-E) . Moreover, ACE2 protein levels in bronchial epithelium were significantly higher in patients with COPD GOLD III-IV, compared to ex-smokers without airflow limitation and ex-smokers with COPD GOLD II ( Figure 4E ).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint

We investigated whether the increased ACE2 levels in subjects with COPD could partially result from cigarette smoking. Although the effect size for the association between COPD and pulmonary ACE2 mRNA expression was affected after adjusting for smoking status in linear regression analyses (unadjusted β 0.28 ± 0.09, p = 0.002; smoke status adjusted β 0.22 ± 0.10, p = 0.036), the association remained statistically significant. Secondly, we tested whether the association between COPD and pulmonary ACE2 mRNA could be confounded by comorbidities or intake of RAAS-inhibitors (ACE-inhibitors or angiotensin receptor blockers (ARB)). Multivariate linear regression analysis demonstrated that current smoking (β 0.20 ± 0.09, p = 0.023) and COPD (β 0.31 ± 0.09, p = 0.001) are both independently associated with increased ACE2 mRNA expression in lung tissue, even after adjustment for covariates including age, sex, diabetes, hypertension and use of RAAS-inhibitors ( Figure 5A ). Similar findings were observed for the association between COPD and ACE2 protein expression in alveolar tissue ( Figure 5B ) or bronchial epithelium and ( Figure E3) . Notably, in these models, diabetes mellitus seemed to be significantly associated with increased ACE2 levels in alveolar tissue and bronchial epithelium.

ACE2 mRNA expression was significantly higher in lung tissue of subjects using oral corticosteroid (OCS), but not in lung tissue of subjects using inhaled corticosteroid (ICS) ( Figure E4 ). To test whether increased ACE2 mRNA levels in OCS users may be partially due to underlying COPD, we performed linear regression analyses. Indeed, the significant age-and sex-adjusted association between OCS use and ACE2 mRNA expression (β 0.48 ± 0.16, p < 0.01) did not persist after additional adjustment for COPD (β 0.30 ± 0.17, p = 0.07).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. ACE2 and TMPRSS2 mRNA expression was determined by quantitative RT-PCR in HBECs, cultured at air liquid interface (ALI) and exposed to air or cigarette smoke (CS). ACE2 mRNA expression was not altered 3, 6 and 24 hours after CS-exposure ( Figure 6A ). In contrast, after 6 and 24 hours the TMPRSS2 mRNA expression in CS-exposed HBECs was significantly increased, compared to air exposed cells (Figure 6B ).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint

As healthcare systems around the world are currently under great pressure due to the COVID-19 outbreak, identification of those at high risk is crucial. There is compelling evidence of a more severe course of COVID-19 in smokers and patients with comorbidities such as COPD.

We clearly demonstrate an increased pulmonary expression of the SARS-CoV-2 entry receptor ACE2 in smokers and COPD subjects at both mRNA and protein level, by RT-PCR and immunohistochemistry respectively. Our study in smokers with and without COPD confirms our hypothesis that their increased risk for severe COVID-19 may be at least partially attributed to increased ACE2 expression. Importantly, we demonstrate higher ACE2 protein levels not only in bronchial epithelium but also in alveolar epithelium of patients with COPD, which can [24, 25] . Importantly, it has been demonstrated in mouse models that transgenic (over)expression of human ACE2 enhances the pathogenicity of SARS-CoV-1 and SARS-CoV-2 [26, 27] . Moreover, human ACE2 was essential for viral replication in the lung. The same is true for the presence of host cell proteases such as TMPRSS2 and furin, that are essential in the viral entry process [28] .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint

We measured mRNA expression of ACE2 and TMPRSS2 in lung tissue samples from 120 never smokers and smokers with and without airflow limitation and demonstrate higher ACE2 mRNA levels in current smokers and patients with moderate (GOLD II) and severe to very severe COPD (GOLD III-IV). Importantly, ACE2 mRNA expression shows an inverse correlation with physiological parameters of airway obstruction and emphysema. Linear regression analysis confirms that smoking and COPD are associated with increased ACE2 mRNA expression, independent of covariables such as age, gender, comorbidities, and medication use. The association between COPD and ACE2 mRNA seems to be partly driven by smoking as the effect size of the association between COPD and ACE2 mRNA decreased after adjusting for smoking. The remaining statistically significant association between COPD and ACE2 mRNA might be explained by other factors such as aggravated pulmonary and systemic inflammation, previous exacerbations or genetic predisposition. Interestingly, a recent large meta-analysis of transcriptomic data confirms the increased ACE2 mRNA expression in lung tissue of smokers and patients with COPD [28] .

TMPRSS2 mRNA expression is only significantly higher in patients with (very) severe COPD.

However, there is a significant correlation between TMPRSS mRNA and ACE2 mRNA expression levels, even in sensitivity analyses omitting (very) severe COPD patients from the analysis. Our data suggest that both ACE2 and TMPRSS2 are expressed on the same cells, and co-expression might further increase the chance of viral entry.

By immunohistochemical (IHC) staining we reveal expression of ACE2 in both bronchial and alveolar epithelial cells, with the latter predominantly in alveolar type II (ATII) pneumocytes.

recently been confirmed by both IHC and single cell RNA sequencing analyses [29] [30] [31] . Similar analyses also confirm the expression of ACE2 in bronchial epithelial cells, including goblet cells [31, 32] .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint By quantification of the ACE2 IHC staining, we demonstrate increased protein levels of ACE2 in both alveolar and bronchial epithelium of current smokers and patients with moderate and (very) severe COPD. Linear regression analyses suggest that these associations are partly driven by smoking. Importantly, we are the first to report quantitative evidence for increased ACE2 protein levels in alveolar epithelium, which is the primary site for pathogenic pulmonary infections and could thus explain why smokers and patients with COPD are more prone to develop bilateral pneumonia and/or acute respiratory distress syndrome (ARDS) in severe COVID-19. Indeed, the preferential expression of ACE2 on alveolar type II epithelial cells might make these cells more vulnerable to SARS-CoV-2 infection and virus-induced cell death.

Since ATII cells produce surfactants which are crucial for preventing alveolar collapse during breathing, SARS-CoV-2 induced targeting and lysis of ATII cells will alter lung compliance and induce ventilation-perfusion mismatch in severe COVID-19, leading to hypoxia and respiratory failure [33] . Our findings of increased ACE2 levels in bronchial epithelial cells complement the recent paper of Leung et al., who reported increased ACE2 mRNA expression in bronchial brushings of smokers and patients with COPD [32] .

In vitro cultures of primary HBECs at air liquid interface reveal increased mRNA expression of TMPRSS2 6 and 24 hours after a single exposure to cigarette smoke, indicative of an acute effect of smoking on the TMPRSS2 levels in the bronchial epithelium. ACE2 expression might be more dependent on chronic cigarette smoke exposure, since no effect on ACE2 mRNA expression is observed in these short-term in vitro experiments.

Inhaled corticosteroids (ICS) are essential in the treatment of asthma and a subgroup of COPD patients (i.e. with a history of frequent exacerbations). Since ICS are often regarded as immunosuppressive, there is a general concern on whether or not to continue the use of ICS during the COVID-19 pandemic. Importantly, our current data shows no association between the ICS use and the expression of ACE2 in the lung. We do report significantly increased ACE2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint expression in subjects using oral corticosteroids (OCS), albeit based on a low number of subjects using OCS, implicating the need to confirm these preliminary findings in larger studies.

By performing linear regression analyses on our data set, we reveal that lifestyle factors and comorbidities that are linked to a more severe course of COVID-19, such as smoking, COPD, and especially diabetes are associated with a higher expression of ACE2 protein in alveolar tissue. However, due to the observational design of this cross-sectional study, we cannot defer any causal relationships from these associations. Moreover, our results need to be replicated in larger studies and in subjects of other ethnicities.

The main strength of this study is the large number of lung tissue samples from well-phenotyped subjects that are included in the both RT-PCR and IHC analyses. Moreover, IHC allowed us to quantify ACE2 protein levels in both bronchial and alveolar epithelium. However, certain limitations should be kept in mind. First, this study consists mainly of lung tissue from lobectomy. Since these patients might not be representative for the general population, we cannot exclude selection bias. Second, differential information bias may have occurred when questioning smoke status at the time of lobectomy, since COPD status was known to the investigator. However, by carefully checking medical records, we ascertained accurateness of the patient characteristics. Last, although this study consisted of a relatively large and wellphenotyped cohort, samples size may be insufficient for multivariate linear regression analyses.

In conclusion, we report higher ACE2 mRNA and protein levels in lung tissue of smokers and subjects with moderate to (very) severe COPD. Importantly, ACE2 protein levels are not only increased in bronchial but also in alveolar epithelium. In addition, TMPRSS2 mRNA expression is higher in lung tissue of (very) severe COPD subjects and increases in in vitro . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission

Comorbidity and its impact on 1590 patients with Covid-19 in China: A Nationwide Analysis

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Clinical features of patients infected with

New insights into the immunology of chronic obstructive pulmonary disease

Severity and Mortality associated with COPD and Smoking in patients with COVID-19: A Rapid Systematic Review and Meta-Analysis

OpenSAFELY: factors associated with COVID-19-related hospital death in the linked electronic health records of 17 million adult NHS patients

Expression in the Small Airway Epithelia of Smokers and COPD Patients: Implications for COVID-19

in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients

Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration

Guidance on Statistical Reporting to Help Improve Your Chances of a Favorable Statistical Review

Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence

Role of B Cell-Activating Factor in Chronic Obstructive Pulmonary Disease

MicroRNA Profiling Reveals a Role for MicroRNA-218-5p in the Pathogenesis of Chronic Obstructive Pulmonary Disease

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Structural basis of receptor recognition by SARS-CoV-2

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Tobacco Smoking Increases the Lung Gene Expression of ACE2, the Receptor of SARS-CoV-2

the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A Potential Adhesion Site for Novel Coronavirus SARS-CoV-2 (Covid-19)

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

Expression in the Small Airway Epithelia of Smokers and COPD Patients: Implications for COVID-19

Eur Respir J 2020.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in per (which was not certified by peer review)The copyright holder for this this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in per (which was not certified by peer review). CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in per (which was not certified by peer review)The copyright holder for this this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.27.20114298 doi: medRxiv preprint