key: cord-0958483-26efnyda
authors: Jacobs, Merel; Van Eeckhoutte, Hannelore P.; Wijnant, Sara R. A.; Janssens, Wim; Joos, Guy F.; Brusselle, Guy G.; Bracke, Ken R.
title: Increased expression of ACE2, the SARS-CoV-2 entry receptor, in alveolar and bronchial epithelium of smokers and COPD subjects
date: 2020-07-16
journal: Eur Respir J
DOI: 10.1183/13993003.02378-2020
sha: b54df46b1878b31451f1b85cffe8460a9f9e333d
doc_id: 958483
cord_uid: 26efnyda

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. Angiotensin-converting enzyme 2 (ACE2) was identified as the cell entry receptor used by SARS-CoV-2 [1, 2]. Importantly, smokers and patients with COPD are at increased risk for severe complications and a higher mortality upon SARS-CoV-2 infection [3]. We hypothesised that ACE2 expression is increased in lungs of smokers and patients with COPD, which may at least partially explain their higher risk for a more severe course of COVID-19. Therefore, we aimed to investigate the expression of ACE2 on both mRNA and protein level in a large number of lung tissue specimens of well phenotyped subjects, including never smokers, current smokers without airflow limitation, and patients with COPD.

identified as the cell entry receptor used by SARS-CoV-2 [1, 2] . Importantly, smokers and patients with COPD are at increased risk for severe complications and a higher mortality upon SARS-CoV-2 infection [3] . We hypothesized that ACE2 expression is increased in lungs of smokers and patients with COPD, which may at least partially explain their higher risk for a more severe course of COVID-19. Therefore, we aimed to investigate the expression of ACE2 on both mRNA and protein level in a large number of lung tissue specimens of well phenotyped subjects, including never smokers, current smokers without airflow limitation, and patients with COPD.

In this cross-sectional observational study, we analyzed lung tissue specimens from 134 subjects from our large lung tissue biobank at Ghent University Hospital and from explant lungs from end-stage COPD patients collected at UZ Gasthuisberg Leuven, Belgium. Ex-smoking was defined as smoking cessation for at least 1 year. COPD severity was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification. Written informed consent was obtained from all subjects. This study was approved by the medical ethical committees of the Ghent University Hospital (2016/0132; 2019/0537) and the University Hospital Gasthuisberg Leuven (S51577).

RNA extraction from lung tissue blocks of 120 subjects was performed with the miRNeasy Mini kit (Qiagen, Hilden, Germany). Next, cDNA was prepared with the EvoScript Universal cDNA Master Kit (Roche), followed by RT-PCR analysis for ACE2 and 3 reference genes as described previously [4, 5] .

Sections from formalin fixed paraffin embedded lung tissue blocks of 87 subjects were stained for ACE2. 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). Quantitative measurements of the ACE2-positive signal in alveolar tissue and bronchial epithelium were performed on images of stained paraffin sections as described previously [6] .

Statistical analysis was performed with Sigma Stat software (SPSS 26.0, Chicago, IL, USA) and R3.5.1, using Kruskal-Wallis tests (on all 6 groups) followed by Mann-Whitney U tests (for the comparison between 2 groups), and multivariable linear regression analyses.

Using RT-PCR, ACE2 mRNA levels were determined in lung tissue from 120 subjects. ACE2 mRNA expression was significantly higher in lung tissue of current smokers without airflow limitation and current smokers with COPD (GOLD stages II and 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. Multivariable linear regression analysis demonstrated that current smoking and COPD are both independently associated with increased ACE2 mRNA expression in lung tissue, even after adjustment for covariates including age, sex, BMI, and arterial hypertension (data not shown).

By immunohistochemical (IHC) staining, ACE2 protein levels were assessed in lung tissue from 87 subjects. ACE2 IHC revealed positive staining in both bronchial and alveolar epithelial cells, with the latter predominantly in alveolar type II cells ( Figure 1B-C) . Quantification of ACE2 protein levels in alveolar tissue revealed a significantly higher percentage of ACE2-positive alveolar tissue in current smokers without airflow limitation and current smokers with COPD (GOLD II and III-IV) compared to never smokers ( Figure 1B) . Moreover, the percentage of ACE2-positive alveolar tissue was significantly higher in patients with COPD GOLD III-IV, compared to ex-smokers without airflow limitation and exsmokers with COPD GOLD II ( Figure 1B) . 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 1C) . 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 exsmokers with COPD GOLD II ( Figure 1C) . The observed association between COPD and ACE2 protein expression in alveolar tissue or bronchial epithelium remained significant after adjustment for possible confounders (data not shown).

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. While our observations complement previous reports on increased ACE2 mRNA and protein levels in whole lung tissue and bronchial epithelium [7] [8] [9] [10] , this is the first study demonstrating increased ACE2 protein in alveolar epithelium of smokers and patients with COPD, which can be directly linked to the site of injury when patients with severe COVID-19 develop dyspnea, hypoxia and pneumonia.

Currently, published data on COVID-19 in patients with COPD is fairly limited [11] .

Nevertheless, an increased risk of developing severe COVID-19 as well as a higher mortality, has been reported in patients with COPD and in current smokers [3, 12] . Although there are several possible explanations for the increased susceptibility for severe COVID-19 in patients with COPD, including older age, comorbidities, dysregulated immune defenses and impaired mucociliary clearance, increased pulmonary expression of the SARS-CoV-2 entry receptor ACE2 is most likely another contributor [13] . 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 [14] .

Moreover, human ACE2 was essential for viral replication in the lung.

The main strength of this study is the large number of lung tissue samples from wellphenotyped subjects that are included in the 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 samples from lung resections for pulmonary tumors. This may introduce selection bias since altered expression of ACE2 in lung cancer has been suggested [15] . Second, samples of (very) severe COPD originate from a different patient population (lung transplantation), for which smoking cessation was an inclusion criterium. Finally, since we did not study lung tissue samples from COVID-19 positive subjects, we can only speculate on the importance of increased ACE2 in the pathogenicity of SARS-CoV-2.

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. Further research is needed to elucidate whether upregulation of ACE2 expression in airways and lungs has consequences on infectivity and clinical outcomes of COVID-19. 

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