key: cord-0825850-xriudb7n authors: Higham, Andrew; Singh, Dave title: Increased ACE2 Expression in the Bronchial Epithelium of COPD Patients who are Overweight date: 2020-05-19 journal: Obesity (Silver Spring) DOI: 10.1002/oby.22907 sha: f4965100e92ba296f832ba1af91ccb8e47c67d5b doc_id: 825850 cord_uid: xriudb7n OBJECTIVE: Mortality from coronavirus disease 2019 (COVID‐19) is increased in COPD patients. Furthermore, higher body mass index (BMI) is related to severe disease. SARS‐CoV‐2 utilises angiotensin converting enzyme 2 (ACE2) to gain cellular entry. METHODS: We have investigated whether ACE2 bronchial epithelial expression is increased in COPD patients who are overweight compared to those who are not by RNA sequencing. RESULTS: We observed increased ACE2 expression in COPD patients who are overweight (mean BMI 29 kg/m(2)) compared to those notoverweight (mean BMI 21 kg/m(2)) (p=0.004). CONCLUSION: Increased ACE2 expression may cause increased SARS‐CoV‐2 infection of the respiratory tract. COPD patients who are overweight may be at greater risk of developing severe COVID‐19. The rapid emergence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the global coronavirus disease 2019 (COVID-19) pandemic. COVID-19 presents with symptoms including fever, cough and dyspnoea (1) . Lower respiratory tract infection is a characteristic feature of COVID-19, with diffuse bilateral ground-glass opacification and consolidation associated with respiratory failure in severe cases (2) . The mortality from COVID-19 is higher in patients with comorbidities including COPD, cardiovascular diseases and diabetes (1, 3, 4) . Furthermore, a higher body mass index (BMI) is related to increased hospital admission rates and a greater need for mechanical ventilation, independent of hypertension and diabetes (5-7). More severe COVID-19 is associated with higher levels of pro-inflammatory cytokines and ferritin in the blood, and lower lymphocyte and platelet counts, consistent with hyper-inflammation or a cytokine storm (8, 9) . SARS-CoV-2 is a beta coronavirus which gains cellular entry via angiotensin converting enzyme 2 (ACE2) (10) . ACE2 is an enzyme of the renin-angiotensin system which degrades angiotensin II, thereby promoting vasodilation and increased water excretion. ACE2 is expressed in many human organs, including the lungs. Leung et.al recently showed increased ACE2 expression in the bronchial epithelium of COPD patients compared to controls, a negative association between This article is protected by copyright. All rights reserved ACE2 expression and the degree of airflow obstruction, and increased expression in current smokers (11) . These findings suggest that current smoking and / or the presence of COPD may facilitate enhanced SARS-CoV-2 cellular entry due to increased access to the virus receptor ACE2. COPD patients often suffer from systemic comorbidities, with some patients showing muscle wasting and weight loss while others have obesity (12) . The expression of ACE2 is increased in the adipose tissue of people with obesity compared to those without (13). As higher BMI and COPD are both associated with more severe COVID-19 (1, 3, 4, 6), we have investigated whether ACE2 bronchial epithelial expression in COPD patients is increased in those who are overweight. 37 COPD patients aged >40 with a smoking history of > 10 pack-years, a post-bronchodilator forced expiratory volume in 1 sec (FEV 1 ) and forced vital capacity (FVC) ratio <0.7, and with no history of asthma were recruited for bronchoscopy. The cohort was separated into two groups based on a BMI threshold of >24.9 kg/m 2 which defines overweight and includes individuals with obesity (at ≥30 kg/m 2 ). There were 14 COPD patients with BMI <24.9 kg/m 2 (mean BMI 21) including 1 individual who was underweight (BMI<18.5), and 23 patients with BMI >24.9 kg/m 2 including 6 individuals with obesity (mean BMI 29). Patients receiving oral corticosteroids or antibiotics within six weeks of the study were excluded. Sample collection was approved by South Manchester REC 06/Q1403/156. All subjects provided written informed consent. The relationship between blood and lung eosinophil counts in these patients have been previously reported (14) . Bronchial brushings were collected in bronchial epithelial basal medium (Lonza, Slough, UK) and stored on ice before centrifugation (400 g for 10 min at 4°C). The cell pellet was re-suspended in RLT buffer (Qiagen) with β-mercaptoethanol added according to manufacturer's instructions. Following removal of the supernatants during sputum processing, the remaining sputum cell pellet was re-suspended in RLT buffer plus β-mercaptoethanol. Total RNA was extracted from bronchial epithelial brushings and sputum cells using ZR RNA MicroPrep kit (Zymo Research, Orange, CA, USA) and RNA-seq libraries were prepared using TruSeq Stranded mRNA Prep kit Accepted Article (Illumina, San Diego, CA, USA) per manufacturers' protocols. To confirm RNA quality, RIN scores were evaluated using the 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA). Paired-end sequencing (75 base pairs per end) with sequencing depth at 80 million reads was performed on the HiSeq2000 platform (Illumina, San Diego, CA, USA) to generate FASTQ files. These were aligned to human genome (version HG19) using HiSAT2 (John Hopkin's University, Baltimore, USA) and SAMtools (Genome Research Limited, Cambridge, UK). Normalized read counts were generated per transcript using DESeq2. Read counts were then transformed to log2 scale (after adding 1 to account for zero read counts). Comparisons between groups were made by Chi-squared tests or unpaired t-tests as the data were normally distributed. Pearson correlations were performed to determine associations. Patient demographics are shown in table 1. There were no differences in clinical characteristics, except for a higher modified Medical Research Council (mMRC) score indicating greater dyspnoea in the >24.9 kg/m 2 group (mean 1.9 versus 1.0, p=0.02). The bronchial expression of ACE2 was significantly higher in the >24.9 kg/m 2 group compared to the <24.9 kg/m 2 group (means = 6.91 vs 7.35 respectively; p=0.004; figure 1A ). There was moderate positive correlation between BMI and ACE2 expression which marginally failed to reach statistical significance (r=0.3 p=0.057; figure 1B ). There was a trend for increased ACE2 expression in bronchial epithelial cells in COPD current versus COPD ex-smokers, but this did not reach statistical significance (p=0.1; figure 1C ). There was no correlation between pack year smoking history and ACE2 expression (data not shown). There was a negative association between FEV 1 % predicted and ACE2 expression (r= -0.4 p=0.02; figure 1D ). There was no association between FEV 1 % predicted and BMI (r= -0.1 p=0.5). There were no differences in ACE2 expression in individuals with hypertension (n=10) and without hypertension (n=27), nor in patients with hypertension receiving ACE inhibitors compared to those not (n=5 each group; data not shown). This article is protected by copyright. All rights reserved The expression of transmembrane protease serine 2 (TMPRSS2) in the bronchial epithelium of the >24.9 kg/m 2 group compared to the <24.9 kg/m 2 group was similar and we found no difference (p=0.2; means = 11.36 vs 11.55 respectively). COPD patients hospitalised with COVID-19 have more severe disease and increased mortality (1, 3, 4). Leung et.al showed increased ACE2 bronchial epithelial expression in COPD patients compared to controls (11) . As SARS-CoV-2 utilises ACE2 for cellular entry (10), then increased ACE2 expression in the bronchial epithelium may enhance opportunities for viral entry and survival. We now demonstrate increased bronchial epithelium ACE2 expression in COPD patients who are overweight compared to those who are not overweight. Obesity is a risk factor for worse clinical outcomes in COVID-19 (5-7). If increased ACE2 expression allows greater virus uptake, then our data suggests that the subgroup of COPD patients who are overweight have a higher risk of developing severe COVID-19. Information regarding BMI has not been included in all cohort studies of patients hospitalised with COVID-19. The relationship between BMI and outcomes may be masked by other co-existing systemic comorbidities (15) . Our study highlights a subgroup of individuals with a co-morbidity (COPD) who are overweight; this subgroup expresses higher levels of the receptor for SARS-CoV-2. Similar to Leung et.al, we observed a negative association between FEV 1 % predicted and ACE2 expression (11) . Additionally, recent studies have shown that current smoking increases ACE2 expression in the bronchial epithelium (16) . Similarly, we observed a numerical increase in ACE2 expression in current smokers compared to ex-smokers with COPD. The proportion of current smokers was numerically higher in the <24.9 kg/m 2 group and the >24.9 kg/m 2 group, suggesting current smoking may have reduced the size of the differences we observed regarding weight. SARS-CoV-2 cellular entry depends upon priming of the spike glycoprotein to facilitate attachment to host cells. The serine protease TMPSSR2 is important for this process (17) . We did This article is protected by copyright. All rights reserved not observe a difference in the expression of TMPRSS2 between the <24.9 kg/m 2 group and the >24.9 kg/m 2 group. A limitation of this study is that we studied gene expression alone, without protein expression studies. However, Leung et.al showed that increased COPD bronchial ACE2 gene expression was replicated in protein expression studies (11) , providing confidence that the ACE2 gene expression results reported, relate to protein levels. We did not observe any differences in ACE2 expression in hypertensive compared to nonhypertensive patients. Simonnet et.al previously showed increased COVID-19 severity in patients with obesity independent of hypertension (6) . Increased ACE2 expression may provide one mechanism whereby SARS-CoV-2 is more virulent and other complex mechanisms may exist in hypertensive individuals. In summary, we have shown increased ACE2 expression in the bronchial epithelium of COPD patients who are overweight compared to those who are not overweight. This may provide increased opportunities for SARS-CoV-2 infection of the respiratory tract. These individuals may be at greater risk of developing severe COVID-19. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved expression was plotted against BMI and a Pearson's correlation coefficient was calculated. 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