Dysbalance of ACE2 levels - a possible cause for severe COVID-19 outcome in COPD

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin-converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS-CoV-2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open-access RNA sequencing datasets. Immunohistochemical and single-cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme-linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS-CoV-2 infection.

Keywords: ACE2; COPD; COVID-19; SARS-CoV-2; TMPRSS2; chronic lung disease; chronic obstructive pulmonary disease; pulmonary fibrosis; pulmonary hypertension.

Figure 1

Local and systemic levels of SARS‐CoV‐2 receptors and processing enzymes in patients and controls. (A) Quantitative real‐time PCR analysis of ACE2, TMPRSS2, Furin (FURIN), Ezrin (EZR), and CD147/Basigin (BSG) expression levels in lung homogenates of donors and transplant patients with COPD, IPAH, and PF. (B) sACE2 levels measured by ELISA in plasma samples of healthy controls (Ctrl), COPD, IPAH, and PF patients. Boxplots with single data points are shown. Statistical analysis was performed using a nonparametric Kruskal–Wallis test with post hoc comparison of multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Expression profiling of SARS‐CoV‐2 receptors and processing enzymes in publicly available datasets of COPD and control lungs. Expression profiling in lung biopsy tissue (A, dataset GSE8500), lung explant tissue (B, D, datasets GSE103174 and GSE47460, respectively), and lung cancer resection tissue (C, dataset GSE37768) compared between COPD patients and donors. Boxplots with single data points are shown. Statistical analysis was performed using a nonparametric Kruskal–Wallis test with post hoc comparison of multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

ACE2 expression in the lung correlates with lung function, but not with mPAP in COPD. Spearman's rank correlation of FVC (% predicted) (A) or mPAP (mmHg) (B) with ACE2 expression levels (ΔΔCt).

Figure 4

scRNAseq reveals cell type‐specific expression of SARS‐CoV‐2 receptors and processing enzymes in COPD and control lungs. (A) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) representation of cell clusters in control and COPD lungs. Dots represent single cells. ATI, alveolar type I cells; ATII, alveolar type II cells; B_Plasma, plasma cells; DC, dendritic cells; ILC, innate lymphoid cells; Lymphatic, lymphatic endothelium; Mast, mast cells; NK, natural killer cells; pDC, plasmacytoid dendritic cells; PNEC, pulmonary neuroendocrine cell; SMC, smooth muscle cell. (B) Expression of ACE2, TMPRSS2, BSG (CD147), FURIN, and EZR in single cells of control and COPD lungs. Cell clusters are shown as indicated in (A). (C) Dot plot representation of ACE2, TMPRSS2, BSG (CD147), FURIN, and EZR expression in specific cell populations within control and COPD lungs. Dot sizes represent the percent of cells within each cluster with positive gene expression and color intensity indicates the average expression of within one cluster.

Figure 5

Immunostaining reveals cellular localization of SARS‐CoV‐2 receptors and processing enzymes in COPD and donor lung tissue. High‐magnification images showing ACE2, TMPRSS2, Furin, Ezrin, and CD147 immunostaining in bronchial, parenchymal, and vascular compartments on corresponding serial sections of donor and COPD tissue. Scalebar = 20 μm. Black arrowhead, bronchial epithelium; black arrow, alveolar epithelial type II cells; white arrowhead, vascular endothelium; gray arrowhead, erythrocytes; asterisks, inflammatory cells.