Asthma Associated Cytokines Regulate the Expression of SARS-CoV-2 Receptor ACE2 in the Lung Tissue of Asthmatic Patients

Abstract

It is still controversial whether chronic lung inflammation increases the risk for COVID-19. One of the risk factors for acquiring COVID-19 is the level of expression of SARS-CoV-2 entry receptors, ACE2 and TMPRSS2, in lung tissue. It is, however, not clear how lung tissue inflammation affects expression levels of these receptors. We hence aimed to determine the level of SARS-CoV-2 receptors in lung tissue of asthmatic relative to age, gender, and asthma severity, and to investigate the factors regulating that. Therefore, gene expression data sets of well-known asthmatic cohorts (SARP and U-BIOPRED) were used to evaluate the association of ACE2 and TMPRSS2 with age, gender of the asthmatic patients, and also the type of the underlying lung tissue inflammatory cytokines. Notably, ACE2 and to less extent TMPRSS2 expression were upregulated in the lung tissue of asthmatics compared to healthy controls. Although a differential expression of ACE2, but not TMPRSS2 was observed relative to age within the moderate and severe asthma groups, our data suggest that age may not be a key regulatory factor of its expression. The type of tissue inflammation, however, associated significantly with ACE2 and TMPRSS2 expression levels following adjusting with age, gender and oral corticosteroids use of the patient. Type I cytokine (IFN-γ), IL-8, and IL-19 were associated with increased expression, while Type II cytokines (IL-4 and IL-13) with lower expression of ACE2 in lung tissue (airway epithelium and/or lung biopsies) of moderate and severe asthmatic patients. Of note, IL-19 was associated with ACE2 expression while IL-17 was associated with TMPRSS2 expression in sputum of asthmatic subjects. In vitro treatment of bronchial fibroblasts with IL-17 and IL-19 cytokines confirmed the regulatory effect of these cytokines on SARS-CoV-2 entry receptors. Our results suggest that the type of inflammation may regulate ACE2 and TMPRSS2 expression in the lung tissue of asthmatics and may hence affect susceptibility to SARS-CoV-2 infection.

Keywords: ACE2; COVID-19; IL-17; IL-19; SARS-CoV-2; TMPRSS2; asthma; cytokines.

Figure 1

Expression of COVID-19 entry genes, ACE2 and TMPRSS2, in airway epithelium and bronchial biopsies of asthmatics with different severity categories. (A, B) The expression of ACE2 and TMPRSS2 in airway epithelium of healthy controls (n = 20), moderate (n = 50), and severe asthmatics (n = 38) (C, D) The expression of ACE2 and TMPRSS2 in the bronchial biopsies of moderate (n = 33) and severe asthmatics (n = 58). Two-way comparison was done using unpaired t-test or Mann–Whitney U test, depending on the skewness of the data. *P < 0.05.

Figure 2

Gene expression of ACE2 and TMPRSS2 in airway epithelium and bronchial biopsies of moderate and severe asthmatics relative to age. (A, C) The expression level of ACE2 in airway epithelium of moderate asthmatics below and above 40 years of age (B, C) The expression of ACE2 in airway epithelium of severe asthmatics below and above 40 years of age. (D, F) The expression levels of TMPRSS2 in airway epithelium of moderate asthmatics below and above 40 years of age (E, F) The expression of TMPRSS2 in airway epithelium of severe asthmatics below and above 40 years of age. (G, I) The expression levels of TMPRSS2 in bronchial biopsies of moderate asthmatics below and above 40 years of age (H, I) The expression of ACE2 in bronchial biopsies of severe asthmatics below and above 40 years of age. (J, L) The expression levels of TMPRSS2 in bronchial biopsies of moderate asthmatics below and above 40 years of age (K, L) The expression of TMPRSS2 in bronchial biopsies of severe asthmatics below and above 40 years of age. Correlation between ACE2 or TMPRSS2 gene expression level and individual’s age was measured using Pearson’s correlation coefficient with a two‐sided test for significance (P < 0.05 significant). Two-way comparison was done using unpaired t-test or Mann–Whitney U test, depending on the skewness of the data. ns, non-significant. *P < 0.05, **P < 0.01.

Figure 3

Gene expression of ACE2 in airway epithelium and bronchial biopsies of moderate and severe asthmatics relative to gender. (A–C) ACE2 expression level in AECs has an increasing trend in elderly males with moderate asthma and young males with severe asthma. (D–F) ACE2 expression level is significantly increased in AECs of elderly females with moderate asthma and young females with severe asthma. (G–I) ACE2 expression level in bronchial biopsies has an increasing trend in elderly males with moderate asthma and young males with severe asthma. (J–L) ACE2 expression level in bronchial biopsies has an increasing trend in elderly females with moderate asthma and young females with severe asthma. ns, non-significant. Correlation between ACE2 gene expression level and individual’s age was measured using Pearson’s correlation coefficient with a two‐sided test for significance (P <0.05 significant). Two-way comparison was done using unpaired t-test or Mann–Whitney U test, depending on the skewness of the data. ns, non-significant. *P < 0.05.

Figure 4

Inflammatory Cytokines associated with ACE2 and TMPRSS2 expression in airway epithelium, bronchial biopsies, and sputum of moderate and severe asthmatics. Linear regression model was used to determine correlations of ACE2 and TMPRSS2 with asthma-associated cytokines in (A) airway epithelium, (B) bronchial biopsies, and (C) sputum samples of moderate to severe asthma patients, respectively. The linear regression model was adjusted for age and gender in airway epithelium; for age, gender and oral corticosteroid use in lung tissue datasets; and for age, gender, smoking, and atopic status of asthmatic patients in sputum dataset. None of asthma patients in sputum dataset were on oral corticosteroid use. The standardized β-coefficient values were plotted to aid in interpretation of the degree of association of each cytokine with ACE2 or TMPRSS2 in respective tissues. All the bar charts showing significant association with P < 0.05.

Figure 5

The effects of Type II cytokines, IFN-γ, and IL-19 stimulation on the lung cell expression levels of ACE2 and TMPRSS2. (A–D) The expression of ACE2 and TMPRSS2 following treatment of BEAS-2B cells with IL-4, IL-13, IFN-γ and IL-17 (GSE37693; for IL-13, and GSE148829 for IFN-γ, IL-17, and IL-4 treatment). (E, F) ACE2 mRNA and protein expression in primary human bronchial fibroblasts of severe asthmatic (n = 3) following in vitro stimulation with IL-19 cytokine. Blots were visualized on a BioRad ChemiDoc™ Touch Imager-ACE2; exposure time of 54 s (signal accumulation) and β-actin; exposure time of 0.4 s (Optimal Auto-exposure). Two-way comparison was done using unpaired t-test or Mann–Whitney U test, depending on the skewness of the data. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.