Inhaled corticosteroids downregulate the SARS-CoV-2 receptor ACE2 in COPD through suppression of type I interferon

Abstract

Background: The mechanisms underlying altered susceptibility and propensity to severe Coronavirus disease 2019 (COVID-19) disease in at-risk groups such as patients with chronic obstructive pulmonary disease (COPD) are poorly understood. Inhaled corticosteroids (ICSs) are widely used in COPD, but the extent to which these therapies protect or expose patients to risk of severe COVID-19 is unknown.

Objective: The aim of this study was to evaluate the effect of ICSs following pulmonary expression of the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme-2 (ACE2).

Methods: We evaluated the effect of ICS administration on pulmonary ACE2 expression in vitro in human airway epithelial cell cultures and in vivo in mouse models of ICS administration. Mice deficient in the type I IFN-α/β receptor (Ifnar1^-/-) and administration of exogenous IFN-β were used to study the functional role of type-I interferon signaling in ACE2 expression. We compared sputum ACE2 expression in patients with COPD stratified according to use or nonuse of ICS.

Results: ICS administration attenuated ACE2 expression in mice, an effect that was reversed by exogenous IFN-β administration, and Ifnar1^-/- mice had reduced ACE2 expression, indicating that type I interferon contributes mechanistically to this effect. ICS administration attenuated expression of ACE2 in airway epithelial cell cultures from patients with COPD and in mice with elastase-induced COPD-like changes. Compared with ICS nonusers, patients with COPD who were taking ICSs also had reduced sputum expression of ACE2.

Conclusion: ICS therapies in COPD reduce expression of the SARS-CoV-2 entry receptor ACE2. This effect may thus contribute to altered susceptibility to COVID-19 in patients with COPD.

Keywords: COPD; COVID-19; inhaled corticosteroids; viral infection.

Graphical abstract

Fig 1

Sputum gene expression of ACE2 and TMPRSS2 in subjects with COPD stratified according to use or nonuse of ICSs. Sputum samples were taken from a cohort of patients with COPD when clinically stable for at least 6 weeks. Patients were stratified according to current use or nonuse of ICSs (n = 18 per group). Sputum cell mRNA expression of ACE2 (A) and TMPRSS2 (B) was measured by quantitative PCR. Box and whisker plots show median (line within box), interquartile range (box) and minimum to maximum (whiskers). Statistical comparisons made by using the Mann-Whitney U test. ∗P < .05.

Fig 2

ICS administration downregulates ACE2 expression in mouse lung. A, C57BL/6 mice were treated intranasally (i.n.) with a single dose of FP or vehicle (VEH) dimethyl sulfoxide control. B, Lung Ace2 mRNA expression was measured by quantitative PCR (qPCR) at the indicated time points following administration of 20 μg of FP. C, Lung Ace2 mRNA expression was measured by qPCR at 24 hours following single-dose administration of FP at doses of 20, 6.7, and 2 μg. D, Lung Ace2 mRNA expression was measured by qPCR at 24 hours following single-dose administration of 20 μg of budesonide (Bud), beclomethasone (Beclo), or VEH control. E, ACE2 protein in lung tissue homogenate measured by ELISA at 24 hours following single-dose administration of 20 μg of FP. Data are shown as means ± SEMs of 4 or 5 mice per treatment group from a single experiment, representative of at least 2 independent experiments. Data were analyzed by using the Mann-Whitney U test or 1-way ANOVA with Bonferroni posttest. ∗P < .05; ∗∗∗P < .001. ns, Nonsignificant.

Fig 3

Downregulation of ACE2 by an ICS is functionally related to suppression of type I interferon. A, Correlation between lung Ace2 mRNA and lung 2',5'-oligoadenylate synthetase and BAL IFN-λ in C57BL/6 mice. B, C57BL/6 mice were treated intranasally with FP (20μg) or vehicle (Veh) dimethyl sulfoxide control and additionally with recombinant IFN-β or PBS control. C, Lung Ace2 mRNA expression and lung ACE2 protein concentrations were measured at 8 hours after FP administration. Data shown as means ± SEMs of 4 or 5 mice per treatment group from a single experiment representative of at least 2 independent experiments. Data were analyzed by using Spearman rank correlation (A) or 1-way ANOVA with Bonferroni posttest (C). ∗P < .05; ∗∗ P < .01. ns, Nonsignificant.

Fig 4

Type I interferon receptor–deficient mice have reduced pulmonary ACE2 expression. Lung tissue was harvested from wild-type or Ifnar1^−/− C57BL/6 mice. A, Lung Ace2 mRNA expression was measured by quantitative PCR. B, Lung ACE2 protein concentration was measured by ELISA. Data are shown as means ± SEMs of 5 mice per treatment group from a single experiment representative of at least 2 independent experiments. Data were analyzed by using a t test. ∗P < .05.

Fig 5

ACE2 expression is increased in COPD and suppressed by fluticasone administration in cultured bronchial epithelial cells (BECs). A, Primary BECs from 6 subjects with COPD and 6 healthy control subjects were cultured ex vivo and treated with 10 nM FP or medium control. Cell lysates were collected. ACE2 mRNA expression was measured at baseline (B) and at 24 hours after FP administration (C) in COPD BECs. Data are shown as medians ± interquartile ranges. Statistical comparisons were made by using the Mann-Whitney U test. ∗∗P < .01.

Fig 6

ACE2 expression is increased in a mouse model of COPD and suppressed by fluticasone administration. A, C57BL/6 mice were treated intranasally with a single dose of elastase (Elas) or PBS as control and lung tissue harvested at 10 or 17 days later. Some mice were treated with FP or vehicle (VEH) control at 10 days, before sampling 24 hours later. B, Lung Ace2 mRNA expression in Elas- versus PBS-treated mice at 10 and 17 days after treatment. C, At 10 days after Elas treatment, C57BL/6 mice were treated with a single dose of 20 μg of FP or VEH control. Lung tissue was harvested at 24 hours after FP administration. Ace2 mRNA was measured by quantitative PCR (left panel), and ACE2 protein was measured by ELISA (right panel). Data are shown as means ± SEMs of 5 mice per treatment group from a single experiment. representative of at least 2 independent experiments. Data were analyzed by 1-way ANOVA with Bonferroni posttest. ∗P < .05; ∗∗P < .01. ns, Nonsignificant.

Fig E1

Sputum gene expression of ACE2 in subjects with COPD stratified according to use or nonuse of ICSs, excluding subjects with bronchodilator reversibility. Sputum samples were taken from a cohort of patients with COPD when clinically stable for at least 6 weeks. Patients were stratified according to current use or nonuse of ICSs (n = 17 ICS users and 15 ICS nonusers). Sputum cell mRNA expression of ACE2 was measured by quantitative PCR. Box and whisker plots show median (line within box), interquartile range (box), and minimum to maximum (whiskers). Statistical comparisons made by using the Mann-Whitney U test. ∗P < .05.

Fig E2

Sputum gene expression of BSG in subjects with COPD stratified according to ICS use. Sputum samples were taken from a cohort of patients with COPD when clinically stable for at least 6 weeks. Patients were stratified according to current use or nonuse of ICSs (n = 18 per group). Sputum cell mRNA expression of BSG was measured by quantitative PCR. Box and whisker plots show median (line within box), interquartile range (box), and minimum to maximum (whiskers). Statistical comparisons made by using the Mann-Whitney U test.

Fig E3

No effect of FP administration on expression of Tmprss2 or Bsg in mouse lung. C57BL/6 mice were treated intranasally with a single 20-μg dose of FP or vehicle dimethyl sulfoxide control. Lung expression of Tmprss2 (A) and lung Bsg (B) mRNA was measured by quantitative PCR at 8 hours following FP administration. Data are from the same experiment as shown in Fig 2 and represent means ± SEMs of 5 mice per treatment group from a single experiment that is representative of at least 2 independent experiments. Data were analyzed by 1-way ANOVA with Bonferroni posttest. ns, Nonsignificant.

Fig E4

No effect of fluticasone administration on TMPRSS2 expression in cultured BECs. Primary BECs from 6 subjects with COPD and 6 healthy control subjects were cultured ex vivo and treated with 10 nM FP or control medium. Cell lysates were collected. TMPRSS2 mRNA expression was measured by quantitative PCR. Data are shown as medians ± interquartile range. Statistical comparisons made by using the Mann-Whitney U test. ns, Nonsignificant.

Fig E5

No effect of fluticasone administration on lung Tmprss2 expression in a mouse model of COPD. A, C57BL/6 were treated intranasally with a single dose of elastase (Elas) or PBS and treated with FP of vehicle (VEH) 10 days later, before sampling 24 hours after. B, Lung Tmprss2 mRNA expression was measured by quantitative PCR. Data shown are means ± SEMs of 5 mice per treatment group from a single experiment representative of at least 2 independent experiments. Data were analyzed by 1-way ANOVA with Bonferroni posttest. ns, Nonsignificant.