infection which can cause a spectrum of disease ranging from a mild self-limiting illness to severe respiratory failure requiring ventilatory support. Current guidance advocates that high-risk individuals including those with chronic lung diseases such as severe asthma and COPD should be shielded to reduce risk of acquisition of the virus (1) . This guidance is based upon extensive prior knowledge that these conditions are exquisitely susceptible to being exacerbated by a range of respiratory virus infections (2) . However, early evidence has indicated that the prevalence of asthma and COPD among hospitalised COVID-19 cases may be lower than the general population, in contrast to other chronic comorbidities such as hypertension and diabetes, raising speculation of a possible protective phenotype (3) . Conversely, COPD (but not asthma) has been shown to be associated with greater risk of COVID-19 related mortality (4, 5) , suggesting that these patients could theoretically be protected from acquisition of the virus but paradoxically at increased risk of complications if they become infected.
Inhaled corticosteroids (ICS) are mainstay therapies for airways diseases and confer beneficial effects including protection against exacerbations (6, 7) , suggesting that these drugs may reduce the risk of virus acquisition or alternatively suppress virus-induced inflammation and prevent symptomatic manifestations. Conversely, we and others, have shown that ICS have the adverse effect of suppressing innate immune responses to rhinovirus and influenza infection, leading to increased virus replication (8) (9) (10) , although the opposite (protective) effect of ICS has been reported in vitro for the seasonal coronavirus 229E (11) and SARS-CoV-2 (12) . It is thus unclear whether, overall, ICS impart a protective or detrimental effect on immune responses to SARS-CoV-2 and the extent to which these widely used drugs protect or expose patients with asthma or COPD to COVID-19 is unknown.
SARS-CoV-2 utilises the entry receptor Angiotensin-converting enzyme (ACE)-2 with priming of the serine protease TMPRSS2 (Transmembrane protease, serine 2) to gain entry into the respiratory mucosa and cause active infection (13) . Increased epithelial ACE2 expression has been recently reported in smokers and subjects with COPD (14) and is postulated to be a factor predisposing these individuals to adverse outcome from COVID-19. Conversely, ACE2 is downregulated in asthma (15) , an effect that may be due to suppressive effects of type 2 cytokines (16) or related to ICS use (17) .
Emerging evidence also indicates that ACE2 expression co-localizes with immune genes involved in interferon signalling pathways.(18) Moreover, Ziegler et al recently elucidated that ACE2 is an interferon-stimulated gene in human respiratory epithelial cells (19) , indicating that antiviral pathways may be important in regulation of pulmonary ACE2 expression. We have previously reported that ICS potently suppress epithelial expression of type I IFNs and interferon stimulated genes (ISGs) in a range of in vitro and in vivo COPD models (8) and it is plausible that ICS-mediated suppression of IFN might drive downregulation of ACE2 in the lungs and thus be an important determinant of susceptibility to SARS-CoV-2 in chronic lung disease patients.
Here, we show that ICS administration attenuates pulmonary expression of ACE2, an effect observed consistently across a range of human and animal COPD models. Using functional experiments, we demonstrate that the downregulation of ACE2 is mechanistically related to suppression of type I interferon by ICS. These data indicate that use of ICS therapies alter expression of the SARS-CoV-2 entry receptor and may thus contribute to altered susceptibility to COVID-19 in patients with COPD.
Recent data indicate that sputum expression of ACE2 mRNA is reduced in asthmatic subjects taking ICS (17) but it is unclear whether similar suppression occurs in the context of COPD. We therefore initially used a community-based cohort of 40 COPD subjects (20) to determine whether ICS use affects ACE2 expression in COPD. 36 out of 40 subjects had sufficient sample for evaluation and were stratified according to current use (n=18) or non-use (n=18) of ICS. There were no significant differences between these groups in terms of age, disease severity, smoking status or other comorbidities known to affect ACE2 expression and/or associated with increased risk of COVID-19 (   Table 1) . Sputum cell ACE2 mRNA expression was detectable in 22/36 COPD subjects (61.1%) and, consistent with prior observations in asthma (17) , significantly reduced in ICS users compared to nonusers (Fig 1a) . Sputum cell expression of the serine protease TMPRSS2 that is used by SARS-CoV-2 for mucosal entry (13) was detectable in all subjects with no significant difference observed between ICS users and non-users (Fig 1b) . Similarly, the alternative SARS-CoV receptor CD147 (21) (gene: Basigin BSG) was detectable in all subjects with no difference observed between ICS users and non-users ( Supplementary Fig 1) .
Given that cause and effect cannot be inferred from a cross-sectional human study, we next evaluated whether experimental pulmonary administration of the ICS fluticasone propionate (FP) in mice, at a dose previously shown to induce lung glucocorticoid receptor (GR) activation (8, 22) , had similar effects on Ace2 expression. A single administration of 20µg FP in mice (Fig 2a) induced significant downregulation of pulmonary Ace2 mRNA expression at 8 hours (a timepoint where we have also previously shown that significant GR activation occurs (8) ). This effect persisted at 24 hours postadministration but had resolved from 48 hours onwards (Fig 2b) . Consistent with effects observed in human sputum, FP administration had no effect expression of Tmprss2 or Bsg in mouse lung ( Supplementary Fig 2) . Suppression of Ace2 by FP occurred in a dose-dependent manner with loss of suppression at a ten-fold lower concentration (2µg) (Fig 2c) , a dose at which effects on GR activation are also lost (8) . We observed similar suppression of lung Ace2 mRNA expression with administration of 20µg of other commonly used ICS budesonide and beclomethasone, suggesting that the effect of ICS on Ace2 is not class dependent (Fig 2d) . To corroborate the effects observed on Ace2 mRNA expression, we subsequently measured protein levels in lung homogenate of ICS-treated mice by ELISA. We observed similar suppression of total lung ACE2 protein occurring at 24 hours postadministration, consistent with effects observed at the mRNA level (Fig 2e) .
ACE2 has recently been reported to co-localise with expression of type I-IFN related genes (13, 18) and has also been shown to be an interferon stimulated gene (ISG) in the respiratory tract (19) , suggesting that type I IFN may be a major regulator of pulmonary ACE2 expression. Consistent with this, we observed that basal lung Ace2 expression in mice positively correlated with mRNA expression of the ISG 2'-5' OAS mRNA and BAL concentrations of IFN-λ in a combined analysis of FP and vehicle treated mice (Fig 3a) . Given our previous data showing that COPD patients treated with ICS have reduced basal airway expression of IFNb (8) , we hypothesized that downregulation of ACE2 by FP may be functionally related to its suppressive effects on type I IFN signalling. Accordingly, recombinant IFNb administration (Fig 3b) could reverse FP-mediated suppression of Ace2 mRNA and ACE2 protein (Fig   3c) , indicating that the effect of FP on ACE2 expression is functionally related to suppressive effects on type I IFN.
To further confirm the functional importance of type I IFN in regulating pulmonary ACE2, we evaluated basal pulmonary expression levels in mice deficient in IFN signalling (Ifnar -/-). Compared to wild-type control mice, Ifnar -/mice had a small, but statistically significant, reduction in lung ACE2 mRNA expression (Fig 4a) with a concomitant trend (P=0.15) towards reduced lung ACE2 protein levels (Fig   4b) . These observations further confirm the key regulatory role played by type I IFN signalling in pulmonary expression of ACE2.
Existing data indicate that ACE2 is expressed primarily in the nasal and bronchial epithelium and is absent from immune cells (16) . Given our prior data indicating that FP also exerts its inhibitory effects on immunity principally at the pulmonary epithelium (8, 23), we next assessed whether suppressive effects on ACE2 were also observed following ex vivo ICS administration in cultured COPD bronchial epithelial cells (BECs, Fig 5a) . Baseline characteristics of the subjects included in these analyses are shown in table 2. In keeping with recent in situ expression studies in COPD patients (14) , we found that basal expression of ACE2 was increased by ~3 fold in BECs from COPD patients compared to healthy non-smokers (Fig 5b) . Consistent with our findings in human ICS users and in the mouse model of ICS administration (Figs 1&2), FP administration (at a clinically relevant concentration of 10nM) induced ~75% suppression in ACE2 expression (Fig 5c) .
To further confirm that ICS administration suppresses ACE2 expression in COPD, we employed a mouse model of elastase-induced emphysema which recapitulates many hallmark features of human disease (24). Mice were treated intranasally with a single dose of porcine pancreatic elastase (Fig 6a) . and lung ACE2 expression was measured at 10 days after administration (timepoint at which COPDlike disease features are established (24)) and a further 7 days later. In keeping with our findings in human COPD cells, elastase-treated mice had significantly increased (~5-fold) lung Ace2 expression at 10 days with further enhancement to >15-fold at 17 days (Fig 6b) . Administration of a single dose of FP at 10 days attenuated the significant upregulation of lung Ace2 mRNA (Fig 6c) and ACE2 protein concentrations measured 24 hours later in elastase-treated mice (Fig 6d) . Therefore, the suppressive effects of ICS on ACE2 also occur in an in vivo model of COPD-like disease.
The mechanisms driving altered susceptibility to COVID-19 in chronic lung diseases and whether the commonly used therapies ICS promote or protect against infection by SARS-CoV-2 is a crucial question for the field. In this study, we demonstrate consistently, across a range of human and animal models, that ACE2, a receptor that facilitates entry of SARS-CoV-2 in the respiratory tract, is upregulated in COPD and suppressed by ICS treatment. Our studies indicate a novel mechanism for downregulation of ACE2 by ICS through suppression of type I interferon.
There is clear evidence to support the premise that ACE2 mediates cell entry of SARS-CoV2 into the respiratory tract and also acts as a major receptor for SARS-CoV1 and NL63 coronaviruses (13, 25, 26 ). Viral entry occurs as a two-step process with initial binding of the N-terminal portion of the viral protein to the ACE2 receptor, followed by viral protein cleavage facilitated by the receptor transmembrane protease serine 2 (TMPRSS2) (13) . Therapeutic blockade of TMPRSS2 inhibits entry of SARS-CoV-1 and -2 into cells, supporting a critical role for this protease in viral pathogenesis (13) . Our ACE2 is expressed primarily in the nasal goblet cells and type II pneumocytes within the respiratory tract (30) and is upregulated in subject groups known to be associated with increased disease severity including elderly individuals (31) and patients with diabetes (17), indicating that it may play a clinically important role in governing susceptibility to virus acquisition and/or development of severe disease in at-risk groups. ACE2 expression has similarly been shown to be increased in COPD: using combined transcriptomic and immunohistochemical analyses, Leung et al recently demonstrated that epithelial ACE2 expression is increased in bronchial brushings/tissue samples from COPD subjects versus healthy controls (32), effects also previously shown in cigarette smoke exposure animal models (33). Our data showing increased ACE2 expression in cultured airway epithelial cells and in an elastase mouse model of COPD are consistent with these findings. Rates of patients with COPD being hospitalized with COVID-19 have been relatively low, ranging from 1.1% in Chinese cohorts to 5% in US cohorts (34) (35) (36) (37) However, patients with COPD have greater risk of severe disease and mortality from COVID-19 (4).
These data suggest that COPD may be associated with possible protection against the need for hospitalisation (possibly due to reduced risk of virus acquisition) but increased propensity to severe disease upon infection. The original SARS-CoV-1 pandemic was also characterised by an extremely low prevalence of chronic lung disease comorbidities (38, 39) , an effect that could also have been driven by ICS-mediated suppression of ACE2 in these subjects. The mechanism underlying these putative alterations in susceptibility has not been extensively explored. Our data suggests that suppression of ACE2 by the commonly used therapies ICS may be one important factor that dictates susceptibility in COPD.
Currently, a direct causal link between ACE2 and increased susceptibility to acquisition of SARS-CoV-2 or subsequent severity has not been proven and we cannot conclude unequivocally that downregulation of ACE2 by ICS is an effect that would confer protection clinically. In asthma, a disease where ICS are more commonly prescribed than in COPD, ACE2 expression is reduced compared to healthy subjects (15) and also further attenuated in ICS users (17) . In contrast to COPD, asthma has not been shown to be associated with increased COVID-19 mortality (5) and the more widespread use of ICS with associated suppression of ACE2 could theoretically be one factor driving this. Conversely, It is important to note that there is evidence to suggest that downregulation of ACE2 could also theoretically worsen outcome. In mouse models of experimentally-induced acid aspiration and sepsis, genetic deletion of Ace2 worsens acute lung injury, an effect that is partially rescued by recombinant ACE2 administration (40) . ACE2 also degrades angiotensin II which can drive production of proinflammatory cytokines (41, 42) which may be detrimental in the context of the hyper-inflammation that is characteristic in severe COVID-19 (43) Understanding the mechanism through which ICS suppress expression of ACE2 is important to delineate how this effect could either be harnessed as a protective factor or reversed if deemed to be detrimental. We have previously shown that ICS can suppress type I IFN both at steady state and during active virus infection (8) . Here, we show that this effect directly drives suppression of ACE2 expression since administration of recombinant IFN-β in combination with FP could reverse the downregulation of ACE2. Furthermore, Ifnar -/mice which lack type I IFN signalling also had reduced ACE2 providing additional evidence that expression is directly regulated by type I IFN. These findings are consistent with recent studies showing that genes relevant to IFN pathways (IFNAR1, IFITM1) are expressed in ACE2+ type 2 pneumocytes and that type I IFNs can upregulate ACE2 in a range of experimental systems (13) . We additionally showed that two other commonly used ICS, budesonide and beclomethasone, have similar effects on ACE2, confirming that, in contrast to impairment of antibacterial immunity (22), the suppressive effect of ICS on ACE2 is not class-dependent. This is also consistent with the mechanism of suppressed IFN as we have also shown previously that budesonide suppresses IFN to a similar degree to FP (8).
In summary, these studies indicate that inhaled corticosteroid use in COPD suppresses the expression of the SARS-CoV-2 receptor ACE2 through a type I interferon-dependent mechanism. These effects are likely to contribute to altered susceptibility to COVID-19 in patients prescribed these therapies.
Further studies are now needed to elucidate the precise effects that altered ACE2 expression has on the acquisition of SARS-CoV-2 infection and associated severity in COVID-19. 
RNA was extracted from cell lysates of primary airway epithelial cells, human sputum cells or mouse lung tissue using an RNeasy kit (Qiagen). 2µg was utilised for cDNA synthesis using the Omniscript RT kit (Qiagen). Quantitative PCR was carried out using previously described specific primers and probes and normalized to 18S rRNA housekeeping gene. (8) Reactions were analysed using the ABI 7500 real time PCR machine (Applied Biosystems).
A commercially available ELISA duoset kit (Abcam) was used to measure total ACE2 concentrations in mouse lung homogenate. The lower limit of detection for this assay is 10 pg/mL. IFN-λ concentrations in mouse BAL were also measured using a commercially available ELISA duoset kit (Bio-techne), as previously reported (8) .
For animal experiments, group sizes of 5 mice per condition were used and data is presented as n/a - Table 1 : Demographic and clinical characteristics of COPD patients included in mRNA analyses in Figure 1 and Supplementary Fig 1  COPD Table 2 : Demographic and clinical variables in COPD and healthy subjects included in primary airway epithelial cell experiments ( Figure 5) 