Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1&#945;-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

(TMPRSS2) triggers fusion of the viral and cell membranes (Hoffmann et al., 2020;Wan et al., 2020). ACE2 is highly expressed in epithelial cells of the respiratory tract as well as those of the kidney and intestine (Hamming et al., 2004;Tipnis et al., 2000;Zhao et al., 2020b). Although COVID-19 is mild in most cases, a defining feature of severe disease is systemic low-oxygen levels (hypoxemia), which is often disproportionate to lung injury. There is evidence to suggest that this profound hypoxemia may alter the ability of SARS-CoV-2 to infect host cells. Hypoxia has been reported to regulate the replication of a number of viruses (Jiang et  (F) siRNAs targeting either HIF-1 or 2a were delivered into Calu-3 cells individually or in combination. Cells were treated with FG-4592 (50 mM) or 1% O 2 for 24 h, and ACE2 mRNA levels were quantified. Data are expressed relative to the normoxic siScramble (siScram) control. Statistical significance was determined by et al., 2020), enhancing the replication of Epstein-Barr virus (Jiang et al., 2006;Kraus et al., 2017), but suppressing HIV and influenza infection (Zhao et al., 2020a;Zhuang et al., 2020), demonstrating that the interaction between hypoxia signaling and viral infection is context specific and dependent on both the host cell and viral species. Furthermore, hypoxia has been reported to either induce or, in some cases, suppress ACE2 expression in lung pulmonary arterial smooth muscle cells (PASMCs) (Zhang et al., 2009(Zhang et al., , 2019, hematopoietic stem cell precursors (Joshi et al., 2019), and hepatocarcinoma cells (Clarke et al., 2014). Because the effects of low oxygen on both ACE2 expression and SARS-CoV-2 replication are likely to be cell context dependent, we evaluated whether hypoxia alters SARS-CoV-2 entry and replication in lung epithelial cells.
Mammalian cells adapt to low oxygen through an orchestrated transcriptional response regulated by hypoxia-inducible factor (HIF), a heterodimeric transcription factor comprising HIF-1a or HIF-2a subunits, which is regulated by oxygen-dependent and -independent stress signals. When oxygen is abundant, newly synthesized HIFa subunits are rapidly hydroxylated by HIF prolyl-hydroxylase domain (PHD) enzymes and are targeted for polyubiquitination and proteasomal degradation. In contrast, when oxygen is limited, HIFa subunits translocate to the nucleus, dimerize with HIF-1b, and activate the transcription of genes involved in cell metabolism, proliferation, pulmonary vasomotor control, and immune regulation (Kaelin and Ratcliffe, 2008;Palazon et al., 2014;Urrutia and Aragones, 2018). Defining how hypoxia or activation of HIF affects the SARS-CoV-2 life cycle in lung epithelial cells will increase our understanding of disease pathogenesis and inform therapeutic strategies. Specifically, this has the potential for pharmacological intervention because drugs that inhibit the PHD enzymes to stabilize HIF (Pugh and Ratcliffe, 2017;Sanghani and Haase, 2019) are either in advanced clinical trials for the treatment of renal anemia or are licensed for clinical use (Roxadustat in China [Chen et al., 2019a[Chen et al., , 2019b and Japan [Akizawa et al., 2020a[Akizawa et al., , 2020c[Akizawa et al., , 2020d and Daprodustat in Japan [Akizawa et al., 2020b]).
The host proteins ACE2 and TMPRSS2 are key determinants of SARS-CoV-2 cell entry (Hoffmann et al., 2020). We screened several commonly used cell lines for ACE2 and TMPRSS2 mRNA, and only four demonstrated notable expression of ACE2: HepG2 (hepatoma), Caco-2 (colonic adenocarcinoma), Calu-3 (lung adenocarcinoma), and Vero E6 (monkey kidney epithelia) ( Figure 1A). We noted that Vero E6 do not express TMPRSS2 mRNA. To assess the role of HIF in regulating these entry factors, we cultured the cells under hypoxic conditions (1% O 2 ) or after being treated with an inhibitor targeting the PHD enzymes (FG-4592/Roxadustat), which stabilizes HIFa subunits and upregulates HIF target gene transcription. Both treatments reduced ACE2 and TMPRSS2 transcripts, with the magnitude of effect varying between cell lines ( Figure 1B). Successful activation of the HIF-signaling pathway was confirmed by induction of the HIF target genes carbonic anhydrase IX (CAIX), N-Myc downstream regulated 1 (NDRG1), and Egl-9 homolog or HIF prolyl hydroxylase 3 (EGLN3 or PHD3) ( Figure S1A). In HepG2 cells, in which transcript suppression was most evident, FG-4592 downregulated ACE2 and TMPRSS2 mRNA levels in a dose-dependent manner concomitant with its induction of CAIX, NDRG1, and EGLN3 transcription ( Figures 1C and S1B). Reoxygenation of cells previously exposed to hypoxia led to a recovery of both ACE2 and TMPRSS2 mRNA to near pre-hypoxic levels ( Figure 1D), suggesting a specific action of the HIF-PHD pathway. To assess whether hypoxia/FG-4592 regulation is evident at the protein level, we also measured ACE2 and TMPRSS2 protein expression in human lung epithelial Calu-3 cells, a more physiologically relevant cell type for studying SARS-CoV-2 infection. Culturing Calu-3 cells under hypoxic conditions or treating with FG-4592 significantly reduced ACE2 protein expression in a dose-dependent manner with maximum suppression >50 mM FG-4592 or <3% oxygen ( Figure 1E) and no effect on cell viability ( Figure S1C). Similar, but more modest, effects were observed with TMPRSS2 expression ( Figure 1E). The hypoxia-induced changes in ACE2 (and, to a lesser extent, TMPRSS2) protein expression were observed in HepG2 cells ( Figure S1D). Any differences between mRNA and protein levels may, in part, reflect the cleavage and secretion of the TMPRSS2 catalytic domain or that additional hypoxia-stimulated factors regulate protein stability and/or expression. To assess the role of HIF, we silenced HIF-1a or HIF-2a expression in hypoxic or FG-4592-treated Calu-3 cells with small interfering RNAs (siRNAs). siRNA-mediated silencing of HIF-1a (either alone or in combination with HIF-2a) restored ACE2 mRNA levels in FG-4592-treated or hypoxic Calu-3 cells ( Figure 1F). In contrast, silencing HIF-2a did not restore ACE2 mRNA levels in either condition tested and resulted in a modest decrease under normoxic conditions ( Figure 1F). siRNA knockdown was verified by quantifying the relevant HIFa transcripts CAIX, NDRG1, EGLN3, and VEGFA ( Figure S2). These data reveal a role for HIF-1a in repressing ACE2 mRNA and protein expression.
To expand these observations to an in vivo setting, mice were treated with hypoxia (10% O 2 ) or FG-4592 for 24 h, with a dosing regimen (oral, 10 mg/kg twice daily) similar to that previously used to induce polycythemia (Schley et al., 2019) and the clinical dose for treating renal anemia (Provenzano et al., 2016). Both treatments reduced Ace2 and Tmprss2 transcripts in the lung, along with an increase in Endothelin 1 (Edn1) mRNA ( Figure 1G), a host gene previously reported to be induced by HIF activation in the respiratory tract (Hickey et al., 2010). Collectively, these data show a role for hypoxia in reducing ACE2 and TMPRSS2 in vitro across multiple cell lines, and this is recapitulated in the lungs of mice after systemic hypoxia or FG-4592 treatment. (C) Calu-3 cells were cultured at 1% O 2 for 16 h and re-oxygenated over a 0.5-6-h period. Cells were infected with SARS-CoV-2pp at the indicated times, and the pp entry levels were measured 48 h after infection. Data are expressed relative to normoxic cells.
(D) siRNAs against HIF-1a and HIF-2a were delivered into Calu-3 cells either individually or in combination. Cells were treated with FG-4592 (50 mM) 24 h after transfection and then infected with SARS-CoV-2pp. Data are expressed relative to an siScrambled (siScram) control. * denotes significance relative to control siRNA (siScram) at 18% O 2 , whereas # indicates significance relative to control siRNA per condition.
(E) Calu-3 cells were treated with FG-4592 (50 mM) or cultured at 1% O 2 for 24 h before inoculation with SARS-CoV-2 (MOI 0.001) for 2 h. Infected cells were washed to remove the residual inoculum, and viral replication was assessed 24 h after infection by measuring intracellular and extracellular viral RNA along with infectious titer (particle infectivity) through quantification of plaque-forming units (PFU)/mL. As a control to measure the cellular response to FG-4592 or 1% O 2 , CAIX mRNA was quantified by qPCR. All data (except particle infectivity) is expressed relative to the UT control.

OPEN ACCESS
We hypothesized that the HIF-dependent reduction in ACE2 expression would limit SARS-CoV-2 entry into naive target cells. To assess that, we used lentiviral pseudoparticles (pp) expressing SARS-CoV-2-encoded spike glycoprotein and confirmed that infectivity was ACE2 dependent by infection of human embryonic kidney cells engineered to express ACE2 ( Figure S3A). Culturing Calu-3 or primary bronchial epithelial cells (PBECs) under hypoxic conditions or treating with FG-4592 significantly reduced SARS-CoV-2pp infection (Figure 2A). In contrast, viral pp expressing the vesicular stomatitis virus glycoprotein (VSV-G) infected Calu-3 cells and PBEC with comparable efficiency at both oxygen levels ( Figure S3B), demonstrating a SARS-CoV-2-specific phenotype. We next sought to test whether hypoxia/FG-4592 limits entry of the novel SARS-CoV-2 spike protein variants; these have emerged throughout the course of the pandemic, with some conferring a fitness advantage to viral entry. The most notable of these to date, D614G, is globally prevalent in the pandemic, consistent with a reported fitness advantage for infecting cells in the upper respiratory tract (Weissman et al., 2021;Korber et al., 2020). Further, deletion of the unique furin cleavage site (which mediates membrane fusion) in the SARS-CoV-2 spike protein has been observed in vitro (Davidson et al., 2020) and in animal models of infection (Peacock et al., 2020). Importantly, hypoxia or FG-4592 treatment of Calu-3 cells reduced infection of pp containing either the spike variant to a similar degree as the wild type ( Figure 2B). Reoxygenation of hypoxic Calu-3 cells induced a recovery of SARS-CoV-2pp entry ( Figure 2C), consistent with our earlier data showing post-hypoxic recovery of ACE2 and TMPRSS2 mRNA levels. Silencing HIF-1a reversed the anti-viral effect of FG-4592 ( Figure 2D), demonstrating that HIF-1a represses SARS-CoV-2 entry, consistent with its role in regulating ACE2. In contrast, we observed a negligible effect of silencing HIF-2a on SARS-CoV-2 entry ( Figure 2D). In summary, these data show that hypoxic/ FG-4592 activation of HIF-1a represses ACE2 and impairs entry of SARS-CoV-2 entry pp.
We next assessed whether our observations with SARS-CoV-2pp translate to authentic viral replication. Infecting hypoxic (1% O 2 ) Calu-3 cells with SARS-CoV-2 (Victoria 01/20 strain) resulted in a 90% reduction in viral RNA compared with that of normoxic cells ( Figure 2E). A similar repression in SARS-CoV-2 RNA levels was also observed when culturing Calu-3 cells in 3% oxygen ( Figure S4A). Importantly, FG-4592 (50 mM) mimicked the hypoxic inhibition of SARS-CoV-2 replication, leading to a significant reduction in the genesis of new particles ( Figure 2E). To define whether hypoxia altered the infectivity of SARS-CoV-2 particles, we assessed the ratio of RNA copies per plaque-forming unit (PFU), finding no significant difference between virus produced from cells at either 18% O 2 or 1% O 2 (9.3 3 10 3 ± 6.7 3 10 3 and 2.6 3 10 3 ± 1.6 3 10 3 means ± SD. RNA copies/PFU, respectively). Notably, we demonstrated comparable antiviral efficacy of FG-4592 treatment against the recently identified B.1.1.7 (United Kingdom) and B1.351 (South Africa) SARS-CoV-2 variants ( Figure 2F). Treating Calu-3 cells with FG-4592 or two additional PHD inhibitors of the same class: Daprodustat and Molidustat, inhibited SARS-CoV-2 replication in a dosedependent manner with maximal inhibition noted at approximately 6 mM ( Figure 2G), which is in the range of reported plasma levels in human subjects after oral administration of these drugs at clinical doses (Provenzano et al., 2016). Efficacy of either PHI treatment or hypoxic culture in the activation of HIF was validated by assessing the induction of CAIX mRNA ( Figures 2E  and S4B). siRNA silencing of HIF-1a, but not HIF-2a, in Calu-3 cells reversed the hypoxic or FG-4592-mediated suppression of viral infection, demonstrating a role for HIF-1a in repressing SARS-CoV-2 RNA replication ( Figure 2H). These data show a key role for HIF-1a in repressing ACE2-dependent, authentic SARS-CoV-2 entry and infection.
To define whether hypoxia signaling regulates additional postentry steps in the SARS-CoV-2 life cycle, we evaluated the effect of hypoxia on viral replication when applied throughout or after virus inoculation. Hypoxia reduced viral RNA levels in both conditions and at all multiplicities of infection (MOIs) tested (Figure 3A). Importantly, treating SARS-CoV-2-infected Calu-3 with FG-4592 or hypoxia for 24 h significantly reduced both intracellular and extracellular SARS-CoV-2 RNA ( Figure 3B). To further define the post-entry effects of HIFs on viral replication, we infected Calu-3 cells and treated them with either FG-4592 or 1% oxygen 8 h later, once replication complexes were established. We noted a significant reduction in intracellular and extracellular viral RNA with both treatments and an induction of CAIX mRNA ( Figure 3C), demonstrating a role for HIFs in the regulation of post-entry viral RNA replication.
Given the marked reduction in the cellular viral RNA burden observed under hypoxic conditions, we sought to understand the effect of hypoxia on the initial establishment of viral replication complexes and quantities of positive genomic-strand viral RNA at the single-cell level. Using single-molecule fluorescence in situ hybridization (smFISH), we measured the effect of hypoxia and FG-4592 on positive-strand viral RNAs within the first 6 h of infection, which represents the first cycle of infection (eclipse phase) before the secretion of infectious particles ( Figure S5). (H) siRNA targeting either HIF-1 or 2a was delivered into Calu-3 cells individually or in combination, and 24 h after transfection was treated with FG-4592 (50 mM) or 1% O 2 before inoculating with SARS-CoV-2 (MOI 0.001). Intracellular RNA was quantified 24 h after infection, and data are expressed relative to the normoxic siScramble (siScram) control. * denotes significance relative to the control siRNA (siScram) at 18% O 2 , whereas # indicates significance relative to control siRNA per condition. Data are presented as means ± SD from (A) n = 4 (Calu-3) and n = 5 (PBEC donors); and (B-G) n = 4. Statistical significance was determined using a one-way (A, B, E, and G) or two-way (D and F) ANOVA. *p or #p < 0.05, **p or ##p < 0.01, ***p or ###p < 0.001, ****p or ####p < 0.0001. See also Figures S2-S4 Figures 4A and 4B). We noted a reduction in the frequency of infected cells, as judged by the detection of genomic RNA ( Figure 4C). Because de novo generated viral particles were first detected at 6 h after infection ( Figure S5), these RNA signals represent primary infection events.
In conclusion, we describe striking inhibitory effects of hypoxia and FG-4592 (Roxadustat) treatment on SARS-CoV-2 entry (including spike variants), replication, and secretion of infectious particles in lung epithelial cells. These effects were mediated by a HIF-1a-dependent repression of SARS-CoV-2 replication, in concert with the reduced expression of ACE2 across a range of cell lines and mouse lung tissue. Of note, there are reports of hypoxic induction of ACE2 gene expression in other cell types, albeit often transient (Clarke et al., 2014;Joshi et al., 2019;Zhang et al., 2009). Although this contrasts with our findings, the discrepancy may reflect the minimal ACE2 expression detected in many cell lines we examined, whereas in this study, we focused on cell lines that express greater levels of ACE2 and are relevant to the clinical sites of infection. Alternatively, the reported differences in ACE2 transcriptional regulation may reflect cell-type-specific metabolic phenotypes that modulate HIF signaling (Codo et al., 2020) or expression of co-regulators that mediate indirect effects of HIF stabilization. For example, a study of hypoxic regulation of ACE2 in PASMCs suggests an indirect mechanism through HIF-1a induction of ACE1 and ANG-II/ ATR1 signaling (Zhang et al., 2009); however, ACE1 was not regulated by hypoxia or FG-4592 in Calu-3 cells ( Figure S2). Interestingly, recent evidence describes a HIF-1a-dependent induction of the microRNA LET7b, which directly targets the ACE2 coding sequence to suppress its expression in hypoxic PASMCs (Zhang et al., 2019). Although the precise mechanism by which HIF-1a represses ACE2 mRNA in lung epithelial cells is unclear, the reversible nature of this repression, combined with the presence of a hypoxia responsive element in the ACE2 promoter (Zhang et al., 2009), may be consistent with direct HIF-mediated repression.
Beyond effects on ACE2-mediated viral entry, we observed marked suppression of SARS-CoV-2 RNA and genesis of infectious particles by hypoxia or pseudohypoxia. Notably, treatment with additional prolyl hydroxylase inhibitors Daprodustat and Molidustat exhibited a comparable antiviral capacity, suggesting a class effect that extends beyond Roxadustat. HIF has been shown to regulate the replication of other RNA viruses through effects on host cell metabolism (Farquhar et al., 2017;Frakolaki et al., 2018;Zhao et al., 2020a). For example, HIF was reported to repress hepatitis C virus replication in the liver via activation of C B A Figure 3. Hypoxia or FG-4592 (Roxadustat) inhibits SARS-CoV-2 replication post-entry (A) Calu-3 cells were treated with 1% O 2 before or after infection with SARS-CoV-2 at the indicated MOIs, and intracellular RNA was quantified by qPCR 24 h later. Data are expressed as RNA copies 3 10 8 /mg of total cellular RNA. (B) Calu-3 cells were inoculated with SARS-CoV-2 (MOI 0.001) for 2 h; unbound virus was removed by washing, and cells were treated with FG-4592 (50 mM) or cultured at 1% O 2 . Viral replication was assessed by measuring intra-and extracellular levels of SARS-CoV-2 RNA. The cellular response to FG-4592 or 1% O 2 was assessed through CAIX mRNA quantification. All data are expressed relative to the UT control. Data are presented as means ± SD from (A and B) n = 4, and statistical significance was determined using a twoway ANOVA. (C) Calu-3 cells were infected with SARS-CoV-2 as detailed above and 8 h later were cultured under 1%O 2 or treated with FG-4592 (50 mM) for 24 h. Intracellular and extracellular viral RNA, along with CAIX transcripts, were measured by qPCR, and data are expressed relative to UT control. Data are presented as means ± SD from (A-C) n = 4, and statistical significance was determined using a two-way ANOVA. *p < 0.05, **p < 0.01. the autotaxin-lysophosphatidic acid signaling pathway to regulate virus particle genesis (Farquhar et al., 2017). Moreover, our understanding of how HIF regulates respiratory viruses is exemplified by influenza A virus, whose replication was enhanced in mice, with HIF-1a inactivation restricted to type II alveolar epithelial cells (Zhao et al., 2020a), highlighting a role for HIF-1a in repressing this respiratory pathogen. Our findings contrast to those reported by Codo et al. (2020) who showed that treatment of monocytes with the HIF prolyl hydroxylase inhibitor BAY 85-3934 (Molidustat) increased SARS-CoV-2 RNA levels in an HIF-1a-dependent manner. This may relate to cell-typespecific differences; for example, monocytes have limited permissivity to support SARS-CoV-2 replication, and viral RNA levels were substantially lower than those measured from infected lung epithelial cells. Further work is needed to characterize the HIF-1a-dependent mechanisms of SARS-CoV-2 repression described here, which are likely mediated via HIF-1a regulation of host factors essential for viral RNA replication and/or stability. Our observations raise clear questions as to how cellular hypoxia translates to humans, both in terms of SARS-CoV-2 susceptibility and clinical progression of COVID-19. There has been some speculation that chronic hypoxia may be protective, with reports of reduced incidence of COVID-19 disease in high-altitude human populations (Pun et al., 2020) (although these observations are complicated by geographic and socioeconomic factors). Some clinical studies suggest that smokers and patients with chronic respiratory diseases (e.g., asthma and COPD) are under-represented co-morbidities in hospitalized patients with COVID-19 (Halpin et al., 2020). However, these conditions are also associated with a higher risk of poor outcomes in established infections (Lippi and Henry, 2020;Sanchez-Ramirez and Mackey, 2020) and, more generally, hypoxemia is a negative prognostic indicator in severe COVID-19 (Berenguer et al., 2020;Petrilli et al., 2020;Yadaw et al., 2020). Although this is seemingly at odds with our findings, clinical hypoxemia is a complex state that reflects multiple pathogenic processes, including vascular inflammation, coagulopathy, and microthrombotic disease (McGonagle et al., 2020;Varga et al., 2020), which may confound any protective effects of hypoxia on SARS-CoV-2 infection.
A key finding from our study is the potential therapeutic application of Roxadustat, and other related HIF prolyl hydroxylase inhibitors, in COVID-19, especially because these act on multiple stages of the viral life cycle (impairing entry and replication) and, as such, may be effective against emerging SARS-CoV-2 variants. These drugs have been developed as erythropoiesis-stimulating agents in patients with anemic and chronic kidney disease and are currently being used in both pre-dialysis and dialysis settings. Thus, it is likely that substantial numbers of patients who are at risk of severe COVID-19 Wu et al., 2020) will be receiving these drugs. Our work highlights the urgent need to monitor these patients for any evidence that PHD inhibitors provide prophylactic and/or therapeutic activity against COVID-19. However, clinical translation of Roxadustat may be complex because HIF has multiple systemic effects that could affect COVID-19 disease progression. Moreover, ACE2 is protective in models of lung injury (Kuba et al., 2005), so it is uncertain whether reducing ACE2 expression would have a net benefit in severe lung disease. Regardless of the potential complexity, the marked effects of Roxadustat in protecting naive cells from SARS-CoV-2 entry and in inhibiting viral replication within infected cells merits further evaluation in animal models and consideration for study in human clinical trials.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.

Data and code availability
The authors declare that all data supporting the findings of this study are available in the article. Original data have been deposited to Mendeley Data: https://doi.org/10.17632/yvgx2sgsf6.1.

Animals
All animal procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. Mice were housed in the Functional Genetics Facility of the Wellcome Trust Centre for Human Genetics (University of Oxford) in individually ventilated cages with food and water provided ad libitum and on a 13h light/11h dark cycle. Wild-type male mice on a C57BL/6 genetic background, approximately 8 weeks old and littermate controlled were used for the experiments. Mice were treated over the course of 24h with 3 oral gavages of 10mg/kg FG-4592 prepared as a 2.5mg/mL solution in 5mg/ mL methyl cellulose, 0.5% Tween80 vehicle (or vehicle alone). Hypoxic mice were housed in a normobaric altitude chamber held at 10% O 2 with controlled temperature, humidity and carbon dioxide levels and compared against mice held in normoxia. Animals were sacrificed by an overdose of Isoflurane (Primal Critical Care) and exsanguination, after which lungs were collected and immediately frozen in liquid nitrogen.
Cell culture RKO, U2-OS, Caco-2 and Vero E6 cell lines were cultured in standard DMEM; SH-SY5Y cell line in DMEM/F-12; Calu-3 in Advanced DMEM; U937 in RPMI; and A549 in F-12K; all supplemented with: 10% fetal bovine serum, 2mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin. EA.hy926 and HepG2 cells were cultured in standard DMEM additionally supplemented with endothelial cell growth supplement or non-essential amino acids, respectively. All cell lines were maintained at 37 C and 5% CO 2 in a standard culture incubator and exposed to hypoxia using an atmosphere-regulated workstation set to 37 C, 5% CO 2 :1%-5% O 2 :balance N 2 (Invivo 400, Baker-Ruskinn Technologies). Human PBECs were obtained using flexible fiberoptic bronchoscopy under light sedation with fentanyl and midazolam from healthy control volunteers. Participants provided written informed consent. The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361). Airway epithelial cells were taken by 2mm diameter cytology brushes from 3rd to 5th order bronchi and cultured in Airway Epithelial Cell medium (PromoCell, Heidelberg, Germany) in submerged culture.