Regulation of angiotensin-converting enzyme 2 isoforms by type 2 inflammation and viral infection in human airway epithelium

SARS-CoV-2 enters human cells through its main receptor, angiotensin-converting enzyme 2 (ACE2), which constitutes a limiting factor of infection. Recent findings demonstrating novel ACE2 isoforms implicate that this receptor is regulated in a more complex way than previously anticipated. However, it remains unknown how various inflammatory conditions influence the abundance of these ACE2 variants. Hence, we studied expression of ACE2 messenger RNA (mRNA) and protein isoforms, together with its glycosylation and spatial localization in primary human airway epithelium upon allergic inflammation and viral infection. We found that interleukin-13, the main type 2 cytokine, decreased expression of long ACE2 mRNA and reduced glycosylation of full-length ACE2 protein via alteration of N-linked glycosylation process, limiting its availability on the apical side of ciliated cells. House dust mite allergen did not affect the expression of ACE2. Rhinovirus infection increased short ACE2 mRNA, but it did not influence its protein expression. In addition, by screening other SARS-CoV-2 related host molecules, we found that interleukin-13 and rhinovirus significantly regulated mRNA, but not protein of transmembrane serine protease 2 and neuropilin 1. Regulation of ACE2 and other host proteins was comparable in healthy and asthmatic epithelium, underlining the lack of intrinsic differences but dependence on the inflammatory milieu in the airways.


INTRODUCTION
Angiotensin-converting enzyme 2 (ACE2) is the major host protein used by SARS-CoV-2 for entering various host cells 1 . There are six ACE2 messenger RNA (mRNA) transcript variants reported to date, which may be translated into four distinct protein isoforms 2 ( Fig. 1a and 1b and Supplementary Fig. 1a and 1b). Long ACE2 isoforms 1-3 contain protein domains responsible for its physiological functions in the renin-angiotensin axis and hold sites responsible for its interactions with SARS-CoV-2 3 . The ACE2 isoform 4, often referred to as short or truncated ACE2, lacks the N-terminal part with the SARS-CoV-2 binding site. ACE2 protein isoforms are encoded by the distinct mRNA transcripts, and they have been reported to be additionally regulated after translation by glycosylation 4 (Fig. 1a and 1b). Functionally, only the short ACE2 mRNA transcript [5][6][7] , but not the other ones as suggested earlier 8 , is an interferonstimulated gene and is increased upon viral infection, although it is not clear whether it is further reflected in protein expression. Since the beginning of the pandemic, several groups 8,9 , including ours 10 , have studied expression of ACE2 and other SARS-CoV-2 receptors and associated host molecules in different cells, organs, and various diseases to predict, to some extent, the impact of SARS-CoV-2 infection. However, these studies analyzed ACE2 mRNA expression based on the single cell or bulk sequencing and microarray techniques, which did not distinguish between distinct ACE2 transcripts. ACE2 and other receptors protein expression or their cellular localization have not been thoroughly assessed. Unfortunately, it led to, at times, dangerous misconceptions, such as perceiving the full-length ACE2 as being increased upon interferons or viral infections 8 and challenging safety and validity of the ongoing interferon clinical trials. Therefore, it is crucial to cautiously revise previous findings and further elucidate the suggested and new mechanisms of its regulation at the mucosal sites.
Interactions between asthma, its viral-or allergen-induced exacerbations, and COVID-19 are still unclear 11,12 . It is suggested that allergic inflammation and type 2 (T2)-high asthma might not constitute a risk factor for severe COVID-19 outcomes or might even convey some forms of protection from SARS-CoV-2 infection 13,14 , whereas T2-low asthma, severe, or uncontrolled asthma might be a risk factor of COVID-19 [15][16][17] . It is based on contrasting epidemiological observations in COVID-19 cohorts from different parts of the world 11,15 , and a few mechanistic studies showing that interleukin (IL)-13, the major type 2 cytokine, decreases ACE2 expression and inhibits SARS-CoV-2 infection in airway epithelium 18,19 . However, IL-13 has also been shown to be a major driver of COVID-19 severity, and anti-IL-4/-13 treatment is showing positive effects in COVID-19 clinical trials 20,21 . Moreover, infection with rhinovirus (RV), the most common exacerbation-inducing factor in asthma, has been shown to inhibit SARS-CoV-2 infection via type I/III interferon (IFN)-dependent mechanism [22][23][24] , which might suggest that this mechanism could constitute some form of protection. However, we recently reported that co-infection of RV and SARS-CoV-2 leads to a greater retinoic acid-inducible gene I inflammasome-dependent damage of airway epithelium in patients with asthma 25 , which is strongly supported by the clinical findings that patients with RV and SARS-CoV-2 co-infection are more prone to severe COVID-19 26 . Finally, some components of house dust mite (HDM) or birch pollen allergens were demonstrated to decrease type I/III IFN responses 25,27 at the mucosal sites, a mechanism that may potentially contribute to the reported correlations between high allergen exposure and increased SARS-CoV-2 infection rates 28 .
Therefore, we aimed to analyze transcriptional, translational, and post-translational ACE2 regulation, together with its spatial localization upon allergic and viral inflammation in human bronchial airway epithelium in health and in asthma. We also analyzed other reported SARS-CoV-2 receptors and host molecules. We found that IL-13 decreased expression of long ACE2 mRNA and reduced glycosylation of full-length ACE2 protein via its effect on the N-linked glycosylation pathway, leading to reduction of apical expression of ACE2 on ciliated cells. RV infection increased expression of short ACE2 mRNA transcript but did not change the short ACE2 protein expression. HDM exposure did not affect ACE2 mRNA or protein. IL-13 and RV significantly regulated transmembrane serine protease 2 (TMPRSS2) and neuropilin 1 (NRP1) mRNA, but not protein. Regulation of ACE2 and other host factors was comparable in health and in asthma.

RESULTS AND DISCUSSION
IL-13 decreased long ACE2 mRNA whereas rhinovirus infection-induced expression of short ACE2 mRNA in human primary bronchial epithelial cells In order to differentiate between the mRNA transcripts encoding for the long ACE2 (isoforms 1-3) and the short ACE2 (isoform 4), we designed primers allowing for such distinction (Fig. 1b, Supplementary Fig. 1b, and Supplementary methods). Primary human bronchial epithelial cells (HBECs) from controls and patients with asthma were differentiated in the air-liquid interface (ALI) for 4 weeks. Then, we treated them with IL-13, HDM, or vehicle (medium) for 24 hours, followed by the successful infection with human RV 16 (RV-A16) or treatment with Ultra Violet (UV)-inactivated RV 16 (UV-RV-A16) for 6 and 24 hours ( Fig. 1c; Supplementary Fig. 2a). We found that the long and short ACE2 mRNA transcripts were expressed in a 1 ∼ 1 ratio at the steady-state, without any differences between controls and patients with asthma (Fig. 1d), therefore, we analyzed them all together in the following experiments. Long ACE2 mRNA expression decreased upon IL-13 stimulation, whereas neither HDM exposure nor RV infection affected its expression at the measured time points (Fig. 1e). IL-13 did not affect the expression of short ACE2 (Fig. 1f). Our findings are in agreement with the initial reports showing a decrease of ACE2 mRNA upon IL-13 29 , with an additional notion that the IL-13 effect indeed refers to the long ACE2 mRNA transcripts which are being translated to the functional full-length ACE2 protein, but it does not regulate the expression of the truncated ACE2 mRNA. In contrast, we observed that short ACE2 mRNA increased slightly, already 6 hours post-infection (hpi), which turned to notably increased levels at 24hpi (Fig. 1f), supporting three other reports of short ACE2 to be an interferon-inducible gene and its responsiveness to infection with the RNA virus [5][6][7] . Once again, our findings confirmed that the long ACE2 transcripts encoding full-length ACE2 did not increase upon RV infection in human primary bronchial epithelium. HDM, IL-13, and UV-RV-A16 did not influence short ACE2 expression. We did not distinguish all different long isoforms by RT-PCR, as most of the sequences share high similarity, making it challenging to find feasible primer pairs. Because the severity of COVID-19 is often associated with age 30 , we also analyzed its correlation with mRNA expression of long and short ACE2 ( Supplementary Fig. 2b) and RNA-seq detectable ACE2 ( Supplementary Fig. 2c), observing only a trend of positive correlation of long ACE2 with age. These results underline the importance of choosing isoform-specific primers for ACE2 when reporting the results and driving relevant conclusions.
IL-13 downregulated N-linked glycosylated ACE2, whereas rhinovirus infection did not affect the expression of any ACE2 protein isoforms Next, we analyzed the expression of ACE2 protein isoforms in the same conditions. We used a C-terminal ACE2 antibody (ab15348), which is able to distinguish isoforms 1, 3, and 4 ( Fig. 1a). We detected three bands of the ACE2 protein in the HBECs in controls and in patients with asthma (Fig. 2a). The band at ∼52.5 kDa corresponded to the short isoform of ACE2 (isoform 4), while the band at ∼92.5 kDa to the full-length ACE2 isoform 1. We hypothesized that the ∼130 kDa band might refer to the glycosylated (N-linked glycosylation on asparagine residues as a post-translational modification) isoform 1, which we confirmed by peptide-N-glycosidase F (PNGase F) treatment, which led to the absence of glycosylated ACE2 and an increase in nonglycosylated ACE2 of ∼92.5 kDa (Fig. 2b). The absence of any (a) Schematic representation of ACE2 protein isoform sequences. The fulllength ACE2, isoform 1, consists of an N-terminal signal peptide, a peptidase domain, and the collectrin homology domain. Isoform 2 has a distinct C-terminal sequence, and isoform 3 lacks the part harboring the ADAM17 cleavage site. Isoform 4 does not possess the N-terminal signal peptide and ends in a unique sequence. N-linked glycosylation sites, SARS-CoV-2 spike interaction, Zn binding site, ADAM17 and TMPRSS2 cleavage sites, and the transmembrane domain are marked accordingly. Created with BioRender.com. (b) Summary of NCBI entries of ACE2ACE2 mRNA transcripts and protein isoforms precursor IDs, predicted molecular weight, amino-acid length, and the detectability by the primers and antibodies used in the manuscript. NP_001358344.1 and NP_068576.1 lead to isoform 1, NP_001373188.1 to isoform 2, NP_001376331.1 and NP_001373189.1 to isoform 3, and NP_001375381.1 to isoform 4, respectively. (c) Schematic representation of the experimental design. Primary human bronchial epithelial cells from healthy controls or patients with asthma were differentiated in ALI cultures for 4 weeks, next they were treated with HDM (200 µg/ml of protein content), IL-13 (50 ng/ml), or vehicle for 24 hours and later infected with RV-A16 (MOI 0.1) or treated with UV-treated RV-A16 or vehicle for 6 hours and 24 hours. (d) Relative mRNA expression of long ACE2 and short ACE2 mRNA in vehicle-treated HBECs from n = 4 controls and n = 5 patients with asthma presented as delta CT (ΔCT) with SEM. (e) Long ACE2 mRNA expression in HBECs from controls (black dots, n = 3) and patients with asthma (red dots, n = 3) upon treatment with either HDM, IL-13, or vehicle, followed by RV-A16, UV-RV-A16, or vehicle, harvested at 6 hpi, left, and 24 hpi, right. RT-PCR values were calculated by 2 −ΔΔCt to unstimulated condition (Veh). (f) Short ACE2 mRNA expression in HBECs from controls (black dots, n = 3) and patients with asthma (red dots, n = 3) upon treatment with either HDM, IL-13, or vehicle, followed by RV-A16, UV-RV-A16, or vehicle and harvested at 6 hpi, left, and 24 hpi, right. d-e, Two technical replicates per donor were performed, and the mean of these measurements for each donor is presented by the individual dot. RT-PCR values were calculated by 2 −ΔΔCt to unstimulated condition (Veh). Bars represent mean ± SEM. One-way analysis of variance with Dunnett's multiple comparison corrections were used to assess statistical significance. *, p < 0.05. **, p < 0.01. ***, p < 0.001.  band at ∼79.4 kDa suggests that isoform 3 is not expressed in primary HBECs. We noted that ACE2 isoform 1, glycosylated isoform 1, and short ACE2 isoform 4 were present in a 1∼1∼1 ratio in a similar fashion in controls and in asthma (Fig. 2c). We observed a significant decrease of glycosylated ACE2 upon IL-13 treatment, whereas unglycosylated full-length ACE2 remained unchanged ( Fig. 2d and 2e) with no differences between asthma and control. It suggests that IL-13, in addition to its transcriptional effect on full-length ACE2, also acts posttranslationally by deglycosylation on ACE2 isoform 1 (Fig. 2b). This phenomenon may also "add up" to the full-length ACE2 pool masking the reflection of the transcriptional decrease of this isoform upon IL-13 treatment. The ratio of ∼130 kDa to ∼92.5 kDa ACE2 also changed significantly in response to IL-13 ( Fig. 2f), which suggests altered availability of unglycosylated versus glycosylated full-length ACE2. Glycosylation of ACE2 overall does not substantially influence the interaction with the receptor-binding domain of SARS-CoV-2 31,32 . However, a recent study showed that N-linked glycosylated ACE2 is predominantly expressed on the cell surface, being accessible for SARS-CoV-2, whereas unglycosylated ACE2 is localized to the endoplasmic reticulum 33 . Therefore, our finding that IL-13 is decreasing full-length ACE2 glycosylation may explain the lower SARS-CoV-2 infection upon IL-13 stimulation observed in other studies.
The expression of short ACE2 protein isoform 4 in HBECs appeared to be very stable (Fig. 2e). We did not find RVinduced upregulation of short ACE2 protein. This lack of an effect on the protein expression might suggest a high turnover of short ACE2 in bronchial epithelium, meaning that the potential increase of short ACE2 translation, mirroring an RV-induced increase of short RNA transcript, is balanced by the increase in protein degradation [34][35][36] . Indeed, it was shown that short ACE2 protein is very unstable and, as such, rarely detectable in a range of cell lines 5,7 . Its more stable expression was demonstrated in the lung airway epithelia and liver bile duct epithelia 37 . These findings may also suggest that short ACE2 mRNA or nonspliced ACE2 RNA itself, may harbor antiviral regulatory functions, e.g. through the possession of circular RNA functions upon viral infections 38 .
Using the same C-terminal ACE2 antibody, we found ACE2 to be localized predominantly but not restricted to the apical side of ciliated cells ( Fig. 2g and 2h). There was also a positive signal localizing to cilia ( Supplementary Fig. 3a), as previously reported 39 , but we found the positive signal in cilia to be rather weak by inspection of transversal sections (Fig. 2h). Using another commonly used N-terminal ACE2 antibody (HPA000288), we observed stronger staining in cilia (Supplementary Fig. 3b). However, using the same antibody for western blot, we found some clear bands at ∼66 kDa and ∼54 kDa, which might suggest an unspecific binding ( Supplementary Fig. 3c). Transversal cryosections of bronchial epithelium revealed a substantial redistribution of ACE2 signal upon IL-13 treatment (Fig. 2h). However, there are contradictory reports on the localization of ACE2 and viral tropism in bronchial epithelial cells in the literature. Although some reports showed goblet cells 40 or secretory type 3 cells 41 to be the target of SARS-CoV-2, other reports clearly demonstrated that viral infection is rarely detected in goblet cells and that the virus primarily infects ciliated cells 42 . These discrepancies could be explained by the usage of disparate ACE2 antibodies, various differentiation statuses of the epithelial cells 41,43 , or syncytia formation by infected cells, complicating the assessment of cellular tropism of the virus 44 . Finally, reporting only the ACE2 mRNA expression, combined with the inconsistency in the annotation of secretory type cells in the single cell sequencing datasets due to their overlapping signatures of ciliated and goblet cells 45 may add to the complexity of this matter.
IL-13 reduced apical ACE2 expression, induced morphological changes in airway epithelium, and altered Nlinked glycosylation gene expression profile Having demonstrated that IL-13 induces profound morphological alterations and reduces the amount of glycosylated fulllength ACE2 in human primary bronchial epithelium, we searched for the underlying mechanisms of the IL-13 effect on ACE2. As we observed ACE2 to be predominantly expressed apically on ciliated cells, where it is accessible as receptor for SARS-CoV-2, we measured apical ACE2 intensities above the tightjunction layer, stained by occludin (Fig. 3a, Fig. 2g, Supplemen-  tary Fig. 3d). Apical ACE2 intensity significantly decreased after IL-13 treatment ( Fig. 3a and b), suggesting that IL-13-induced deglycosylation of full-length ACE2 led to its redistribution from the apical membrane to other cytosolic organelles 33 , as observed in Fig. 2h. IL-13 is known to induce time-dependent morphological changes in airway epithelium such as goblet cells meta-and hyperplasia, reduced cilia cell numbers, and it leads to the epithelial barrier impairment [46][47][48] , all of which might play a role in reduced availability of ACE2 for SARS-CoV-2. Therefore, we assessed cell numbers of MUC5AC (goblet) and alphatubulin (ciliated) positive cells, finding no significant change at 48 hours post IL-13 stimulation (Fig. 3c, Supplementary  Fig. 3e). We also studied the IL-13-dependent changes in the cell type-specific marker genes according to the recently proposed N. Stocker, et al. nomenclature 45 at 24 hours after IL-13 treatment. We indeed found some alterations in the secretory club and goblet cells but with different directionality of changes, whereas most of the markers for ciliated cells remained unchanged (Supplementary Fig. 3f). These results imply that at these timepoints of IL-13 treatment, there are no substantial shifts in the numbers of cell subpopulations. We nonetheless noted morphological changes like size increase of non-ciliated cells, such as goblet/secretory cells (Fig. 3a). Therefore, we also quantitatively assessed the changes in apical cell shape and cell area on the planar view. Solidity and circularity of ciliated cells decreased significantly upon IL-13 treatment, while non-ciliated cells' area increased and circularity decreased (Fig. 3d). Reduced solidity of ciliated cells in response to IL-13 might be caused by the hypertrophy of goblet cells and might also contribute to lower availability of ACE2 on the apical surface. We also observed and confirmed a more pronounced IL-13-induced disruption of tight junctions in asthma, as previously reported (Fig. 3a) 49 .
To deeper examine the mechanistic influence of IL-13 on bronchial epithelium, we performed RNA sequencing of HBECs from controls (n = 5) and patients with asthma (n = 5) treated with/without IL-13. IL-13 induced a significant change in the transcriptomic profile in both groups with the similar directionality of changes (Fig. 3e). The unbiased enrichment analysis confirmed previous observations in similar models (GSE106812; GSE37693) 18 , showing that the top IL-13-affected pathways include ion and transmembrane transport, lipid metabolic processes and, interestingly protein glycosylation (Fig. 3f). Having found this, plus observing that IL-13 induced deglycosylation of full-length ACE2 and redistribution of ACE2 from the apical side of ciliated cells to the cytosol, we analyzed in more detail the N-linked glycosylation process (GO-term: 0006487). Indeed, we found that IL-13 significantly changed expression of several genes involved in this process (Fig. 3g), suggesting that it might be a mechanism by which IL-13 regulates ACE2 glycosylation. Interestingly, IL-13 downregulated STT3B-a part of the oligosaccharyltransferase complex-which was recently reported to suppress SARS-CoV-2 infection when blocked 50 . Also, TUSC3, which is an accessory protein of the oligosaccharyltransferase complex, was downregulated by IL-13 51 . MAN1C1, also downregulated by IL-13 in our dataset, has been described to be increased in severe COVID-19 patients and to be associ-ated with the risk of disease progression 52 . We did not find any significant differences in IL-13-induced changes in Nlinked glycosylation comparing asthma over control. It all suggests that IL-13 released from T helper 2 and type 2 innate lymphoid cells at the mucosal sites during type 2 inflammation in allergic asthma, on top of other known morphological and transcriptional changes, might potently induce ACE2 deglycosylation and redistribution, which might alter SARS-CoV-2 infection efficiency. Recent publications show that IL-13 can inhibit SARS-CoV-2 infection and can reduce intracellular viral load and cellto-cell transmission in airway epithelial cells 18,19 . IL-13-induced mucus hyperproduction can inhibit SARS-CoV-2 infection by forming a physical barrier, but even after mucus removal, viral loads remained to be lower upon IL-13, suggesting further mechanisms to be involved 18 .
IL-13 and rhinovirus infection regulated mRNA expression of other SARS-CoV-2-entry-related molecules in bronchial epithelium but did not change TMPRSS2 and NRP1 protein expression Finally, as we previously analyzed mRNA expression of SARS-CoV-2-related host molecules in patients with asthma and other COVID-19 comorbidities 10 at the steady-state, here, we focused on further characterization of their expression upon viral and allergic inflammation and localization in primary bronchial epithelium. From the constantly growing list of SARS-CoV-2entry-related host molecules (Supplementary Fig. 4a) 53 , in our RNA-seq dataset, we identified that, in addition to downregulation of ACE2, IL-13 also downregulated NRP1 (Fig. 4a), which could serve as an additional SARS-CoV-2 receptor 54,55 . In contrast, syndecan 2 (SDC2) mRNA, encoding a heparan sulfate proteoglycan, was upregulated upon IL-13, which might affect the initial attachment and internalization of SARS-CoV-2 56 . Also, GNE [Glucosamine (UDP-N-Acetyl)-2-Epimerase/N-Acetylmanno samine Kinase] was upregulated, which is crucial for sialic acid biosynthesis 57 and potentially influences SARS-CoV-2 infection 58 . Interestingly, DPP4 was highly upregulated in these conditions. Although DPP4 seems not to bind SARS-CoV-2 directly in humans, it continues to be controversially discussed, especially in terms of obesity and severe COVID-19 59 . TMPRSS2, encoding a host protease facilitating fusion of SARS-CoV-2 with the cellular membranes 1 , was upregulated upon IL-13 stimulation, as  previously reported 29 . We and others also found TMPRSS2 to be upregulated at baseline in bronchial biopsies of patients with asthma 10,29 . There were no differences in the expression of these genes between controls and patients with asthma after IL-13 treatment. Functional consequences of the increase in TMPRSS2, SDC2, GNE, and DPP4 expression need to be further investigated, as they might play a role in non-T2 asthma in the absence of IL-13 effects on ACE2 and NRP1.
To analyze the effect of RV infection, we performed data mining of previously published datasets of HBECs infected with RV-A16 (GSE61141 60 ). Upon RV infection, IFIH1 (encoding MDA5) was one of the top upregulated SARS-CoV-2-associated genes (Fig. 4b). MDA5 is a pattern recognition receptor that, once activated, induces an interferon response. SARS-CoV-2 RNA is recognized by MDA5 61 . Infection with RV initiates type I/III IFN response; therefore, pre-infection with RV reduces SARS-CoV-2 replication 22 , but RV and SARS-CoV-2 co-infection leads to greater damage of the airway epithelium, especially in patients with asthma 25 . An observed ACE2 increase was presumably caused by an increase in short ACE2 mRNA, as we demonstrated above. Finally, RV infection led to an increase in mRNA expression of EXT1, ADAM17, TMPRRS2, FURIN, HSPA3, and NRP1, which potentially might affect a few mechanisms involved in SARS-CoV-2 infection. All analyzed genes of SARS-CoV-2 associated molecules upon IL-13 and RV infection are shown in Supplementary Fig. 4b and 4c. Interestingly, we have not observed any correlations of TMPRSS2, NRP1, or FURIN with age of the study participants ( Supplementary Fig. 4d).
Because TMPRRS2 and NRP1 mRNA were regulated by IL-13 and RV infection, we analyzed their protein localization and expression in these conditions and upon HDM exposure. Surprisingly, we did not observe significant changes in TMPRSS2 or NRP1 protein expression in any of the experimental settings ( Fig. 4c and 4d). This again suggests a high protein turnover, translation repression, mRNA degradation, or that the transcriptional change in expression might be reflected on the protein level later than 24 to 48 hours, respectively [34][35][36] . Interestingly, we detected a difference in NRP1 protein expression between asthma and controls in RV-A16 + HDM and IL-13 conditions (Fig. 4d), which requires further investigation. TMPRSS2 localized apically on ciliated cells, but we could also observe positive signal localizing to nuclei (Fig. 4c). Similar to TMPRSS2, we detected NRP1 signal to localize apically on ciliated cells and nuclei, and we found a remarkably strong positive signal in cilia (Fig. 4d). Altogether, we found ACE2, NRP1, and TMPRSS2 proteins to be predominantly localized apically on the ciliated bronchial epithelial cells, which suggests that these cell types might be the most susceptible to SARS-CoV-2 infection.
A limitation of the current study is that, here, we have not analyzed SARS-CoV-2 infection in the studied conditions, although we and others performed such experiments for the HDM stimulation and for SARS-CoV-2 and RV co-infection in separate studies 18,19,25 . In addition, the shed or soluble ACE2 was not investigated, and the isoform 2 was not distinguished by RT-PCR and was not analyzed on the protein level. Also, protein measurements performed by western blot are semi-quantitative and may not resemble the actual protein content in the cells. Nonetheless, despite these limitations, our study constitutes the most thorough overview of mRNA and protein regulation and spatial localization of ACE2 and other SARS-CoV-2-related host molecules in differentiated primary HBECs upon allergic and virus-induced inflammation in health and in asthma.

Detailed methods are described in the Supplementary files.
ALI culture Primary HBEC from control individuals and patients with asthma (Epithelix, Plan-les-Ouates, Switzerland; Lonza, Basel, Switzerland, and indicated patient cohort, Jagiellonian University Medical Collage, Cracow, Poland) were passaged in bronchial epithelial basal medium (Lonza) with all supplements (even T3 and retinoic acid?) (Bronchial Epithelial SingleQuots, Lonza). Cells were grown up to 80%-90% confluency and then seeded into transwell inserts at a density of 1.5 × 10 5 cells per well (0.40 µm pore size, 0.33 cm 2 , Corning Inc., New York, USA). After reaching confluence, the apical medium was removed, and the basal medium was changed to 1:1 supplemented bronchial epithelial basal medium (Lonza) with Dulbecco's modified eagle's medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), containing 0.06% wt/vol all-trans retinoic acid (Sigma-Aldrich, Schnelldorf, Germany). The medium was changed every 2-3 days, and excess mucus was removed until cells were fully differentiated (∼28 days). HDM extract (Allergopharma, Reinbek, Germany) was introduced apically on HBECs at a dose of 200 µg/ ml of the protein concentration of the extract. Human IL-13 (Lubio Science, Zürich, Switzerland) was diluted in OptiMem (Thermo Fisher Scientific) and used at 50 ng/ml. Cells were incubated at 37°C with 5% CO 2 .
Viruses Viral titer of human RV-A16 (Microbiologics Global Virology Center, San Diego, CA, USA) was determined by plaque assay in H1-Hela cells (ATTC, Manassas, VA, USA). Inactivation of virus was performed with UV light at 254 nm for 60 minutes. UV-treated RV-A16 was included in the experiments to control for the effects of viral proteins, not depending on replication of the live virus 62,63 . Virus was diluted in OptiMem (Thermo Fisher Scientific) and applied apically at a multiplicity of infection (MOI) of 0.1 for 2 hours at 34.5°C with 5% CO 2 .  Reverse transcription-quantitative polymerase chain reaction (RT-PCR) RNA was isolated by RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Yield and purity were measured by Nanodrop 2000 (Thermo Fisher Scientific). RT was performed with RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed with Maxima SYBR Green/ROX qPCR Master-Mix (Thermo Fisher Scientific) on Quantstudio 7 Real-Time PCR System (Thermo Fisher Scientific). Relative quantification was calculated by 2 −ΔΔCt method described previously 64 . Sequences of primers used are summarized in the Supplementary methods.

Transcriptome analyses
Next-generation sequencing from differentiated HBECs from control and patients with asthma, with or without IL-13 stimulation at 50 ng/ml for 24 hours was performed as previously described 10 and uploaded on the Gene Expression Omnibus platform (https://www.ncbi.nlm.nih.gov/geo) and is publicly available under the accession number GSE206510. Nextgeneration sequencing from differentiated HBECs from control and patients with asthma, infected with RV-A16, were obtained from the Gene Expression Omnibus platform under accession number GSE61161.