key: cord-0979942-ec387vzs
authors: Scagnolari, Carolina; Bitossi, Camilla; Viscido, Agnese; Frasca, Federica; Oliveto, Giuseppe; Scordio, Mirko; Petrarca, Laura; Mancino, Enrica; Nenna, Raffaella; Riva, Elisabetta; De Vito, Corrado; Midulla, Fabio; Antonelli, Guido; Pierangeli, Alessandra
title: ACE2 expression is related to the interferon response in airway epithelial cells but is that functional for SARS-CoV-2 entry?
date: 2021-01-15
journal: Cytokine
DOI: 10.1016/j.cyto.2021.155430
sha: b003c83329c6eb4fed7bd9436b9a9b87fc055a87
doc_id: 979942
cord_uid: ec387vzs

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS‐CoV‐2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs’ activation, ISG15, in children (n=59) and adults (n=48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 concentration, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs’ activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS‐CoV‐2 infection in the respiratory tract.

Starting from December 2019 in China, a novel virus of the Coronaviridae family, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide [1] . SARS-CoV-2 infections range from completely asymptomatic to mild symptoms, up to a severe acute respiratory distress syndrome (ARDS) and death. SARS-CoV-2 entry into respiratory tract cells starts with the binding to its main receptor, the angiotensin-converting enzyme 2 (ACE2) [2] , the same receptor used by SARS-CoV-1 [3] . Differently from SARS-CoV-1, the spike protein of the novel CoV is activated by the serine protease furin that enhances viral entry into human lung cells [4] . The efficiency of SARS-CoV-2 entry may be related to the number and type of cells programmed to express ACE2 and/or furin other than to the rate of their expression in the specific epithelial cells. Besides being SARS-CoV-2 binding/entry receptor, ACE2 exerts a protective role in acute lung injury through its involvement in renin-angiotensin system (RAS) signaling and its down regulation in SARS-CoV infections might also play a causal role in disease progression to ARDS [5] .

ACE2 has been recently proposed to be an interferon (IFN)-stimulated gene (ISG) in epithelial tissues [6, 7] coherently with the notion that its upregulated expression may be functional in preventing ARDS during viral infections [5, 8] . Accordingly, we sought to verify this ISG-type induction of ACE2 expression in upper airways cells, collected during children's and adult's respiratory infections caused by pathogens other than SARS-CoV-2.

Subsequent studies [9] [10] [11] demonstrated the existence of truncated ACE2 isoforms (dACE2) of unknown functions, not able to serve as SARS-CoV-2 entry receptor. Moreover, the authors observed that IFNs and viruses induced dACE2, but not full-length ACE2, in several, but not all, the studied cell models [9] [10] [11] . Therefore, to clarify this issue, we performed experiments of in vitro stimulation of the A549 and Calu3 cell lines with type I and III IFNs, to characterize the activation of ACE2/dACE2 and to investigate the potential induction of the furin gene as well.

Fifty-nine previously healthy children admitted to the Paediatric Department of the University Hospital "Policlinico Umberto I" with a clinical diagnosis of bronchiolitis and pneumonia, and 48 adults with respiratory symptoms attending the same Hospital were enrolled over the 2019/2020 winter season. The local ethics committee approved the study protocol (Sapienza University of Rome, University Hospital "Policlinico Umberto I"); all adult patients and parents/guardians gave informed consent to participate in the study and patients' data were anonymized. Respiratory specimens (nasopharyngeal washings for children, nasopharyngeal swabs for adults) were tested for 14 respiratory viruses and for SARS-CoV-2 [12] ; remaining aliquots were centrifuged, and RNA extracted from cell pellet for gene expression analysis [13] .

The ACE2 mRNAs levels, of the serine protease furin and that of the well-known marker of IFNs' activation, ISG15, were measured in respiratory cells by quantitative RT-Real time PCR assays ACE2 was tested with a commercial qPCR Assay (Hs.PT.58.27645939. IDT, Iowa, USA), targeting exons 14-15 that are common to ACE2 isoforms; furin was tested with a commercial assay to normalize the amount of total RNA, using the threshold cycle relative quantification (the 2 −ΔCt method) as previously reported [13] . Each raw Ct (threshold cycle) value was tagged as undetermined when fell between levels of 40 and 45. For mRNA copies that were undetectable by the RT-PCR assays, the Ct was set to 45, for calculating the fold changes to the GUS Ct of the same sample.

The experiments of IFNs exogenous administration in A549 and Calu-3 (human lung carcinoma derived cell lines) were performed treating, for 24 hours, 200.000 cells/well cultured in 24-well plates, with natural IFNα (Alfa Wassermann SpA, Bologna, Italy) at 5x10 4 IU/mL, IFNβ1a (Avonex Pen, Biogen Idec Ltd. Cambridge, Massachusetts, USA) and IFNλ1-3 (R&D systems, Minneapolis, MN, USA) all at 500 ng/mL. The comparative threshold cycle method was applied to calculate the fold changes, compared with non-stimulated cell (2 -ΔΔCT ) of the genes of interest: furin and ISG15 were tested as described above. In addition, the full-length ACE2 isoform (f-lACE2) and dACE2 were quantified using specific primers and probes [9] , and two other ISGs were tested: ISG56 (F 5'- transcription levels were significantly elevated in both cell lines, with fold-changes about ten-times higher in A549 than in Calu-3 cells (Fig. 1) .

Furin levels of expression were not augmented in either cell lines following stimulation with any IFN (Fig. 1) ; undoubtedly, IFN treatments could have altered furin intracellular trafficking, glycosylation and/or enzymatic activation, that we did not address. Previous studies observed that furin mRNA and protein expression is negatively regulated by IFNγ and that two ISGs (GBP2 and GBP5) are able to inhibit furin proteolytic activity, as summarized in a recent review [14] .

After treatments by IFNs, the f-lACE2 levels were not increased, or were even diminished (in the A549 by type III IFNs) (Fig. 1) . Differently, dACE2 transcripts were significantly elevated, especially after type I IFNs treatments (Fig. 1) , in the A549 (IFNα=11.4-fold and β=31.1-fold) and

Calu-3 cells (IFNα=6.8 fold and β=10.6-fold). Hence, these in vitro experiments further supported that the mRNA(s) for the truncated isoform dACE2, and not that for the full-length ACE2, is induced by IFNs [9] [10] [11] . Compared to 'traditional' ISGs, dACE2 regulation by IFNs appeared weaker and its expression might be also affected by different cell-specific factors. The latter aspect is not a novelty since the exact set and number of genes that are regulated by IFN is known to largely vary between different cell types, often depending on the constitutive level of expression of the IFN genes [15] . In this respect, Onabajo et al [9] reported that dACE2 transcript was not inducible by IFNs in Vero E6, and moderately stimulated in HEK293T cells. Notably, Ng et al [10] found that dACE2 is more 

Funding: This work was supported by a Sapienza University grant to AP "Ricerca 2019" n.

RM11916B893FC0D0 and by grant to GA from the Italian Ministry of Health: COVID-2020-12371817.

Induction of the Interferon (IFN)-stimulated genes (ISG), IRF-7, ACE2 and furin after type I and III IFN stimulation of the A549 and Calu-3 epithelial cell lines.

Expression of ISG15, ISG56, IRF7, full-length (f-l) dACE2 and furin in A549 (panel A) and Calu-3 (panel B) cells treated with IFNα (5x10 4 IU/mL), IFNβ1a (500 ng/mL), IFNλ1 (500 ng/mL), IFNλ2 (500 ng/mL) and IFNλ3 (500 ng/mL) for 24 h. Data are expressed as fold change (ΔΔCt method), from the equation 2 -ΔΔCt used to calculate the mRNA levels of the stimulated genes, compared to baseline values in untreated cells (set to 1 and indicated in the graphs as CTRL). Differences in mRNA expression levels between untreated and treated cells were evaluated using paired T-test. *=p<0.05.

Correlation of ACE2 levels with the Interferon (IFN)-stimulated gene (ISG) 15 and with patients' age.

The correlation between ACE2 mRNA copy number, expressed as fold change (2 -ΔCt ) with respect to the β-Glucuronidase (GUS) gene, and ISG15 mRNA copy number is shown in panel A (ρ=0.414; p<0.001). The correlation between the ACE2 mRNA copy number and all patients' age (in years) is shown in panel A (total, ρ= -0.2466; p=0.0105), B (children, ρ= -0.3220; p=0.0129) and C (adults, ρ= 0.1495; p=0.3105). 

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Male (%)

Age is expressed as mean ± SD. **Ct (threshold cycle) values of GUS (beta-glucuronidase gene) are indicated as mean ± SD. ***Transcript levels of ISG15, ACE2 and furin, calculated using 2 -ΔCt , are indicated as median

001 by Chi-Squared test; ▼ p<0.001 by Mann-Whitney U test