key: cord-0703991-scqkyr5g
authors: Sharif‐Askari, Fatemeh Saheb; Sharif‐Askari, Narjes Saheb; Goel, Swati; Fakhri, Samer; Al‐Muhsen, Saleh; Hamid, Qutayba; Halwani, Rabih
title: Are patients with chronic rhinosinusitis with nasal polyps at a decreased risk of COVID‐19 infection?
date: 2020-08-05
journal: Int Forum Allergy Rhinol
DOI: 10.1002/alr.22672
sha: da64b1bdbeddd9b5988196225304b2bfd023b487
doc_id: 703991
cord_uid: scqkyr5g

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Corresponding author: Rabih Halwani; Address: College of Medicine, University of Sharjah, Sharjah, United Arab Emirates; Email: rhalwani@sharjah.ac.ae.

The authors have no conflicts of interest to declare.

SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry to the cell and the serine protease TMPRSS2 for S protein priming [1, 2] . Higher ACE2 expression was recently reported in nasal compared to throat tissue [3] . In fact, higher SARS-CoV-2 viral load was detected in nasal compared to throat swabs obtained from COVID-19 infected patients [4] , and that was attributed to the difference in ACE2 expression between both tissues. In fact, higher SARS-CoV-2 viral load was detected in nasal compared to throat swabs obtained from COVID-19 infected patients. This was attributed to the difference in ACE2 expression between both tissues [4] . Recently, we have also shown that the upper airway expresses more SARS-CoV-2 entry genes, ACE2 and TMPRSS2 compared to the lower airway [5] . Moreover, Hou et al [6] have recently established that multiciliated cells are the main cell types expressing ACE2 in nasal tissue and infected with SARS-CoV-2. Moreover, Sungnak et al [7] by analyzing data of single-cell RNA-sequencing from healthy human nasal epithelial cells showed that ACE2 and TMPRSS2 are co-expressed in nasal epithelium with genes involved in host innate immunity, referring to the potential role of these cells in initiating SARS-CoV-2 infection. Therefore, the level of SARS-CoV-2 receptors in nasal tissue may determine the level of viral infectivity given the fact that these receptors are not upregulated following infection [1] . With that in mind, we decided to investigate potential factors that may affect the expression of SARS-CoV-2 receptors and hence the risk of infectivity with COVID-19 in various phenotypes of sinonasal inflammation.

To achieve this, gene-expression data sets were used to determine the expression pattern of SARS-CoV-2 cell entry genes, ACE2 and TMPRSS2 in the inflamed uncinate process from patients with CRS without NPs (CRSsNPs) and nasal polyp from patients with CRSwNPs (supplementary methods). A reduction in ACE2 expression was observed in the inflamed uncinate tissue of CRSsNPs patients but not to a significant level ( Figure 1A ). Interestingly, a significant reduction in the expression of ACE2 and TMPRSS2 was observed in the nasal polyps of CRSwNPs patients compared to healthy controls ( Figure 1A ). This data implies that patients with CRSwNPs might have a lower risk of SARS-CoV-2 infection due to lower expression levels of SARS-CoV-2 cell entry genes.

To our knowledge, the impact of allergic inflammation on the level of expression of ACE2 and TEMPRSS2 remains poorly understood. In an in-vitro study, IL4 has been shown to reduce ACE2 gene expression levels in SARS-CoV infected cells [8] . Moreover, ACE2 was shown to be less activated in eosinophilic and allergic airway inflammation conditions [9] which may indicate potential regulatory effect of such conditions on these receptors. In our study, we investigated the expression levels of IFN, IL17, and type 2 cytokines in the tissues of these CRS patients. Significantly higher levels of IL4, IL5, and IL13 cytokines were observed in nasal polyp tissue from CRSwNP; while IFN and IL17 cytokines were more elevated in the uncinate tissue ( Figure 1A ). This suggest that type 2 inflammation may downregulate ACE2 and TEMPRSS2 expression.

To further investigate that, we used another data set of nasal polyp tissue from eosinophilic and non-eosinophilic CRSwNPs patients. Interestingly, a reduction in expression of ACE2 and TMPRSS2 receptors was observed in the eosinophilic nasal polyp tissue compared to healthy controls. In contrast, an increase was observed in both receptors in non-eosinophilic nasal polyp tissue compared to controls ( Figure 1B ). In addition, ACE2 and TEMPRSS2 expression in non-eosinophilic polyp tissue was significantly higher than in eosinophilic tissue (p=0.0378 and p=0.0039, respectively).

We then assessed the level of expression of IFN, IL17, and type 2 cytokines in eosinophilic versus non-eospinophilic CRSwNP tissue. As expected, eosinophilic polyp tissue had higher levels of expression in IL4, IL5, and IL13 cytokines, while higher levels of IFN and IL-17 were observed in noneosinophilic polyp tissue ( Figure 1B) . It was also observed that ACE2 has positive correlation with IFN (r=0.86, P=0.003), but not with IL17. ACE2 levels negatively correlated with IL5 (r=0.709, P=0.033) and IL13 (r=0.682, P=0.043) (Supplementary Figure 1A -C). No significant correlation was found between ACE2 and IL4 levels. Correlations of TMPRSS2 with the above mentioned cytokines were not significant. Next, the differential expression of ACE2 and TMPRESS2 was assessed in datasets of human ethmoid sinus cells treated independently with IL4, INF, or IL17 [6] . INF and IL17 increased the expression of both ACE2 and TMPRESS2 in these cells ( Supplementary Figure 2A and B) ; while IL4 suppressed their expression levels (Supplementary Figure 2C) . We further evaluated the expression of ACE2 and TMPRESS2 in a data set of IL13 treated human nasal epithelial cells isolated from nasal turbinates. IL13 also suppressed ACE2, but increased the expression of TMPRESS2 in these cells (Supplementary Figure 2D) , consistent with recent reports [6, 11] .

This data suggest that eosinophilic inflammation and the associated type 2 cytokines downregulate the expression of ACE2 in nasal tissue of CRS patients and thus may have a protective role against COVID-19 infection. In contrast, our data indicates that IFN increases ACE2 expression , in line with previously published data [10] . This may suggest that neutrophilic inflammation and the associated cytokines such as IFN and IL17 may upregulate COVID-19 receptors levels and thus facilitate COVID-19 infectivity. This data is also in line with our previous finding [5] indicating that patients with chronic obstructive pulmonary disease, characterized with neutrophilic inflammation, have significantly elevated ACE2 and TMPRSS2 lung tissue levels.

CRS patients are usually under prolonged exposure to corticosteroids which may regulate ACE2 expression. A recent report suggested that in asthmatic subjects, sputum ACE2 expression is significantly lower in patients who are on inhaled corticosteroids compared to those who are not on inhaled corticosteroids [12] . Here, we hypothesized that corticosteroids may regulate ACE2 gene expression. To test this hypothesis, we examined the expression of ACE2 in relation to steroid treatment in 2 publicly available data sets of gene expression data from airway epithelial cells as well as bronchial biopsies. Treatment with budesonide downregulated the expression of ACE2 in both bronchial epithelial cells ( Figure 1C ) and bronchial biopsies ( Figure 1D ). To confirm this observation, we treated healthy primary bronchial fibroblasts with dexamethasone (100nM for 24 hours) and analyzed ACE2 expression using RT-PCR. Dexamethasone significantly suppressed ACE2 expression in these cells ( Figure 1E ). This suggests that prolonged exposure to corticosteroids may suppresses This article is protected by copyright. All rights reserved. 6 ACE2 gene expression levels in nasal tissue of CRS patients. As the effect of corticosteroids on COVID19 patients is still highly debatable, more research is required to confirm our findings and observations. Moreover, a recent study by Jackson et al [13] , suggested that atopic asthmatics might be at lower risk of COVID-19-related illness as respiratory allergen exposure and type 2 cytokine, IL13, reduces ACE2 gene expression in both nasal and bronchial epithelial cells. The analysis from our study provide further evidence that type 2 or eosinophilic inflammation are biased more towards lowering ACE2 gene expression, in the context of CRS as well; and show for the first-time that steroids may have a protective effect by downregulating ACE2 expression in the nasal tissues of CRS.

The limitation of our study is that although the difference in type 2 cytokines observed between polyp and uncinated tissue may contribute to the regulation of these receptors, this does not exclude the possibility that the difference in receptors expression may reflect tissue differences rather than endotype.

In conclusion, as presented in Figure 2 , our data suggest that the type of inflammation underlying CRS, as well as corticosteroid treatment, may modulate ACE2 and TEMPRSS2 gene expression levels in the nasal polyps of CRSwNPs patients. , and uncinate tissues of healthy controls (n=3). The data presented shows lower expression of ACE2 and TMPRSS2 in the nasal polyps of CRSwNPs compared to normal uncinate tissue. Accordingly, gene expression levels of type 2 cytokines, IL4, IL-5, and IL13 were higher in the nasal polyps of CRSwNPs; while IL17 and IFN cytokines were more elevated in the uncinate tissue of CRSsNPs. (B) Gene expression of ACE2 and TMPRSS2 as well as proinflammatory cytokines in nasal polyps of eosinophilic (N=3), nasal polyps of non-eosinophilic CRSwNPs (n-3), and sphenoid sinus mucosa of healthy controls (n=3). The data presented shows decreased level of ACE2 and TMPRSS2 expression in the nasal polyps of eosinophilic compared to non-eosinophilic CRSwNPs. Additionally, it shows increased gene expression levels of type 2 cytokines, IL4, IL5, and IL13 in the nasal polyps of eosinophilic compared to non-eosinophilic CRSwNPs. In other hand, the gene expression levels of IL17 and IFN was higher in the nasal polyps of non-eosinophilic compared to eosinophilic CRSwNPs. (C) Gene expression level of ACE2 in the human bronchial airway epithelial BEAS-2B cells untreated or treated with budesonide (100 n) for 18 hours. The data shows that treatment with budesonide downregulated ACE2 expression levels in the treated compared to untreated BEAS-2B cells.

Gene expression level of ACE2 in bronchial biopsies from healthy male, non-smoker, non-allergic volunteers treated with placebo or budesonide (1600 g). The data shows that treatment with budesonide downregulated ACE2 expression levels in the budesonide treated compared to placebo treated subjects. (E) Gene expression level of ACE2 in the primary bronchial fibroblasts untreated or treated with dexamethasone (100 nM) for 24 hours. The data shows that treatment with dexamethasone downregulated ACE2 expression levels in the treated compared to untreated fibroblasts. 

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