key: cord-0793167-n30b1ki9
authors: Plaas, M.; Seppa, K.; Gaur, N.; Kasenomm, P.
title: Age- and airway disease related gene expression patterns of key SARS-CoV-2 entry factors in human nasal epithelia
date: 2021-05-25
journal: nan
DOI: 10.1101/2021.05.23.21257673
sha: 1a10befa0608331a9edc213d7148b451e445e8d4
doc_id: 793167
cord_uid: n30b1ki9

The global COVID-19 pandemic caused by SARS-CoV-2 predominantly affects the elderly. Differential expression of SARS-CoV-2 entry genes may underlie the variable susceptibility in different patient groups. Here, we examined the gene expression of key SARS-CoV-2 entry factors in mucosal biopsies to delineate the roles of age and existing chronic airway disease. A significant inverse correlation between ACE2 and age and a downregulation of NRP1 in patients with airway disease were noted. These results indicate that the interplay between various factors may influence susceptibility and the disease course.

The global COVID-19 pandemic caused by SARS-CoV-2 predominantly affects the elderly.

Differential expression of SARS-CoV-2 entry genes may underlie the variable susceptibility in different patient groups. Here, we examined the gene expression of key SARS-CoV-2 entry factors in mucosal biopsies to delineate the roles of age and existing chronic airway disease. A significant inverse correlation between ACE2 and age and a downregulation of NRP1 in patients with airway disease were noted. These results indicate that the interplay between various factors may influence susceptibility and the disease course.

ACE2 -Angiotensin-converting enzyme 2 TMPRSS2 -Transmembrane protease, serine 2 AGTR2 -Angiotensin II Receptor Type 2 NRP1 -Neuropilin 1 CRS -Chronic rhinosinusitis . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint

A novel coronavirus (SARS-CoV-2) was first identified in Wuhan, China in November 2019 and has since spread worldwide causing the current COVID-19 pandemic [1, 2] . The disease mostly affects the elderly [3] and as of January 2021, it had caused more than 1.9 million deaths globally [2] . SARS-CoV-2 enters the upper airways via the nasal epithelium and, like other coronaviruses, uses the ACE2 receptor for cellular entry. Other co-receptors, like TMPRSS2 also reportedly facilitate viral entry [4, 5] . While both ACE2 and TMPRSS2 are expressed in a broad range of tissues, co-expression only occurs in certain cellular populations, indicating that other receptors may mediate viral susceptibility [4, 6] . This is reinforced by clinical observations of a discrepancy between affected organs and ACE2 expression and the fact that not all cells with ACE2/TMPRSS2 co-expression are susceptible to the virus [7] .

AGTR2 (angiotensin II receptor type 2) may be a potential co-entry factor as it was shown to have a higher binding affinity to SARS-CoV-2 spike (S) protein than ACE2 in a 3D structural simulation [8] , although this is yet to be confirmed with in/ex-vivo experimental data. Neuropilin-1 (NRP1) has also been shown to potentiate SARS-CoV-2 entry and host-cell susceptibility [9, 10] . Following cleavage of the Spike (S) protein, a polybasic Arg-Arg-Ala-Arc C terminal sequence is generated which can then bind to the cell surface via the NRP1 receptor [9] . High NRP1 expression has been reported in pulmonary and olfactory cells, with the highest expression in endothelial cells. In addition, NRP1 may be an attractive entry factor for viruses owing to its high expression on epithelia in contact with external environment [10] .

Although additional factors, including age and the presence of chronic airway disease (including for instance asthma and chronic rhinosinusitis (CRS)) might influence susceptibility to their contribution is yet to be rigorously studied and results have been inconsistent. For instance, while Bunyavanich et al [11] , reported that ACE2 expression in nasal epithelia increases with age, similar studies observed no association between age and ACE2 expression [12] . Lower ACE2 expression levels have also been noted in patients with asthma [13] , while lower levels of TMPRSS2 were noted . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint 5 in patients with chronic rhinosinusitis (CRS) [14] . These observations might suggest altered susceptibility for COVID-19 in these patient groups.

At present, there are relatively few studies that have comprehensively examined the roles of age and airway disease on the expression of key SARS-CoV-2 entry factors. Therefore, the objectives of the present study were to 1) investigate the expression of key entry factors ACE2, TMPRSS2, NRP1 and AGTR2 and 2) delineate the influence of age and chronic airway disease within a single clinically characterized cohort.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Between-group comparisons for chronic airway disease (defined as having asthma and/or CRS in history) vs. no airway disease group were performed using either the independent samples t-test or the Mann-Whitney U test. A one-way ANOVA (with post-hoc Tukey test) was performed to assess the effect of age on target gene expression. Assumptions for sphericity and homogeneity of variances and co-variances were met unless stated otherwise. All outliers were retained for analyses. Summary data are reported as the mean with either 95% confidence intervals (CI) or the standard deviation (SD). All . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint analyses were performed on fold changes calculated using the 2−∆Ct method. Two-tailed statistical significance was set at p < 0.05.

While 49 samples were initially collected, the final cohort included 46 individuals; three were excluded owing to insufficient RNA amount. The cohort age ranged from 2-66 years. Further analyses were performed by stratifying the cohort based on either 1) age or 2) history of chronic airway disease. For the age sub-division, the cohort was divided into 3 age groups (Table 1) . Normality testing for all continuous variables showed that age and AGTR2 expression levels were non-normally distributed (p < 0.05). Sex distribution was equally split across all 3 age groups.

For the chronic airway disease sub-division, the cohort was divided into 2 sub-groups (Table 2) ; no significant differences in sex distribution or age were noted (p = 0.073; t = 1.273).

While no significant differences were noted for AGTR2, TMPRSS2 or NRP1 expression, ACE2 expression significantly differed between age sub-groups (p = 0.002; F (2, 43) = 7.514), as seen in Table 1 . Post-hoc analysis showed a significant decrease in ACE2 expression levels in both younger 

No statistically significant differences were noted for ACE2, TMPRSS2 or AGTR2 expression between airway disease and no-airway disease groups. Interestingly, NRP1 expression was significantly lower in the airway disease group (3.67, SD = 1.14) relative to the no-disease group (4.78, SD = 1.81) (p < 0.05), as seen in Figure 2 . However, after controlling for age as a co-variate, this result did not retain statistical significance (p = 0.08).

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 

Despite the changing demographics of COVID- 19 and increased infection rates in the young population, mortality rates are primarily driven by the elderly. For example, in the United States, while people over 50 years account for ~35% of the total case number, they represent 95% of total fatalities. In contrast, children accounted for only 0.2% of total deaths as of January 2021 [3] .

Age-related differences in the expression of SARS-CoV-2 entry factors, in particular ACE2, may underlie these distinctive susceptibility profiles. In contrast to Bunyavanich et al. [11] , we found that enzyme can bind to SARS-CoV-2, therefore competitively reducing the amount of virus that can potentially bind to the membrane-localized ACE2 receptor. The increased ACE2 expression noted in children in the present study may therefore also explain why they are typically asymptomatic and/or have a much milder disease course [16] [17] [18] [19] .

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint It is worth noting that conflicting results between the present and previous studies may also be due to methodological differences, in particular sample collection and processing e.g. cytology brush samples vs mucosal biopsies.

Of note, three subjects in our cohort eventually contracted COVID-19. Curiously, these three individuals also had the lowest values observed for ACE2 expression within this cohort, further suggesting that higher ACE2 expression may confer protection against COVID-19 (individual values indicated in Figure 1 and 2) . No other relevant expression trends for the other gene targets were noted . Naturally however, the limited sample size and statistical power preclude any quantitative analyses and we present this as a retrospective observation.

No age-associated differences in expression were noted for the other receptors investigated in this study, which is in keeping with previous reports. However, we found that the presence of chronic airway disease is associated with lower NRP1 expression. Disease-associated downregulation has also been reported for ACE2 and TMPRSS2 in the presence of asthma and CRS [13, 14] . While no significant disease-associated changes in TMPRSS2 expression were noted in the present study, this may be because of the aforementioned sampling and methodological differences. Although the downregulation of SARS-CoV-2 entry genes might suggest that these patients are less susceptible to disease, our clinical statistics show that prevalence rates within hospitalized COVID-19 patients who had either asthma, CRS or both, were similar to those within the general population.

With regard to other comorbidities, we observed that mean ACE2 expression was lower in individuals with hypertension; however, this effect did not remain significant after the inclusion of age as a covariate (data not shown). Although no other correlations were noted between gene expression and clinical indices, this may also be a result of limited sample size.

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The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint To summarize, while there is already a sizeable body of literature on SARS-CoV-2, its cellular entry mechanisms remain to be fully clarified; existing reports do not fully explain the clinical pattern of COVID-19. The present study uses a clinically characterized cohort to demonstrate how age, via its effect on putative entry receptors, can influence the disease course. Our data also reiterates the central role of ACE2 in mediating COVID-19 pathology and highlights its paradoxical role, wherein lower levels may potentially increase susceptibility.

An additional strength of the study is its use of full mucosal biopsies as these are more physiologically representative of the upper airway and its complex cellular milieu. Limitations include the restricted sample size, single centre design and the fact that analyses were restricted to transcriptomic expression only. We recommend that future studies confirm if the results observed here extend to the protein level, assess expression in confirmed COVID-19 patients, undertake a comprehensive evaluation of associated risk factors and further clarify the role of NRP1 in COVID-19 pathophysiology.

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The authors have no relevant conflicts of interest to declare . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. * this value did not retain statistical significance after controlling for age as a covariate.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.23.21257673 doi: medRxiv preprint

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