key: cord-0996685-nsm1ku3e
authors: Tao, Y.; Yang, R.; Wen, C.; Fan, J.; Ma, J.; He, Q.; Zhao, Z.; Song, X.; Chen, H.; Shi, G.; Yin, M.; Fang, N.; Zhang, H.; Mo, X.
title: SARS-CoV-2 entry related genes are comparably expressed in children's lung as adults
date: 2020-05-26
journal: nan
DOI: 10.1101/2020.05.25.20110890
sha: c120419126e0af097bfd2d5e764c739d6230cc1e
doc_id: 996685
cord_uid: nsm1ku3e

To explore whether the expression levels of viral-entry associated genes might contribute to the milder symptoms in children, we analysed the expression of these genes in both children and adults' lung tissues by single cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC). Both scRNA-seq and IHC analyses showed comparable expression of the key genes for SARS-CoV-2 entry in children and adults, including ACE2, TMPRSS2 and FURIN, suggesting that instead of lower virus intrusion rate, other factors are more likely to be the key reasons for the milder symptoms of SARS-CoV-2 infected children.

Available data, although limited, suggest that the SARS-CoV-2 infection in children is relatively rare and less severe compared to that in adults 4, 5 . However, a recent study pointed out that children are just as likely to be infected by SARS-CoV-2 as adults when exposed to similar environment. Among all children under 10 who had close contact with confirmed cases, the infection rate was 7.4%, which is comparable to that of the whole population average (7.9%) 6 . The latest guidelines also state that all individuals, including children, are generally susceptible to SARS-CoV-2 7 . In the meantime, the low incidence of critical illness in children has been reported repeatedly and accepted widely while the underlying mechanism remains to be elucidated.

Currently, there are two major speculations regarding why children display milder symptoms during the infection: relatively lower expression of viral-entry associated genes compared to adults and immune-related factors 8 . Angiotensin converting enzyme 2 (ACE2), a type I membrane protein expressed in various types of organs, has been proven to be the key receptor of SARS-CoV-2 for cellular entry via its interaction with the spike (S) protein 9 of the virus, which is cleaved by transmembrane protease serine 2 (TMPRSS2) 10 . But it is worth noting that . CC-BY-NC-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 26, 2020. . https://doi.org/10.1101/2020.05.25.20110890 doi: medRxiv preprint simultaneously blocking the activity of TMPRSS2 and the cysteine proteases CATHEPSIN B/L cannot completely inhibit the entry of SARS-CoV-2 in vitro, suggesting the possible involvement of additional proteases in the priming of SARS-CoV-2 10 . A FURIN cleavage site has been identified at S1/S2 boundary in the SARS-CoV-2 S protein 11 , which is suggested to be potentially cleaved by FURIN 12 as an additional possible mechanism for the priming of the virus. Therefore, in the present study, the expression levels of viral-entry associated genes (i.e., ACE2, TMPRSS2

and FURIN) in both children and adults' lung tissues were analyzed by single-cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC) to explore whether the expression levels of these genes might contribute to the milder symptoms in children.

Non-affected lung tissues from four children with congenital heart disease combined with lung diseases requiring lobectomies were collected ( Similarly, the AT2, AT1 and club cells were the major cell types with relatively high expression of TMPRSS2 in both adults and children. The expression pattern of FURIN, however, differed from ACE2 and TMPRSS2, with EC cells and monocyte being the major expressing cell types in adults but AT2 and club cells in children (Fig. 1D ). In general, no significant changes were observed in the expression levels of ACE2, TMPRSS2 and FURIN between adults and children, although the percentage of cells expressing TMPRSS2 and FURIN were higher in adults than children (Fig.   1C ).

To further verify the expression of ACE2, TMPRSS2 and FURIN at the protein level, IHC was performed in the lung biopsy specimens from children and adults in two independent cohorts (Supplementary Table S2 ). Consistent with the scRNA-seq analysis, the overall expression levels of ACE2 in children and adults were comparable in both cohorts, with a very small portion of the lung cells expressing ACE2. However, TMPRSS2 and FURIN both showed higher expression . CC-BY-NC-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 26, 2020. . https://doi.org/10.1101/2020.05.25.20110890 doi: medRxiv preprint level in children compared to adults, although the former did not reach statistical significance (Fig.   2, Supplementary Fig. S1 ). The underlying reasons for the partial discrepancy between the results from scRNA-seq and IHC could be the complicated processes downstream of transcription including post-transcriptional, translational and degradation regulations 16 . Meanwhile, the semi-quantitative characteristics of IHC and its limitation in identifying cell types may also contribute to such discrepancies, which further emphasize the necessity to combine these methods for an impartial interpretation of the results. Besides, in accordance with the previous report, the ACE2 is enriched in the heart, kidney and testis, and is also broadly distributed in the lung, liver, intestine and brain 23 . It is well accepted that the lung is the primary target organ of SARS-CoV-2 infection 24, 25 . An analysis of the scRNA-seq data obtained from 43,134 human lung cells showed that ACE2 was expressed in 0.64 % of all cells in lungs and 83% of the ACE2 expression was found in AT2 cells, indicating that AT2 is the primary target of SARS-CoV-2 in the lungs 26 , which is consistent with our results. Although children of all ages are susceptible to COVID-19, accumulating clinical data indicated better clinical outcomes in children compared to adults. However, the expression and the activity of ACE2 during the development of children and teenagers are largely unclear 24, 27 . Data from animal models remain controversial so far. ACE2 expression in the lungs was reported to decrease during aging in a study performed in aged rats 28 and another study using sheep as model revealed significantly lower expression of ACE2 in the neonatal sheep comparing to that in the adult ones 29 .

. CC-BY-NC-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 26, 2020. . https://doi.org/10.1101/2020.05. 25.20110890 doi: medRxiv preprint The present study showed comparable expression levels of ACE2, as well as other factors involved in SARS-CoV-2 cellular entry, in children and adults, suggesting that the expression level of viral-entry associated genes is unlikely to be the key reason for milder symptoms in children. Instead, other factors such as unique features in children immunity may play a more important role. Children have a distinct immune profile during infections because their immune system is still under development. It has been shown that compared to adults, decreased mononuclear and polymorphonuclear chemotaxis is found in children and such decrease remains significant until the age of 16 30 . In the meantime, frequent exposure to a plethora of other pathogens, such as respiratory syncytial virus (RSV), would repeatedly challenge the innate immunity including the age dependent maturation of the interferon response [31] [32] [33] [34] , possibly leading to an enhanced innate immune function which is one feature of trained immunity 8, 35 . Taken together, unique features in children immunity could be a possible explanation for the milder symptoms and lower mortality rate in children SARS-CoV-2 infections.

In summary, the present study, for the first time, described the features of children's lungs by scRNA-seq. Both scRNA-seq and IHC analyses showed comparable, if not higher, expression of the key genes for SARS-Cov-2 entry, including ACE2, TMPRSS2 and FURIN, suggesting that instead of lower virus intrusion rate, other factors are more likely to be the key reasons for the milder symptoms of SARS-CoV-2 infected children, which awaits further investigation. 

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. CC-BY-NC-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 26, 2020. . CC-BY-NC-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 26, 2020. . https://doi.org/10.1101/2020.05.25.20110890 doi: medRxiv preprint . CC-BY-NC-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 26, 2020. . https://doi.org/10.1101/2020.05.25.20110890 doi: medRxiv preprint