key: cord-0828885-74o1iobx
authors: Xu, K.; Shi, X.; Husted, C.; Hong, R.; Wang, Y.; Ning, B.; Sullivan, T.; Rieger-Christ, K. M.; Duan, F.; Marques, H.; Gower, A. C.; Xiao, X.; Liu, H.; Liu, G.; Duclos, G.; Spira, A.; Mazzilli, S. A.; Billatos, E.; Lenburg, M. E.; Campbell, J. D.; Beane, J.
title: Smoking modulates different secretory subpopulations expressing SARS-CoV-2 entry genes in the nasal and bronchial airways
date: 2021-04-04
journal: nan
DOI: 10.1101/2021.03.30.21254564
sha: f91e2511731dbdffefb45347e444b62410238dfa
doc_id: 828885
cord_uid: 74o1iobx

Coronavirus Disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells with help from the Viral Entry (VE) proteins ACE2, TMPRSS2, and CTSL. Proposed risk factors for viral infection, as well as the rate of disease progression, include age, sex, chronic obstructive pulmonary disease, cancer, and cigarette smoking. To investigate whether the proposed risk factors increase viral infection by modulation of the VE genes, we examined gene expression profiles of 796 nasal and 1,673 bronchial samples across four lung cancer screening cohorts containing individuals without COVID-19. Smoking was the only clinical factor reproducibly associated with the expression of any VE gene across cohorts. ACE2 expression was significantly up-regulated with smoking in the bronchus but significantly down-regulated with smoking in the nose. Furthermore, expression of individual VE genes were not correlated between paired nasal and bronchial samples from the same patients. Single-cell RNA-seq of nasal brushings revealed that an ACE2 gene module was detected in a variety of nasal secretory cells with the highest expression in the C15orf48+ secretory cells, while a TMPRSS2 gene module was most highly expressed in nasal keratinizing epithelial cells. In contrast, single-cell RNA-seq of bronchial brushings revealed that ACE2 and TMPRSS2 gene modules were most enriched in MUC5AC+ bronchial goblet cells. The CTSL gene module was highly expressed in immune populations of both nasal and bronchial brushings. Deconvolution of bulk RNA-seq showed that the proportion of MUC5AC+ goblet cells was increased in current smokers in both the nose and bronchus but proportions of nasal keratinizing epithelial cells, C15orf48+ secretory cells, and immune cells were not associated with smoking status. The complex association between VE gene expression and smoking in the nasal and bronchial epithelium revealed by our results may partially explain conflicting reports on the association between smoking and SARS-CoV-2 infection.

TMPRSS2 gene modules were most enriched in MUC5AC+ bronchial goblet cells. The CTSL gene module was highly expressed in immune populations of both nasal and bronchial brushings.

Deconvolution of bulk RNA-seq showed that the proportion of MUC5AC+ goblet cells was increased in current smokers in both the nose and bronchus but proportions of nasal keratinizing epithelial cells, C15orf48+ secretory cells, and immune cells were not associated with smoking status. The complex association between VE gene expression and smoking in the nasal and bronchial epithelium revealed by our results may partially explain conflicting reports on the association between smoking and SARS-CoV-2 infection.

As of February 14 th , 2021, over 108 million confirmed cases and 2.39 million deaths have been reported globally for COVID-19 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports). SARS-CoV-2 infects cells by utilizing host cell-surface proteins for viral entry (VE). VE proteins include angiotensin-converting enzyme 2 (ACE2), which provides a binding site for the virus; proteases such as transmembrane protease serine 2 (TMPRSS2) that can cleave the viral S glycoprotein; and cathepsin L (CTSL), which can also aid with S glycoprotein priming [1] [2] [3] [4] . Prior studies have attempted to establish associations between the expression of VE genes and clinical variables to identify factors that may influence infection incidence or COVID-19 severity.

However, some conflicting findings have been reported. Smith et al 14 19 reported that the expression level of ACE2 is elevated with smoking only in the small airway epithelium but not in the large airway epithelium.

Bunyavanich et al 17 reported that ACE2 expression levels were associated with age but did not control for other important clinical variables; however, Smith et al 14 found that ACE2 expression was equivalent between young and elderly individuals. Moreover, earlier studies with limited numbers of subjects led to the conclusion that race was associated with ACE2 expression 20 , which was not found by Smith et al in a larger dataset 14 . Lastly, lung airway expression of both ACE2 and TMPRSS2 was found to be significantly up-regulated in patients with chronic obstructive pulmonary disease (COPD) compared with healthy subjects or in smokers compared with nonsmokers 21 , but these associations were found without adjusting for other important factors such as age and sex. The goal of this study is to resolve these discrepancies by studying gene expression profiles in a large number of airway samples from multiple cohorts and to compare and contrast the biological processes associated with the expression of VE genes in the nasal and bronchial compartments. Table 1) . Study participants were current or former smokers undergoing bronchoscopy as part of either a diagnostic workup or a screening process for lung cancer.

Clinical data collected on samples included smoking status, sex, age, forced expiratory volume during the first second (FEV1) percent Predicted, and, in the AEGIS study, lung cancer status (Supplemental Tables 1). Notably, we observed different patterns of association with smoking between ACE2 and TMPRSS2 gene expression between the nasal and bronchial epithelium.

ACE2 was down-regulated in the nasal epithelium of current smokers in 1 out of 2 cohorts (p < 0.01) but strongly up-regulated in the bronchus in all four cohorts (p < 0.01; Figs. 1a and 1b; Supplemental Fig. 1a and 1b) . TMPRSS2 expression was not associated with smoking in the nose but its expression was up-regulated in current smokers in the bronchus in 3 out of 4 cohorts (p < 0.001). CTSL expression was down-regulated in current smokers in the nasal epithelium in 1 out of 2 cohorts (p < 0.01) and the bronchial epithelium in 3 out of 4 cohorts (p < 0.05). These genes were not strongly associated with other clinical variables (sex, age, FEV1% Predicted) across cohorts in either the nasal or bronchial samples in contrast to some previous studies 14, 17, 20, 21 . Adjusting for lung cancer diagnosis in the AEGIS cohort did not change these observed correlations, although positive lung cancer status negatively correlated with ACE2 expression in the nasal epithelium (p<0.05, Supplemental Fig. 1b) . A lack of correlation in the expression of each VE gene between the nose and bronchus was observed in paired samples from the same subjects (p > 0.05; Fig. 1c; Supplemental Fig. 1c, Supplemental Table 3 ),

suggesting that the biological processes that influence the expression of these genes may differ across airway sites.

To further explore the biological pathways associated with VE genes in each site of the airway, we developed co-expression networks using Weighted Gene Correlation Network Analysis (WGCNA) 26 . We identified 58 and 48 gene modules within the nasal and bronchial cohorts, respectively ( Fig. 2a and 2b, Supplemental Fig. 2 ; Supplemental Table 2 ). The consensus modules eigengenes containing the VE genes significantly correlated with their respective VE gene expression within each cohort (Supplemental Fig. 3a , Pearson correlation, p < 2.2E-16, average R = 0.584), suggesting that the eigengene can be used as a surrogate for each individual VE gene. The VE gene module eigengenes showed similar associations with the clinical covariates (Supplemental Fig. 3b ). While the nasal VE gene module eigengenes were not highly correlated, ACE2-and TMPRSS2-associated module eigengenes in the bronchus were highly correlated across all four datasets (Supplemental Figs. 2a and 2b) , suggesting that similar biological processes control these two gene modules in the bronchial compartment.

Comparing biological pathways enriched among VE gene modules between the nose and bronchus revealed ( Fig. 2c ; Supplemental Table 3 ) that genes of the nasal ACE2 module were enriched in the inflammatory response, interferon-alpha signaling, and interferon-gamma signaling pathways while the bronchial ACE2 gene module was enriched in genes associated with protein secretion and androgen response. Genes of the TMPRSS2 module in the nose were enriched in estrogen response and KRAS signaling pathways while the module in the bronchial epithelium was enriched in MTORC1 and TNFα-NFκB signaling pathways. The lack of shared biological pathways mirrors the small number of genes shared by the nasal and bronchial modules for ACE2 (n = 5) and TMPRSS2 (n = 12). In contrast, the nasal and bronchial CTSL modules shared 177 genes (Fisher's exact test, p = 2.23E-10) and were both enriched in genes related inflammatory response and TNF-alpha signaling pathways. Furthermore, in the bronchus, ACE2 and TMPRSS2-associated modules share enrichment in genes involved in the p53 pathway, adipogenesis, androgen response, MTORC1 signaling, and estrogen response (Supplemental . We also identified two bronchial goblet-like secretory populations: one characterized by high expression of CEACAM5 but not MUC5AC, previously described as peri-goblet cells 27 , and All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Figs. 3a-b, Supplemental   Fig. 4) . In the nose, we discovered two club-cell-like secretory cell clusters (STATH+ and C15orf48+), as well as a novel cell cluster of "keratinizing epithelial cells" expressing genes involved in cornification, epidermis development, and keratinocyte differentiation, such as SPRR3 and SPRR2A. Immune populations represent <1% and 26% of total cell populations in the nasal and bronchial epithelium, respectively.

We found low expression of VE gene in the single cell data, consistent with other reports (Supplemental Fig. 5 ) 28,29 , so we calculated VE gene module metagene scores in each cell to understand how biological processes associated with the VE genes in the bulk RNA-seq data are distributed across nasal and bronchial cell populations (Figs. 3c-e) . In the nose, the ACE2 module was moderately expressed across many cell types but was most highly expressed in C15orf48+ secretory cells and club cells. The TMPRSS2 module was predominantly expressed in the keratinizing epithelial cells, followed by C15orf48+ secretory cells. The expression of gene modules of ACE2 and TMPRSS2 is different from the gene expression pattern reported by Sungnak et al in that TMPRSS2 gene expression was limited to ACE2+ cells 30 . In the bronchus, both the ACE2 and TMPRSS2 modules were expressed at the highest levels in the goblet cell population, in agreement with previous findings 31 . The highest median metagene score for the CTSL module was in the neutrophils and macrophages in both the nose and bronchus; however, expression was also high in T cells in the nose and dendritic cells in the bronchus. We found very few cells co-expressing ACE2, CTSL, and TMPRSS2 at high levels -114 out of 34833 cells in the nose and no cells in the bronchus. On the other hand, we found that ACE2 and TMPRSS2 modules were more like to be highly co-expressed in nasal keratinizing epithelial cells (odds ratio = 7.56), nasal C15orf48+ secretory cells (N = 518, odds ratio = 7.36), and bronchial goblet cells (odds ratio = 16.80) (N = 806, Supplemental Table 6 , FDR q < 0.001). Thus, ACE2 and TMPRSS2 were found to be expressed in different nasal and bronchial cell populations, which All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 April 4, 2021. ; https://doi.org/10.1101/2021.03.30.21254564 doi: medRxiv preprint may be influenced by smoking in ways that lead to their divergent correlation with smoking in the two airway compartments.

To investigate how smoking modulates different cell populations, we computationally deconvolved cell population proportions in the bulk RNA-seq data using gene markers identified from the single-cell data (Supplemental Table 7 While goblet cell proportions were significantly higher in smokers in both the nose and bronchus, the ACE2 module was highly expressed only in bronchial, not nasal goblet cells. These results may explain the lack of association of ACE2 expression with smoking in the bulk RNA-seq data.

Additionally, in the AEGIS nasal data (Supplemental Fig. 6 ) the fraction of keratinizing epithelial cells is increased and that of STATH+ secretory cells is decreased in current smokers, suggesting that shifts in these populations may be partially responsible for the differential correlation with smoking between ACE2 and TMPRSS2 in the nose. With respect to CTSL, both nasal brushings showed a significant decrease in the proportion of the neutrophil/macrophage population with respect to smoking. While this may partially explain the down-regulation of CTSL in smokers in the nasal bulk RNA-seq data, the overall proportions of these populations estimated in the bronchial data by the deconvolution were close to zero for most samples, which could have prevented us from confirming the decrease of this population with smoking in the bronchus.

The current study investigated the relationship between the expression of genes encoding proteins important for SARS-CoV-2 entry (ACE2, CTSL, and TMPRSS2) and clinical factors including age, sex, COPD, and smoking in both the nose and bronchus. In our datasets, only smoking status showed a consistent association with the expression of VE genes across cohorts All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 April 4, 2021. ; https://doi.org/10.1101/2021.03.30.21254564 doi: medRxiv preprint within each airway compartment. In the bronchus, current smoking status was associated with higher ACE2 and TMPRSS2 expression; however, in the nose, current smoking status was inversely correlated with ACE2 expression and was not associated with TMPRSS2 expression as in prior studies [14] [15] [16] 19 . The cohorts used in this study contain subjects at high risk for developing lung cancer and are thus composed of older subjects (mean age = 63, SD = 8); therefore, we did not find an association between nasal ACE expression and age as previously reported by Bunyavanich et al 17 in a study comparing children and adults. We also did not find sex to be associated with ACE2 expression as was reported by another study with a lower sample size 18 and could not confidently assess differences in expression profiles between racial subgroups as our cohorts are predominantly (> 70%) composed of White subjects. Our analyses did not find significant VE gene expression correlations between paired nasal and bronchial samples suggesting that there are differences in biological pathways or cell types between the airway compartments.

Our WGCNA analysis demonstrated that genes co-expressed with ACE2 and TMPRSS2 in the bulk RNA-seq data were more highly correlated to one another in the bronchus than in the nose and that pathway enrichment was different for these modules in the upper and lower airways.

Similarly, scRNA-seq data showed that ACE2 and TMPRSS2 modules were co-expressed in bronchial goblet cells but had distinct patterns of expression across nasal cell populations. Our high-resolution nasal scRNA-seq dataset containing over 30,000 cells allowed us to differentiate between secretory cell populations to show that C15orf48+ and keratinizing epithelial cells had the highest expression of ACE2 and TMPRSS2 modules, respectively. In contrast, we found that bronchial goblet cells had the highest expression of both ACE2 and TMPRSS2 modules and only the goblet cell population was increased in smokers in our deconvolution analysis. Of note, ACE2

and TMPRSS2 modules were also co-expressed highly within the peri-goblet cells in the bronchus, which was also observed by Lukassen et al 29 .

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Interestingly, the nasal and bronchial CTSL modules showed enrichment of immune-associated biological programs and were highly expressed in similar immune cell populations. Despite these similarities, CTSL gene or module expression was not correlated between paired bronchial and nasal samples potentially due to the low abundance of immune populations in the scRNA-seq and bulk RNA-seq data.

Our study leveraged bulk and single-cell gene expression profiles from large cohorts to show that current cigarette smoking status is consistently associated, in a site-specific manner, with the expression of genes required for SARS-CoV-2 entry in the nose and bronchus. Future work investigating other putative viral entry genes such as FURIN and ATRNL1 29-31 may build upon these findings to enhance our understanding of the effect of smoking on SARS-CoV-2 infection.

The results of our study of the expression of ACE2 and TMPRSS2 suggest that smoking is unlikely to impact the likelihood of SARS-CoV-2 infection in the upper airways, but that it may play a significant role in COVID-19 disease progression and severity.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 April 4, 2021. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 April 4, 2021. (c) Expression of VE genes is not significantly correlated (p > 0.05, Pearson correlation) between paired DECAMP nasal (x-axis) and bronchial (y-axis) epithelial samples (N = 123) . Residual counts adjusted for sex, age, percentage of predicted FEV1, batch, and mTIN were used for the comparison. The blue line is the line of best fit and the gray shading represents the 95% confidence level interval for predictions from the linear model. (a) WGCNA was used to identify consensus co-expression modules in nasal and bronchial samples from the DECAMP cohort (n=58 and 48 modules, respectively). A heatmap of the correlation of module eigengenes computed from each module demonstrates that ACE2 and TMPRSS2 modules were more highly correlated with each other in the bronchus than in the nose. The CTSL module was not correlated with ACE2 or TMPRSS2 modules in either the nose or bronchus. (b) Scatterplots comparing the overrepresentation of MSigDB Hallmark pathway gene sets in each VE module in the nose and bronchus. The -log10(FDR q) values denoting the degree of overrepresentation of each gene set in each module in the nose and bronchus are shown on the x-and y-axes, respectively. The overrepresentation of gene sets in the ACE2 and TMPRSS2 modules is largely discordant between the nose and bronchus, whereas several immune-related pathways are overrepresented in the CTSL module in both sites. (c) VE module eigengenes were not significantly correlated (Pearson p > 0.05) between paired nasal (x-axis) and bronchial (y-axis) samples in DECAMP (N = 114) . The blue line is the line of best fit and the gray shading represents the 95% confidence level interval for predictions from the linear model. (which was not certified by peer review) 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 April 4, 2021. ;  gene modules (ACE2, top; CTSL, middle; TMPRSS2 bottom) across different cell types in the nasal (left) and bronchial (right) epithelium. The cells are colored gray for low expression and red for high expression of metagene scores of each VE gene module. Immune cells with high expression of the CTSL modules were encircled in blue (d-e). Violin plot showing the metagene score for each VE gene module across the cell types in (d) nasal and (e) bronchial epithelium. For each violin plot, metagene expression is designated as elevated (light brown) or highly elevated (pink) if it is greater than one or two standard deviation above the mean metagene score, respectively. (f) UMAP projections showing cells greater than 1 standard deviation above the mean for both ACE2 and TMPRSS2 modules (i.e., double positive). Double positive cells were overrepresented in nasal secretory (C15orf48+) and keratinizing epithelial cells (left) and bronchial goblet cells (right). (a-b) Top: boxplots of cell type proportions estimated by AutoGeneS in bulk RNA-seq data from nasal (N = 281) and bronchial brushings (N = 355) obtained from current and former smokers in the DECAMP cohort. Significant cell proportion differences between current and former smokers were determined by Wilcoxon test (* indicates FDR q < 0.05). Bottom: the heatmap displays the average VE module score for each cell population from the single-cell RNA-seq data (first three rows), as well as the proportion of each cell type that is ACE2+/TMPRSS2+ (i.e., the expression of both genes is one standard deviation above the average). Cell types that express both ACE2 and TMPRSS2 modules are in red. While goblet cell proportions were significantly higher in smokers in both the nose and bronchus, the ACE2 module was only highly expressed in goblet cells from the bronchus, which may explain the lack of association of ACE2 expression with smoking in the bulk RNA-seq data. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 April 4, 2021. ; https://doi.org/10.1101/2021.03.30.21254564 doi: medRxiv preprint

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus

Evolving epidemiology and transmission dynamics of coronavirus disease 2019 outside Hubei province, China: a descriptive and modelling study

Evolving Epidemiology and Impact of Non-pharmaceutical Interventions on the Outbreak of Coronavirus Disease

Clinical Characteristics of Coronavirus Disease 2019 in China

Comorbidity and its impact on 1590 patients with Covid-19 in China: A Nationwide Analysis

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity

COVID-19 prevalence and mortality in patients with cancer and the effect of primary tumour subtype and patient demographics: a prospective cohort study

COVID-19 and smoking: A systematic review of the evidence

Smoking Is Associated With COVID-19 Progression: A Meta-analysis

COVID-19 and Smoking

Active smoking is not associated with severity of coronavirus disease 2019 (COVID-19)

Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A Potential Adhesion Site for Novel Coronavirus SARS-CoV-2 (Covid-19)

Nasal Gene Expression of Angiotensin-Converting Enzyme 2 in Children and Adults

Expression of the SARS-CoV-2 ACE2 Receptor in the Human Airway Epithelium

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity

Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways. medRxiv (2020)

Single-Cell RNA Expression Profiling of ACE2, the Receptor of SARS-CoV-2. Am J Respir Crit Care Med

Airways Expression of SARS-CoV-2 Receptor, ACE2, and TMPRSS2

Is Lower in Children Than Adults and Increases with Smoking and COPD

Detection of early lung cancer among military personnel (DECAMP) consortium: study protocols

A Bronchial Genomic Classifier for the Diagnostic Evaluation of Lung Cancer

Airway gene expression in chronic obstructive pulmonary disease

Molecular subtyping reveals immune alterations associated with progression of bronchial premalignant lesions

WGCNA: an R package for weighted correlation network Authors contributions

contributed equally to this work (co-senior authors). K. X. is the guarantor of this paper and takes full responsibility for the integrity of the work as a whole

edited and revised manuscript; all the others approved the final version of manuscript

The DECAMP study is supported by funds from the Department of Defense

Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The IDA study is supported by a Department of Defense Idea Development Award W81XWH-14-1-0234 (J. B.). Part of the work on method development was funded by the National Library of Medicine (NLM) RO1LM013154-01 (JDC)

Financial/nonfinancial disclosures: The authors have reported the following

Inc, and has received personal fees from Veracyte Inc outside the submitted work. M. E. L. reports grants and personal fees from Johnson and Johnson and is a shareholder in Metera Pharmaceuticals; he also has a patent US PTO 9

We would like to thank Yuriy Alekseyev, Ashley LeClerc, and Kangning Zhang of the Single Cell Sequencing Core at Boston University School of Medicine for their help with data generation.