key: cord-345058-jfzzngce
authors: Baughn, Linda B.; Sharma, Neeraj; Elhaik, Eran; Sekulic, Aleksandar; Bryce, Alan H.; Fonseca, Rafael
title: Targeting TMPRSS2 in SARS-CoV-2 infection
date: 2020-07-19
journal: Mayo Clin Proc
DOI: 10.1016/j.mayocp.2020.06.018
sha: 
doc_id: 345058
cord_uid: jfzzngce

Abstract SARS-coronavirus 2 (SARS-CoV-2) has rapidly caused a global pandemic associated with a novel respiratory infection now termed coronavirus disease-19 (COVID-19). ACE2 is necessary to facilitate SARS-CoV-2 infection, but due to its essential metabolic roles, it may be difficult to target it in therapies. TMPRSS2, which interacts with ACE2, may be a better candidate for targeted therapies. Using publicly-available expression data, we show that both ACE2 and TMPRSS2 are expressed in many host tissues, including lung. The highest expression of ACE2 is found in the testes, whereas the prostate display the highest expression of TMPRSS2. Given the increased severity of disease among older males with SARS-CoV-2 infection, we address the potential roles of ACE2 and TMPRSS2 in their contribution to the sex differences in disease severity. We show that expression levels of ACE2 and TMPRSS2 are overall comparable between males and females in multiple tissues suggesting that differences in the expression levels of TMPRSS2 and ACE2 in the lung and other non-sex-specific tissues may not explain the gender disparities in SARS CoV-2 severity. However, given their instrumental roles for SARS-CoV-2 infection and their pleiotropic expression, targeting the activity and expression levels of TMPRSS2 is a rational approach to treat COVID-19.

In December 2019, a novel member of the Coronaviridae family, SARS-coronavirus 2 (SARS-CoV-2), has spread rapidly throughout the world, probably from Wuhan, Hubei province, China resulting in an infectious respiratory infection termed coronavirus disease-19 (COVID-19) [1] [2] [3] . As of May 1, 2020, over 3.2 million individuals are confirmed to have contracted SARS-CoV-2, and nearly 250,000 have died from this global pandemic 4 . This global public health emergency requires a rapid and effective response to mitigate COVID-19-related morbidity and mortality 2 . Elucidating the mechanism of SARS-CoV-2 infection is necessary for the rational design of therapeutics, the use of novel or re-purposed therapeutics, effective vaccine development and understanding the clinical course of COVID-19.

Infections by the SARS coronaviruses, SARS-CoV and SARS-CoV-2, are dependent on host proteins angiotensin converting enzyme 2 (ACE2) receptor, which has been the subject of multiple investigations in the literature 5, 6 . However, viral entry requires not only binding to the ACE2 receptor but also priming of the virus's spike (S) protein by the transmembrane protease serine 2 (TMPRSS2) protease by cleavage of the S proteins at the S1/S2 and S2' sites. This cleavage step is necessary for the virus-host cell membrane fusion and cell entry 6, 7 . One striking observation made across most countries is the increased severity of disease among older males with SARS-CoV-2 infection 8 , although there might also be differences in infectivity. While early data of 140 SARS-CoV-2 patients from Wuhan, China demonstrated a nearly equal male to female ratio for infection 9 , subsequent studies of hospitalized and/or deceased patients have identified an increased male prevalence from about 55% to 85% in China 1, 10-16 , to 82%

in Italy 17 and about 63% in New York City of hospitalized patients 18 . Interestingly, this skewed sex difference in disease severity was also reported in studies of respiratory infections caused by related coronaviruses, SARS-CoV and MERS-CoV, suggesting a potentially shared underlying mechanism responsible for the high disease severity among infected males [19] [20] [21] [22] . Host susceptibility to severe COVID-19 disease is also associated with older age, hypertension, heart failure, chronic kidney disease, diabetes, obesity and the use of ACE inhibitors 22-24 . While gender can be associated with these additional comorbidities, male sex remains an independent variable associated with severe COVID-19 infection in multiple studies 22, 23 . It is possible that expression differences in ACE2 and TMPRSS2 explain the increased severity of disease among males. ACE2 is located at Xp22.2 within the non-pseudoautosomal portion of the X chromosome (15,561,033-15,602,069, GRCh38). It displays incomplete X-chromosome inactivation and shows a male-biased expression pattern in several tissues 25 .

TMPRSS2 is located at 21q22.3 within chromosome 21 (41,464,305-41,508,158, GRCh38) and its expression is modulated by androgen signaling via multiple androgen receptor elements upstream of the gene's transcriptional start site 26, 27 . In this study, we evaluate the expression levels of both ACE2 and TMPRSS2 in many host tissues, including lung. Given the increased severity of disease among older males with SARS-CoV-2 infection, we also address the potential roles of ACE2 and TMPRSS2 in their contribution to the sex differences in disease severity.

ACE2 and TMPRSS2 expression data were obtained directly from the Genotype-Tissue Expression (GTEx) Project (https://gtexportal.org) or from The Human Protein Atlas GTEx data (RNAseq based on RSEMv1.2.22 (v7). Expression from all tissue samples available were plotted using the box plots available from the GTExPortal website with plots shown as median and 25 th and 75 th percentiles and dots displayed as outliers if they are above or below 1.5 times the interquartile range. Comparison of ACE2 and TMPRSS2 expression was performed using GTEX tool multiGeneQueryPage.

From The Human Protein Atlas GTEx data (RNAseq based on RSEMv1.2.22 (v7), box plots were created using SPSS Software (IBM) and extreme outliers in each type of sample were identified (data not shown). If extreme outliers were present in the sample population, then the significance of the difference was calculated using the Mood's median test. For all other samples without extreme outliers, Kruskal Wallis was used to calculate the significance.

Allele frequency of two missense variants rs75603675 and rs12329760 in TMPRSS2 gene were calculated using the Geography of Genetic Variants Browser 28 .

Given the necessity of ACE2 and TMPRSS2 genes for SARS CoV-2 infection, we evaluated their expression in human tissues using data from the Genotype-Tissue Expression (GTEx) Project (https://gtexportal.org). Expression of ACE2 and TMPRSS2 was detectable in multiple tissues. Since the common symptoms of COVID-19 involve cough, sore throat, gastrointestinal issues, anosmia and dysgeusia 14, 29 , we analyzed the levels of ACE2 and TMPRSS2 in tissues associated with these sequelae. Both ACE2 and TMPRSS2 were expressed in the lung, with expression levels of TMPRSS2 higher than ACE2 ( Figure 1 ). Tissues associated with the gastrointestinal system with elevated ACE2 expression include small intestine (terminal ileum), colon (transverse), esophagus (mucosa), minor salivary gland and pancreas, esophagus, liver, colon (sigmoid) and stomach ( Figure 1A ). TMPRSS2 expression was also detected in gastrointestinal tissues, including stomach, colon (transverse), pancreas, small intestine (terminal ileum), minor salivary gland, esophagus (mucosa), liver and colon (sigmoid) ( Figure 1B ). Some patients with COVID-19 experience anosmia and dysgeusia, findings that may be a result of olfactory nerve abnormalities 29 . While GTEx does not have expression data specifically from olfactory tissues, the overall expression levels of both (Table 2) ; however, these findings were not considered significant. No significant differences in ACE2 or TMPRSS2 expression were observed in lung tissue (Table 2 ).

The rapid development of novel approaches or re-purposing of existing therapies is critical in mitigating the coronavirus pandemic. Due to the centrality of ACE2 and c.589G>A p.Val197Met) whose frequencies vary by ancestry and geography ( Figure 4 ).

Targeting TMPRSS2 protease activity through protease inhibitors or indirectly through ADT will require additional studies to determine the role of rs75603675 and rs12329760 on TMPRSS2 expression, protease activity and in response to protease inhibition.

Our observations are consistent with previous studies evaluating TMPRSS2 expression in lung tissue using GTEx data 45 , although, due to the limitation of the data, they do not consider age, menopausal status, or ancestry. While a very slight increase in TMPRSS2 expression was observed in the bronchial epithelial cells of males compared to females using GSE66499 microarray data; 45 , it is unclear whether this small increase in TMPRSS2 expression explains the increase in disease severity in males. Our results are also consistent with previous studies supporting no significant differences in ACE2 expression in lung tissue in association with age (>60 and <60 years), race (Caucasian and Asian) and sex using RNAseq, microarray and GTEx datasets 46 . Combined, these findings suggest that differences in the expression levels of TMPRSS2 and ACE2 in the lung and other non-sex-specific tissues likely do not explain the gender disparities in SARS CoV-2 severity. However, the observation of high ACE2 expression in the testes is an intriguing observation, and a recent study hypothesized that the testes could serve as a reservoir for SARS-CoV-2 30 . However, the expression of genes in the testes has been associated with "leaky expression" without evidence of a functional consequence of the expressed genes 47 . Therefore, the expression of ACE2 in the testes warrants additional investigation, including demonstration of SARS-CoV-2 in semen. Additional studies are also needed to evaluate whether androgens (or even estrogens) contribute to sex-associated disease severity independently of ACE2 or TMPRSS2 functions.

SARS-CoV-2 has rapidly caused a global pandemic. Both ACE2 and TMPRSS2 are expressed in many host tissues, but the expression levels of ACE2 and TMPRSS2 are overall comparable between males and females in multiple tissues. This suggests that expression differences of TMPRSS2 and ACE2 in the lung and other non-sex-specific tissues may not explain the gender disparities in SARS CoV-2 severity. However, targeting the activity and expression levels of TMPRSS2 is a rational approach to treat COVID-19 and should be explored further.

The Genotype-Tissue Expression (GTEx) Project was supported by the Common 

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

A novel coronavirus outbreak of global health concern

A Novel Coronavirus from Patients with Pneumonia in China

An interactive web-based dashboard to track COVID-19 in real time

A pneumonia outbreak associated with a new coronavirus of probable bat origin

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

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study