key: cord-0809553-t8odetzw
authors: Pinto, Bruna G G; Oliveira, Antonio E R; Singh, Youvika; Jimenez, Leandro; Gonçalves, Andre N A; Ogava, Rodrigo L T; Creighton, Rachel; Peron, Jean Pierre Schatzmann; Nakaya, Helder I
title: ACE2 Expression is Increased in the Lungs of Patients with Comorbidities Associated with Severe COVID-19
date: 2020-06-11
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa332
sha: 1a0bed2e948407806e9f64651b2c1d298c3b30a2
doc_id: 809553
cord_uid: t8odetzw

Patients who died from COVID-19 often had comorbidities, such as hypertension, diabetes, and chronic obstructive lung disease. Although angiotensin-converting enzyme 2 (ACE2) is crucial for SARS-CoV2 to bind and enter host cells, no study has systematically assessed the ACE2 expression in the lungs of patients with these diseases. Here, we analyzed over 700 lung transcriptome samples of patients with comorbidities associated with severe COVID-19 and found that ACE2 was highly expressed in these patients, compared to control individuals. This finding suggests that patients with such comorbidities may have higher chances of developing severe COVID-19. Correlation and network analyses revealed many potential regulators of ACE2 in the human lung, including genes related to histone modifications, such as HAT1, HDAC2, and KDM5B. Our systems biology approach offers a possible explanation for increase of COVID-19 severity in patients with certain comorbidities.

A c c e p t e d M a n u s c r i p t 3 

Recent studies of the epidemiological characteristics of COVID-19 have revealed that severe infection is more likely in people with an existing chronic medical condition. Two independent studies of infected populations in Wuhan, China found that approximately half the subjects infected with COVID-19 had an existing comorbidity (1, 2). In a study of 1099 patients across mainland China, 38.7% of patients with comorbidities progressed to severe infection (3) . 2020), and in a study of 52 inpatients in Wuhan, 67% of patients with comorbidities died (2) . The most common comorbidities reported in these studies were hypertension, diabetes, cerebrovascular disease, chronic obstructive lung disease, and coronary heart disease (1-3). Other comorbidities such as carcinoma, chronic kidney disease, chronic liver disease, digestive system disease, and nervous system disease have also been reported in patients with 2, 4) . A better understanding of the link between these conditions and COVID-19 infection is required to inform better treatment and prevention interventions.

The molecular mechanism responsible for the increased disease severity in patients with these comorbidities is not fully understood, but previous studies suggest a role for angiotensin-converting enzyme 2 (ACE2) (5) . ACE2 is a membrane protein required for SARS-CoV2 to bind and enter cells (6) (7) (8) . After binding, viral entry is facilitated by the activation of the viral spike glycoprotein and cleavage of the C-terminal segment of ACE2 by proteases like TMPRSS2 and FURIN that are readily expressed in lung tissue (9) (10) (11) . ACE2 is only moderately expressed in healthy lung tissue compared to the heart, kidneys, and testes (12) , but A c c e p t e d M a n u s c r i p t 4 staining of lung tissue sections from adults with pulmonary hypertension has revealed increased ACE2 protein in the endothelium of pulmonary arteries, compared to healthy controls (13) . A comprehensive analysis of single-cell RNA-seq datasets revealed that ACE2 was co-expressed with TMPRSS2 within ileal absorptive enterocytes, nasal goblet secretory cells, and lung type II pneumocytes (14) . ACE2 upregulation has also been observed in animal models of liver fibrosis (15). However, the reason for this upregulation remains unclear, and a link to other COVID-19 comorbidities has not been determined.

Here, we showed that the expression of the gene encoding the ACE2 receptor in lung tissue is upregulated by diseases representing comorbidities along with COVID-19. We also used systems biology approaches including co-expression analysis, meta-analysis, and network analysis to determine a potential cause of the ACE2 upregulation. From this analysis, we found that ACE2 expression could be regulated by enzymes that modify histones, including KDM5B. This identification of a common molecular mechanism of increased COVID-19 severity in patients with diverse comorbidities could direct the development of interventions to reduce the infection risk and disease severity in this population.

A c c e p t e d M a n u s c r i p t 5

Relevant scientific literature related to key COVID-19 morbidities was retrieved from PubMed on March 16, 2020 using the query terms "pulmonary hypertension," "chronic obstructive pulmonary disease," "hypertension," "smoking," "pulmonary fibrosis," and "asthma". For terms returning more than 100,000 papers ("hypertension," "smoking," and "asthma"), only the most recent 100,000 papers were analyzed. Abstracts were annotated to identify all genes, diseases, and species appearing in the title or abstract using the PubTator Central API (16) . This open source tool uses TaggerOne for disease annotations, GNormPlus for gene annotations, and SR4GN for species annotations (16) . The data were filtered to retain only papers containing a human species annotation. Annotation of the abstract text identified 6 relevant disease MeSH terms: "Autoimmune Diseases," "Cardiovascular Diseases," "Familial Primary Pulmonary Hypertension," "Hypertension," "Hypertension, Pulmonary," and "Renal Insufficiency, Chronic", which were used in Figure 1 . Next, every possible combination of gene and disease annotation within the title and abstract of each paper was generated. Only genedisease associations supported by at least four documents, and those with a proximity less than or equal to the median sentence length of the paper section were retained.

Gene IDs were converted to gene symbols using the biomaRt R package (17, 18) , and disease IDs were converted to disease MeSH terms using the Entrez Programming Utilities to query the Entrez database provided by the National Center for Biotechnology Information. The data was then further filtered to retain disease A c c e p t e d M a n u s c r i p t 6 MeSH terms relevant to reported clinical COVID-19 comorbidities (3). Redundant terms were collapsed using fuzzy string matching. The final gene-disease data set was used to generate a network utilizing Gephi software where the nodes were genes and diseases, and the edge weight was determined by the number of analyzed papers containing the gene-disease combination (19) .

We manually curated Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/) on March 16, 2020 to find lung transcriptome datasets related to "Pulmonary Arterial Hypertension" (PAH), "Chronic Obstructive Pulmonary Disease" (COPD), and "Smoking". Author-normalized expression values and metadata from these datasets were downloaded using the GEOquery package (20) . We performed differential expression analyses between patients with a disease and the control individuals (see Table S1 ) using the limma package (21) . The gene symbol for each probe was obtained from the annotation file (22) . Probes that matched the same gene symbol were collapsed by taking the one with the lowest pvalue. Meta-analysis was performed with the MetaVolcanoR package (23) by combining the p-values using Fisher's method. To adjusting for multiple comparisons, we calculated the False Discovery Rate (FDR) to identify the differentially expressed genes (FDR < 0.05). For enrichment analyses, we utilized the EnrichR tool (24) with the "GO Biological Process 2018" and "BioPlanet 2019" databases. We then selected pathways with a P-value adjusted for multiple comparisons lower than 0.05. The network was created in Cytoscape (25) . 

To identify the genes highly associated with key comorbidities of severe COVID-19

(1, 3), we mined all relevant scientific literature of these human diseases.

Specifically, over 8,000 abstracts were gathered from PubMed by querying titles and abstracts for the terms "pulmonary hypertension," "chronic obstructive pulmonary disease," "hypertension," "smoking," "pulmonary fibrosis," or "asthma" (Figure 1a ).

Several relevant terms, such as "autoimmune diseases" and "cardiovascular diseases" were excluded from the PubMed query because of the breadth of literature published in these fields. Annotation of the abstract text identified 6 relevant disease MeSH terms: "Autoimmune Diseases," "Cardiovascular Diseases," "Familial Primary Pulmonary Hypertension," "Hypertension," "Hypertension, Pulmonary," and "Renal Insufficiency, Chronic." (3) (Figure 1a) . Our text-mining analysis revealed 804 genes highly associated with one or more COVID-19 morbidities (Figure 1b) . Among those genes, 26 were associated with four or more diseases (Figure 1c) . Although ACE2 was known to be related to "Cardiovascular Diseases", "Familial Primary Pulmonary Hypertension", "Hypertension, Pulmonary", and "Hypertension", none of the articles containing this gene-disease association studied how ACE2 expression was altered in the lungs of patients with these diseases.

Based on the list of key comorbidities of severe COVID -19 (1, 3) , we searched for lung transcriptome datasets available at GEO repository. We identified seven lung transcriptome studies of patients with either Chronic Obstructive Pulmonary Disease (COPD) or Pulmonary Arterial Hypertension (PAH), as well as smoking volunteers, compared to individuals who were non-smoking volunteers, were downloaded and used in our meta-analysis (Table S1) . For each study, we performed differential expression analysis between patients and control individuals (Table S1) . By We then decided to investigate whether the gene encoding the ACE2 receptor was specifically up-regulated in the lungs of patients having one of these morbidities (Figure 3a) . In a lung RNA-seq dataset (Table S1) , we compared ACE2 expression between patients with COPD and subjects with normal spirometry (30) . Again, the expression of ACE2 was significantly up-regulated in the disease compared to controls (Figure 3b ). In fact, ACE2 was significantly up-regulated in 6 out of 7 lung transcriptome studies (Figure 3c (Table S1 ), combined the p-values using Fisher's method, and applied an FDR correction (Figure 4a ). This approach identified 544 and 173 genes with positive and negative correlation with ACE2, respectively (Figure 4a) . Several of these genes were related to histone modifications, such as HAT1, HDAC2, KDM5B, among others (Figure 4a ). Among the positively correlated genes, we found ADAM10 that regulates ACE2 cleavage in human airway epithelia (32) and TLR3 that plays a key role in the innate response to SARS-CoV or MERS-CoV infection (33) .

Pathway enrichment analysis revealed that several of the genes positively associated with ACE2 were regulated by KDM5B, and by specific histone acetylation (H3K27ac) and histone methylation (H3K4me1 and H3K4me3) (Figure 4b ). In fact, KMD5B demethylates lysine 4 of histone H3 (i.e. H3K4) and is involved in transcriptional regulation and DNA repair (34) . We then checked in the Roadmap Epigenomics Project database (26) to see whether ACE2 locus contained ChIP-seq information for these histone markers. In the human lung, peaks for H3K4me1 and H3K4me3, as well as H3K27ac, were identified in ACE2 locus (Figure 4c ), suggesting that ACE2 may be epigenetically regulated in the lung. 

We showed here that patients with co-morbidities which have very distinct mechanisms have increased expression of ACE2 in the lungs. Although our findings did not include COVID-19 infection data, we suggest that the higher expression of ACE2 in the lungs is associated with higher chances of developing the severe form of COVID19, by facilitating the SARS-CoV-2 entry into lung cells during the infection.

In fact, COVID19 patients classified as severe cases displayed higher viral loads in nasopharyngeal swab samples during the early stages of disease onset compared to mild patients (35) .

The current diabetes pandemic (36) could be worsening the SARS-CoV-2 pandemic by increasing the comorbidities associated with severe COVID-19. As we did not find lung transcriptome samples from patients with type 2 diabetes, we could not directly test whether ACE2 expression was increased in patients with diabetes, compared to healthy controls. However, our text-mining approach revealed that IL-6 and INS genes were associated with all the diseases we searched. The INS gene encodes the insulin hormone, and insulin is associated with the NAD-dependent histone deacetylase Sirtuin 1 (SIRT1) (37) . We found that SIRT1 was up-regulated in the lung of patients with severe COVID-19 comorbidities in 4 out 7 studies (data not A c c e p t e d M a n u s c r i p t 12 shown). Clarke et al (38) have demonstrated that, under conditions of cell energy stress, SIRT1 can epigenetically regulate ACE2. Others too have shown that nonsteroidal anti-inflammatory drugs may inhibit the SIRT1 deacetylase activity (39) , which in turn could impact ACE2 expression.

The "viral life cycle" pathway that was enriched with up-regulated genes in patients with severe COVID-19 comorbidities contains several genes other than ACE2 that can be potentially important for SARS-CoV-2 cell cycle and invasion/attachment. These include RAB1A gene, whose product promotes the replication of Vaccinia virus (40). Also, RAB1A is important for Herpes Simplex Virus 1 Secondary Envelopment (41) , and is required for assembly of classical swine fever virus particle (42) . It is possible that SARS-CoV-2 utilizes RAB1A as well.

The fact that ACE2 gene is located in the X chromosome, and the initial findings showing that older males with comorbidities are more likely to be have severe COVID-19 compared to females (1), indicate that ACE2 expression in the lung may be sex-biased. Although no significant sexual differences was found in the activity of ACE2 in mouse lung (43) , in rats, the levels of ACE2 were dramatically reduced with aging in both genders, but with significantly higher ACE2 expression in old female rats than male (44) .

Although the mechanisms by which ACE2 is up-regulated in patients with severe COVID-19 comorbidities were not addressed, our analysis may shed some light on the subject. Among the genes whose expression was positively correlated with ACE2, we detected genes associated with epigenetic regulation of gene transcription. For instance, HAT and HDAC modulate chromatin and DNA condensation by changing histone acetylation status, thus permitting gene A c c e p t e d M a n u s c r i p t 13 transcription. This could be happening in lung tissue, facilitating ACE2 expression, as observed during lung cancer and COPD.

KDM5B is associated with infection of hepatitis B virus (45) . In breast cancer cells, blockage of KDM5 triggers a robust interferon response that results in resistance to infection by DNA and RNA viruses (46) . This finding suggests that KDM5 demethylases are potential targets for preventing SARS-CoV-2 infection.

COVID-19 may kill between 5.6% and 15.2% of people infected with SARS-CoV-2 (47) . Drug treatments that lower this mortality rate may save many thousands of lives. Our systems biology approach offers putative gene targets for treating and preventing severe COVID-19 cases. (Table S1) The original studies are indicated and can be found in Table S1 . 

The author reports no conflicts of interest in this work.

Not applicable as we utilized publicly available data.

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

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

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

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

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirusinduced lung injury

Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2

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

Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry

Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase

Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

Upregulation of angiotensin-converting enzyme (ACE) 2 in hepatic fibrosis by ACE inhibitors

PubTator central: automated concept annotation for biomedical full text articles

Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt

BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis

the International AAAI Conference on Weblogs and Social Media

GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor

limma powers differential expression analyses for RNA-sequencing and microarray studies

Long noncoding RNAs are involved in multiple immunological pathways in response to vaccination

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

Cytoscape: a software environment for integrated models of biomolecular interaction networks

The Human Epigenome Browser at Washington University

Viral interactions with host cell Rab GTPases

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

The transcriptional landscape and mutational profile of lung adenocarcinoma

Comprehensive Analysis of Transcriptome Sequencing Data in the Lung Tissues of COPD Subjects

webCEMiTool: Co-expression Modular Analysis Made Easy

Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia

Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection

Small Molecule Inhibitors of KDM5 Histone Demethylases Increase the Radiosensitivity of Breast Cancer Cells Overexpressing JARID1B

Viral dynamics in mild and severe cases of COVID-19

The diabetes pandemic and associated infections: suggestions for clinical microbiology

Epigenetic regulation of angiotensinconverting enzyme 2 (ACE2) by SIRT1 under conditions of cell energy stress

Inhibition of SIRT1 deacetylase and p53 activation uncouples the antiinflammatory and chemopreventive actions of NSAIDs

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

Analysis of Rab GTPase-activating proteins indicates that Rab1a/b and Rab43 are important for herpes simplex virus 1 secondary envelopment

Rab1A is required for assembly of classical swine fever virus particle

Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17beta-oestradiol-dependent and sex chromosome-independent

Age-and gender-related difference of ACE2 expression in rat lung

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B

KDM5 histone demethylases repress immune response via suppression of STING

Real estimates of mortality following COVID-19 infection

We would like to thank Tiago Lubiana for his valuable inputs. 

A c c e p t e d M a n u s c r i p t Query: "pulmonary hypertension", "chronic obstructive pulmonary disease", "hypertension", "smoking", "pulmonary fibrosis", or "asthma"