key: cord-0719951-wjsshj7l
authors: Jimenez, L.; Codo, A. C.; Sampaio, V. S.; Oliveira, A. E. R.; Ferreira, L. K.; Davanzo, G. G.; Monteiro, L. B.; Virgilio-da-Silva, J. V.; Borba, M. G.; Souza, G. F.; Zini, N.; Gandolfi, F. A.; Murano, S. P.; Proenca-Modena, J. L.; Val, F. A.; Melo, G. C.; Monteiro, W. M.; Nogueira, M. L.; Lacerda, M. V.; Moraes-Vieira, P. M.; Nakaya, H. I.
title: The influence of pH on SARS-CoV-2 infection and COVID-19 severity
date: 2020-09-11
journal: nan
DOI: 10.1101/2020.09.10.20179135
sha: f17374461c45f90b8be4054eb88698df35791fa2
doc_id: 719951
cord_uid: wjsshj7l

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of human tissues by using the host receptor angiotensin-converting enzyme 2 (ACE2). Individuals with comorbidities associated with severe COVID-19 display higher levels of ACE2 in the lungs compared to those without comorbidities, and conditions such as cell stress, elevated glucose levels and hypoxia may also increase the expression of ACE2. Here we showed that patients with Barrett's esophagus (BE) have a higher expression of ACE2 in BE tissues compared to normal squamous esophagus, and that the lower pH associated with BE may drive this increase in expression. Human primary monocytes cultured in reduced pH displayed increased ACE2 expression and viral load upon SARS-CoV-2 infection. We also showed in two independent cohorts of COVID-19 patients that previous use of proton pump inhibitors is associated with 2- to 3-fold higher risk of death compared to those not using the drugs. Our work suggests that pH has a great influence on SARS-CoV-2 Infection and COVID-19 severity.

As of August 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected over 20 million people worldwide (World Health Organization). The new coronavirus disease 2019 caused by SARS-CoV-2 is characterized by a broad range of symptoms, from respiratory to neurological and digestive problems (1, 2) . Although a small fraction of patients develop highly lethal pneumonia, at least 20% of COVID-19 patients may display one or more gastrointestinal (GI) symptoms (1), such as diarrhea, vomiting, and abdominal pain (2, 3) .

SARS-CoV-2 tissue tropism can be directly linked to the diverse clinical manifestations of COVID-19. The receptor used by the virus to enter the cells is the angiotensin-converting enzyme 2 (ACE2), which is found in several tissues, including the GI epithelial cells and liver cells (4, 5) . SARS-CoV-2 was detected in biopsies of several tissues, including esophagus, stomach, duodenum and rectum, and endoscopy of hospitalized patients revealed esophageal bleeding with erosions and ulcers (2, 6) .

Higher levels of ACE2 in the tissues may explain in part some of the comorbidities associated with severe COVID-19. Recently, we showed that ACE2 was highly expressed in the lungs of people with pulmonary arterial hypertension and chronic obstructive diseases (7) . Since the expression of ACE2 changes under conditions of cell stress, elevated glucose levels and hypoxia (8, 9) , other comorbidities related to the GI tract can be associated with different forms of COVID-19.

Here we suggest that gastroesophageal reflux disease (GERD) and Barrett's esophagus (BE) may represent novel comorbidities associated with COVID-19. In the United States, it has been estimated that 5.6% of adults have BE, a disease where GERD damages the esophageal squamous mucosa (10) . Here we demonstrated that ACE2 is highly expressed in the esophagus of patients with BE and that the acid pH associated with this condition is a key inducer of ACE2 expression. Human primary monocytes cultured in reduced pH display increased expression of ACE2 and increased viral load upon SARS-CoV-2 infection. We also show that patients using proton pump inhibitors, which are recommended for GERD treatment, have a higher risk of developing severe COVID-19, observed by an increased risk of ICU admittance and death. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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We manually curated the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/) to find esophagus transcriptome datasets related to "Barrett's esophagus" and cell line transcriptome datasets related to "acidosis" and "pH reduction". Author-normalized expression values and metadata from these datasets were downloaded using the GEOquery package (11) . We performed differential expression analyses using the limma package (12) . The GEO study ID and the groups of samples compared are listed in Supplementary Table 1 . The MetaVolcanoR package (13) was used to combine the P values using the Fisher's method. To adjust for multiple comparisons, we calculated the false discovery rate (FDR) using the Benjamini-Hochberg procedure. For enrichment analyses, we utilized the EnrichR tool (14) and fgsea R package (15) with gene sets from the Gene Ontology Biological Process database. We then selected pathways with a P value adjusted for multiple comparisons lower than 0.10.

The single cell RNA-seq (scRNA-seq) data from esophagus, Barrett's esophagus, gastric and duodenum cells from patients with BE were acquired from Owen et al. 2018 (16) . Cells with less than 1,000 genes were excluded from analysis using Seurat v3 (17) . Raw UMI counts were log transformed and variable genes called on each dataset independently based on the VST method. The AddModuleScore function was used to remove batch effects between samples and based on C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, VPS29 genes. We assigned scores for S and G2/M cell cycle phases based on previously defined gene sets using the CellCycleScoring function. Scaled z-scores for each gene were calculated using the ScaleData function and regressed against the number of UMIs per cell, mitochondrial RNA content, S phase score, G2/M phase score, and housekeeping score. Scaled data was used as an input into PCA based on variable genes.

These PCA components were used to generate the UMAP reduction visualization. To identify the number of clusters, UMI log counts were used as input to SC3 (18) . Technical variation was tested using BEARscc (19) , which models technical noise from ERCC spike-in measurements. The clusters were then annotated based on genes previously characterized (16) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Buffy coats provided by the Hematology and Hemotherapy Center of the University of Campinas (SP-Campinas, Brazil) were used for PBMC isolation as described (9) . The study was approved by the Brazilian Committee for Ethics in Human Studies (CAAE: 31622420.0.0000.5404). Briefly, buffy coats were mixed and then diluted in Phosphate Buffer Saline (PBS) (1:1) and carefully to 50 mL tube containing Ficoll (Sigma-Aldrich) and

centrifuged. PBMCs were cultured in RPMI 1640 for 2-3h to allow cell adhesion. Next, cells were washed twice with PBS and adherent cells, enriched in monocytes, were further incubated until infection in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1%

Penicillin-Streptomycin (Pen-Strep) at 37ºC with 5% CO2. Monocytes were maintained in different pH levels (6, 6.5, and 7.4) during 24h and subsequently infected with SARS-CoV-2, as described below.

HIAE-02 SARS-CoV-2/SP02/human/2020/BRA (GenBank MT126808.1) virus was isolated as described (9) . Stocks of Sars-CoV-2 were prepared in the Vero cell line. The supernatant was harvested at 2-3 dpi. Viral titers were obtained by plaque assays on Vero cells.

Monocytes were infected with SARS-CoV-2 at MOI 0.1 under continuous agitation at 15 rpm for 1 h. Next, monocytes were washed twice and incubated in RPMI with 10% FBS and 1% Pen-Strep for 24h at 37°C with 5% CO2 for 24 hours.

Total RNA extraction was performed using TRIzol Reagent (Sigma-Aldrich). RNA concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific).

RNA was reverse-transcribed using GoScript™ Reverse Transcriptase cDNA synthesis kit following manufacturer's instructions. SARS-CoV-2 viral load was determined with primers targeting the N1 region and a standard curve was generated as described (20) . Viral load and gene expression were made using SYBR Green Supermix in BIO-RAD CFX394 Touch is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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We retrieved clinical data from two independent cohorts of 551 and 806 RT-qPCR confirmed COVID-19 patients aged 18 years or older that went to reference hospitals for COVID-19 in Manaus, Amazonas, Brazil (North region cohort) and in São José do Rio Preto city, São Paulo, Brazil (Southeast region cohort), respectively. They were followed for at least 28 days (North region cohort) or 120 days (Southeast region cohort) after recruitment. Adjusted hazard ratios and risk ratios with respective 95% confidence intervals (CI) were estimated for time to death and ICU admittance, respectively by Cox regression and log-binomial generalized linear model models. To adjust for confounders, ages higher than 60 years old and obesity, defined by both BMI and fat percentage, were used as covariables in the multivariable analyses. Wilcoxon Rank-Sum analysis was used to test differences in the days of hospitalization. A 2-tailed P < 0.05 was considered significant. The statistical analyses were carried out using Stata v. 13.0 (StataCorp LP, College Station, TX).

To evaluate whether people with BE may have higher chances of being infected with SARS-CoV-2 when compared to people without the disease, we performed a meta-analysis of 8 transcriptomic studies of BE ( Figure 1A , Table S1 ). A total of 304 and 256 genes displayed, respectively higher and lower expression BE compared to normal esophagus tissue in at least 7 of these studies ( Figure 1B ). ACE2 was among the genes consistently upregulated in the BE compared to normal esophagus ( Figure 1C ). While pathways related to keratinocyte differentiation and epidermis development were enriched with down-regulated genes, we found that bicarbonate transport and regulation of intracellular pH pathways were enriched with up-regulated genes ( Figure 1D ), suggesting that pH may influence ACE2 expression. In fact, when human coronary artery endothelial cells were treated with proton is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. . https://doi.org/10.1101/2020.09.10.20179135 doi: medRxiv preprint pump inhibitorsomeprazole or lansoprazolethe expression of ACE2 decreased in comparison to untreated cells ( Figure 1E ). Gene set enrichment analysis (GSEA) confirmed that Barrett's esophagus tissues have higher expression of genes related to pH alterations ( Figure 1F ). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. Barrett's samples have ACE2 expression above 0 ( Figure 2B ). However, among the cells expressing ACE2, higher levels of the gene were found in gastric, Barrett's, and duodenum cells when compared to esophagus cells ( Figure 2C ). Using GSEA, we found that genes associated with regulation of cellular pH were enriched among the up-regulated genes in gastric, Barrett's and duodenum cells when compared to esophagus cells ( Figure 2D ). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted September 11, 2020. The adjusted P-value of the enrichment is displayed right next to the corresponding bar.

To further evaluate whether pH may influence the expression of ACE2, we analyzed publicly available transcriptomic studies of cells under experimentally-induced acidosis. Cells cultured at lower pH displayed higher expression levels of ACE2 when compared to those cultured under higher pH ( Figure 3A and B). We validated this finding with human primary monocytes cultured at pH 7.4, 6.5 and 6.0 under normoxia. ACE2 expression was significantly increased at pH 6.5 and 6.0 compared to pH 7.4 ( Figure 3C ). The reduction of pH alone also significantly increased SARS-CoV-2 infection of human monocytes ( Figure   3D ), indicating that pH plays a role in ACE2-mediated SARS-CoV-2 infection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. . https://doi.org/10.1101/2020.09.10.20179135 doi: medRxiv preprint control media or with 25mM lactic acid for 1, 4, and 12 hours (x-axis). Each point represents the mean log2 expression and the error bars the standard deviation of biological replicates.

were incubated in medium at 3 different pH (6, 6.5, 7.4) for 24h. Each boxplot represents the fold change ACE2 expression. D. Acid pH increases SARS-CoV-2 viral load. Human peripheral blood monocytes were incubated in medium at 3 different pH (6, 6.5, 7.4) for 24h.

The cells were infected with CoV-2 (MOI 0.1) for 1h under continuous agitation. The RNA viral load was measured by qPCR.

Proton pump inhibitors (PPI) decrease the amount of acid produced in the stomach and are often utilized to treat subjects with GERD symptoms or with certain stomach and esophagus problems (22) . The use of PPIs prior to COVID-19 may serve as a proxy for identifying subjects with tissue irritation and inflammation caused by stomach acid. In two independent cohorts of 551 and 806 RT-qPCR confirmed COVID-19 patients from North and Southeast regions of Brazil, respectively, we investigated the effects of gastrointestinal discomfort and COVID-19 severity. Survival curve analysis showed that people using PPIs had a 2-to 3-fold higher risk of death compared to those not using the drug ( Figure 4A ).

When controlling for potential confounders (i.e. age above 60 years old, diabetes, and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. Although evidence indicates that hypoxic conditions can increase the expression of ACE2 (8, 9) , the expression of neither SIRT1 nor HIF1A seem to be associated with Barrett's esophagus (Table S2) . We found that known regulators of ACE2 -HNF1B (23) and FOXA2 (24)were up-regulated in 6 out of 8 Barrett's esophagus transcriptomic studies (Table S2 ), suggesting that they may be involved with the pH-induced ACE2 expression in Barrett's esophagus. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. . https://doi.org/10.1101/2020.09.10.20179135 doi: medRxiv preprint Pulmonary damage, one of the main features of severe COVID-19, may lead to acute hypoxia and further respiratory acidosis. It is possible that the acidosis in the blood of some patients with severe COVID-19 (25) worsen the disease by increasing the levels of ACE2 and facilitating the entry of SARS-CoV-2 into human cells. Hypoxia itself may contribute to the regulation of ACE2 (9, 26) . In addition, elevated levels of the enzyme lactate dehydrogenase (which converts lactate from pyruvate) has been associated with worse outcomes in patients with COVID-19 (27) . We showed here that the previous use of PPIs is associated with unfavorable outcomes, such as the time of hospitalization, ICU admittance, and death. To the best of our knowledge, none of these associations were previously reported. Almario et al. (29) recently described that individuals using PPIs had higher chances for testing positive for COVID-19 when compared to those not using PPIs. Their hypothesis is that PPIs might increase the risk for COVID-19 by undermining the gastric barrier to SARS-CoV-2 and reducing the microbial diversity in the gut (29) . Rather, we believe that PPIs are important markers of hidden comorbidities that involve the damage caused by the excess stomach acid in GI tissues.

By going from disease (Barrett's esophagus) to molecule (ACE2) to cells (in vitro experiments) and back to clinical findings (COVID-19 patients), we showed that pH may have a great influence on SARS-CoV-2 infection and COVID-19 severity. Additional studies should be performed to not only confirm the clinical findings on a larger scale but also to assess the molecular mechanism related to pH-induced ACE2 expression. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. 

Tables S1 and S2

. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 11, 2020. . https://doi.org/10.1101/2020.09.10.20179135 doi: medRxiv preprint

Prevalence and Mortality of COVID-19 Patients With Gastrointestinal Symptoms: A Systematic Review and Meta-analysis

Diarrhea and altered inflammatory cytokine pattern in severe coronavirus disease 2019: Impact on disease course and in-hospital mortality

Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis

SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19)

Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection

Gastrointestinal symptoms of 95 cases with SARS-CoV-2 infection

ACE2 Expression Is Increased in the Lungs of Patients With Comorbidities Associated With Severe COVID-19

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

Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-1α/Glycolysis-Dependent Axis

Barrett's esophagus

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

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

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

Network enrichment analysis: extension of gene-set enrichment analysis to gene networks

Single cell RNA-seq reveals profound transcriptional similarity between Barrett's oesophagus and oesophageal submucosal glands

SC3: consensus clustering of single-cell RNA-seq data

BEARscc determines robustness of single-cell clusters using simulated technical replicates

Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues

The impact of proton pump inhibitors on the human gastrointestinal microbiome

Identification of target genes of the transcription factor HNF1beta and HNF1alpha in a human embryonic kidney cell line

Forkhead Box Transcription Factors of the FOXA Class Are Required for Basal Transcription of Angiotensin-Converting Enzyme 2

Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study

Role of HIF-1alpha in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells

Lactate dehydrogenase levels predict coronavirus disease 2019 (COVID-19) severity and mortality: A pooled analysis

Famotidine Use is Associated with Improved Clinical Outcomes in Hospitalized COVID-19 Patients: A Propensity Score Matched Retrospective Cohort Study

Increased Risk of COVID-19 Among Users of Proton Pump Inhibitors