key: cord-0846051-yqbhkzcy
authors: Jin, Ramon U.; Brown, Jeffrey W.; Li, Qing Kay; Bayguinov, Peter O.; Wang, Jean S.; Mills, Jason C.; Whelan, Sean P.J.; Fitzpatrick, James A.J.; Meeker, Alan K.
title: Tropism of SARS-CoV-2 for Barrett’s Esophagus may Increase Susceptibility to Developing COVID-19
date: 2021-01-20
journal: Gastroenterology
DOI: 10.1053/j.gastro.2021.01.024
sha: e1e86c10557b866a5cc2d57560e1138fba6ace81
doc_id: 846051
cord_uid: yqbhkzcy

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To test whether intestinal-type metaplasias of the proximal GI foregut might be receptive to SARS-CoV-2 infection, we determined ACE2 and TMPRSS2 expression in a series of esophageal and gastric biopsies harboring intestinal metaplasia (Table S1 ).

Unlike normal esophageal and gastric tissue, intestinal-type metaplasia of the esophagus and stomach strongly expressed both ACE2 and TMPRSS2 on epithelial cells with ACE2 predominantly localized to the apical plasma membranes ( Fig. 1A ; Table S1 ). The findings were corroborated by analyses of previous global surveys of RNA expression e.g. 5 showing that unlike normal esophageal and gastric tissue, intestinal-type metaplasias of these tissues aberrantly express ACE2 and TMPRSS2 mRNA.

To test whether Barrett's metaplasia is susceptible to infection by SARS-CoV-2, we chose an ex vivo model: organoids derived from patients with Barrett's esophagus.

We first demonstrate that organoids generated from biopsies of Barrett's esophagus faithfully reproduce the in vivo histopathologic characteristics (Fig. S1B) . Specifically, they form a columnar monolayer that is low in expression of the foregut transcription J o u r n a l P r e -p r o o f factor SOX2, high in expression of the mid and hindgut transcription factor CDX2, reactive to the histochemical goblet cell mucin stain Alcian blue and, importantly for this study, maintain the apical expression of ACE2 and TMPRSS2 ( Fig. 1B ; S1B,C).

We incubated three Barrett's esophageal organoid lines (WU014, WU002, and WU012) harboring unique intestinal characteristics ( S1D ). We observed in an initial experiment (Experiment 1, Fig. 1D ) that, while both patient organoid lines were efficiently infected, the line with increased intestinalization (i.e. lower SOX2 to CDX2 ratio, Fig. S1B ) was more extensively infected. We thus repeated the experiment (Experiment 2; Fig. 1D ) but added an additional line (WU0014) featuring even greater intestinal features (Fig. S1B ). In Experiment 2, we also reasoned virus in patients would encounter intact epithelium, so we scored only those infected cells within organoids above a threshold size. Together, these organoid results show that Barrett's esophagus may serve as a novel in vivo niche for SARS-CoV-2, and that viral entry may increase with increasing intestinalization (Fig.1D ).

Esophageal and gastric metaplasia are caused by chronic injury and predispose individuals to developing adenocarcinoma of the respective tissue 6, 7 ; however, the data presented here suggest that these metaplasias may also have other unanticipated consequences.

We show that intestinal-type metaplasia of the esophagus and stomach aberrantly express the proteins critical for binding and invasion by SARS-CoV-2: ACE2 and TMPRSS2. Recent studies have demonstrated that the low pH of the stomach as well as the luminal fluid of the small intestine and large intestine greatly attenuate the infectivity of SARS-CoV-2 that is present in swallowed oral secretions and refluxed respiratory sputum 3, 4 . Thus, the ectopic, proximal expression of ACE2 and TMPRSS2 may become salient as it means host receptors for SARS-CoV-2 are located in a region of the digestive tract where higher viable viral titers exist following ingestion. Moreover, individuals with known Barrett's esophagus are almost universally treated with proton pump inhibitors and those with gastric intestinal metaplasia almost invariably have loss of acid secreting parietal cells 6, 7 . Thus, the decreased ability for the stomach to produce acid either by pharmacologic inhibition or loss of acid-secreting cells may compound the risk due to increased gastric pH which could result in increased viral titers and prolonged exposure. In addition, epidemiologically, intestinal metaplasia increases with age and male sex, paralleling demographics of those most susceptible to COVID-19 8 .

interaction with apical ACE2 receptor. Two factors in our cultures increased apical exposure of cells to virus: a) we administered virus immediately after passaging (ie after dissociating organoids to single cells exposing all cell surfaces); and b) as new Barrett's J o u r n a l P r e -p r o o f organoids form, many wholly or partially orient "apical-out", ie with cell apices exposed to the culture medium (Fig. 1B) . Importantly, all three organoid lines had equivalent distribution of apical-out, basal-out, and hybrid orientations (data not shown) indicating orientation did not affect infectivity.

Overall, our data suggest that individuals with intestinal-type metaplasias of proximal 

Staining Human Barrett's esophagus organoids were pelleted and fixed in 10% formalin at 4C for one hour. Organoids were washed, moved to 70% ethanol, mounted in 3% agar and embedded in paraffin. Blocks were cut into 7-micron sections. For immunofluorescence, tissue microtome sections underwent a standard deparaffinization with xylene and rehydration protocol and were antigen retrieved in sodium citrate buffer (2.94 g sodium citrate, 500 uL Tween 20, pH 6.0) using a pressure cooker. Sections were blocked in 1% BSA, 0.3% Triton X-100 in PBS and incubated with primary antibodies: anti-ACE2 (1:100 dilution; catalog# Ab15348, Abcam), anti-TMPRSS2

(1:100 dilution; catalog# HPA035787, Sigma-Aldrich), and anti-β-Actin (1:200 dilution; catalog# A1978, Sigma-Aldrich). Fluorescent secondary antibodies were applied, and slides were mounted using ProLong Gold antifade reagent with DAPI (Molecular Probes). For J o u r n a l P r e -p r o o f immunohistochemistry, the same manual staining procedure for anti-ACE2 staining and auto staining procedure for TMPRSS2 was used as described above. 

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