Epithelial regeneration after gastric ulceration causes ...

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alterations. Eitaro Aihara, Andrea L. Matthis, Rebekah A. Karns, Kristen A. Engevik, Peihua. Jiang, Jiang Wang, Bruce R. Yacyshyn, Marshall H. Montrose. PII:.
Accepted Manuscript Epithelial regeneration after gastric ulceration causes prolonged cell type alterations Eitaro Aihara, Andrea L. Matthis, Rebekah A. Karns, Kristen A. Engevik, Peihua Jiang, Jiang Wang, Bruce R. Yacyshyn, Marshall H. Montrose

PII: DOI: Reference:

S2352-345X(16)30045-5 10.1016/j.jcmgh.2016.05.005 JCMGH 137

To appear in: Cellular and Molecular Gastroenterology and Hepatology Accepted Date: 6 May 2016 Please cite this article as: Aihara E, Matthis AL, Karns RA, Engevik KA, Jiang P, Wang J, Yacyshyn BR, Montrose MH, Epithelial regeneration after gastric ulceration causes prolonged cell type alterations, Cellular and Molecular Gastroenterology and Hepatology (2016), doi: 10.1016/j.jcmgh.2016.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Cellular and Molecular Gastroenterology and Hepatology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

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Epithelial regeneration after gastric ulceration causes prolonged cell type alterations

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Short title: Imperfect gastric ulcer healing Eitaro Aihara1*, Andrea L. Matthis 1†, Rebekah A. Karns2, Kristen A. Engevik1, Peihua Jiang3, Jiang Wang4, Bruce R. Yacyshyn5, and Marshall H. Montrose1 †

Equal contribution to first author

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Affiliation:

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1. Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 2. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 3. Monell Chemical Senses Center, Philadelphia, Pennsylvania 4. Department of Pathology and Lab Medicine, University of Cincinnati, Cincinnati, Ohio 5. Division of Digestive Disease, Department of Internal Medicine, University of Cincinnati, Ohio

Grant: The National Institutes of Health Grants, RO1 DK54940 (MHM)

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The National Institutes of Health Grants, P30 DK078392 (DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati)

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Abbreviations: CFU; colony forming units, DCLK1; doublecortin like kinase 1, GIF; gastric intrinsic factor, GSII; Griffonia simplicifolia lectin II, HK-ATPase; hydrogen potassium exchanger, NHE2; sodium hydrogen exchanger 2, NSAID; nonsteroidal antinflammatory drug, PDX1; pancreatic and duodenal homeobox 1, SPEM; spasmolytic polypeptide expressing metaplasia, TFF2; trefoil factor 2, TGF; transforming growth factor, UEA-1; ulex europaeus *Correspondence: Eitaro Aihara Department of Molecular and Cellular Physiology University of Cincinnati, ML0576 231 Albert Sabin Way, Cincinnati OH 45267 E: [email protected] T: +01-513-558-3791 F: +01-513-558-5738 1

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Disclosures: BRY: Advisory board for Seres Health, Inc., Consultant to Procter & Gamble Pharmaceuticals, Speaker’s Bureau for UCB, Janssen Biotech, Inc., Shire Pharmaceuticals, and Actavis Pharmaceuticals, Grant funding from Merck/Cubist

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Pharmaceuticals. The remaining authors declare no conflict of interest.

Author Contributions:

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EA, MHM designed the project, EA, ALM, KAE conducted experiments and analyzed data. RAK performed RNAseq analysis. EA, ALM, RAK, PJ contributed to interpretation

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of data. JW, BRY assisted in pathological assessments. EA, ALM wrote the manuscript. All authors critically read and contributed critical revision of the manuscript for important intellectual content. MHM obtained the funding. EA performed the study supervision.

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Transcript Profiling:

Accession number of repository: GSE76565 (NCBI's Gene Expression Omnibus)

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Word count: 5,157 words (excluding methods, figure legends, and references)

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Synopsis: This study shows that prominent histologic, structural and gene expression

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abnormalities are observed in macroscopically healed gastric epithelium. Results suggest that the site of ulcer healing is the site of ulcer recurrence and potential source

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of further disease.

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Abstract Background & Aim: The peptic ulcer heals through a complex process, although the

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ulcer relapse often occurs several years later after healing. Our hypothesis is that even after visual evidence of healing of gastric ulceration, the regenerated epithelium is aberrant for an extended interval, increasing susceptibility of the regenerated epithelium

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to damage and further diseases.

Methods: Gastric ulcers were induced in mice by serosal topical application of acetic

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acid.

Results: Gastric ulcers induced by acetic acid visually healed within 30 days. However, regenerated epithelial architecture was poor. The gene profile of regenerated tissue was abnormal, indicating increased stem/progenitor cells, deficient differentiated gastric cell

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types, and deranged cell homeostasis. Despite upregulation of PDX1 in the regenerated epithelium, no mature antral cell type was observed. Four months after healing, the regenerated epithelium lacks parietal cells, TFF2 and SOX9 remain upregulated deep in

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the gastric gland, and the Na/H exchanger 2 (a TFF2 effector in gastric healing) remains downregulated. Gastric ulcer healing was strongly delayed in TFF2 knockout mice, and

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re-epithelialization was accompanied with mucous metaplasia. Following H. pylori inoculum 30 days after ulceration, we observed that the gastric ulcer selectively relapses at the same site where it was originally induced. Follow up at 8 months showed that the relapsed ulcer was not healed in H. pylori infected tissues. Conclusion: These findings reveal that this macroscopically regenerated epithelium has prolonged abnormal cell distribution and is differentially susceptible to subsequent damage by H. pylori. 4

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Key words: Gastric ulcer healing; metaplasia; H. pylori; SOX9; TFF2; NHE2

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Introduction Gastric ulcers, excavations that extend from the epithelial lining to the muscle

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layer of the stomach, heal through a dynamic process that seeks to restore normal architecture and function to a site of deep tissue erosion. Depending on the size of the erosion, it can take days or months for healing to occur. The mechanisms underlying ulcer healing are not fully understood, although the beneficial effects of H. pylori

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eradication or inhibition of acid secretion are well-established1-3 to accelerate

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macroscopic healing of the epithelium over an ulcer bed. Many investigators have focused on ulcer healing by observing the closure of the ulcer hole in short term (90% of genomic microsatellite markers were from C57BL/6J), and in-house bred NHE2 (Slc9A2) KO mice (FVBN background). Animals were fed a standard rodent chow diet, and had free

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access to water. All mice experiments were conducted according to both Animal Welfare Act Regulations and Public Health Service Policy on Humane Care and Use of

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Laboratory Animals. Mice were maintained in an AAALAC approved facility and all animal studies followed protocol 04-03-08-01 that was approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Gastric ulcers were produced by acetic acid, according to a previously described

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method with slight modifications1. In brief, under isoflurane anesthesia, the abdomen was incised and the intact stomach exposed. A microcapillary tube (0.7 mm in diameter: Drummond Scientific Co. Broomall, PA) filled with acetic acid (99%) was placed in

Buprenorphine

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contact with the exterior surface of the stomach corpus region and left in place for 25 s. hydrochloride

(0.75

mg/kg

ip

Buprenex,

Rechkitt

Benckiser

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Pharmaceuticals Inc, Richmond, VA) was given as preemptive analgesia. After the acid was removed, the treated exterior of the stomach was wiped with gauze, the abdomen was closed, and the animals were routinely maintained with food and water. Using this procedure, no incision was made to the stomach as part of ulcer induction. To examine ulcer healing, animals were sacrificed at 9 days, 30 days, 44 days, 4 months, or 8 months after ulceration, and the stomach was removed and opened along

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the greater curvature. The ulcer induced area was found by serosal observation of white discolored muscle tissue. The area (mm2) of ulceration on the mucosal side was measured by digital caliper (Mitutoyo, Kanagawa, Japan). In some cases, H. pylori were

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gavaged at day 30 after the ulceration. Control uninfected animals received Brucella broth vehicle.

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Preparation of H. pylori

As we described previously11, H. pylori strain Sydney strain 1 were grown on

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Columbia blood agar plates (Remel, Lenexa, KS, USA) containing 5% defibrinated horse blood (Colorado Serum, Denver, CO, USA), 0.2% ß-cyclodextrin (Sigma, St. Louis, MO, USA), 50 µg/ml cyclohexamide (Sigma, St. Louis, MO, USA), 5 µg/ml vancomycin (Sigma, St. Louis, MO, USA), 10 µg/ml trimethoprim (Sigma, St. Louis, MO,

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USA) for 4 days. Bacteria harvested from the plate were grown in Brucella broth (BD Diagnostic Systems, Franklin Lakes, NJ, USA) supplemented with 10% fetal bovine serum and CO2 gas pack (BD Diagnostic Systems, Franklin Lakes, NJ, USA) in a

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humidified microaerophilic chamber (BBL Gas System, with CampyPak Plus packs, BD Microbiology, Sparks, MD, USA) in an incubator at 37 ºC for 16 to 18 hours without

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shaking. Bacteria were collected by centrifuge at 2000 rpm for 5 min, and resuspended in Brucella broth without serum. H. pylori were diluted in 50% glycerol (1:100 dilution) and counted in haemocytometer. Each mouse received 200 µl Brucella broth containing 106 H. pylori bacteria. Control mice (uninfected group) received 200 µl of Brucella broth alone.

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Quantification of levels of H. pylori in mouse stomach tissue The wet weight was measured for the mouse gastric tissue collected from intact and ulcerated tissue. Tissue was homogenized by Tissue Tearor (Model: 985370-

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395BioSpec Products) in 1 ml saline and 1:10 dilutions were spread on a Columbia blood agar plate containing 5% defibrinated horse blood, 50 µg/ml cyclohexamide, 5 µg/ml vancomycin, and 10 µg/ml trimethoprim. Plates were incubated for 5 days at 37

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ºC in a humidified microaerophilic chamber with CO2 gas pack. Colonies were counted

(CFU)/g tissue.

Quantitative real-time PCR

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and data normalized using the tissue weight and expressed in colony forming units

Total RNA was isolated from either the intact or regenerated epithelium of the

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stomach using TRI Reagent (Molecular Research Center) and single-stranded cDNA was synthesized by iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was performed using SYBR Green PCR Master Mix

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(Applied Biosystems) with the following specific primers: H,K-ATPase (Forward: 5’AGATGGTGTTTGCTCGAACC-3’ and Reverse: 5’-TCCAGCAAGATCATGTCAGC-3’), (Forward:

5′-GCAGTGCTTTGATCTTGGATGC-3′

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TFF2

TCAGGTTGGAAAAGCAGCAGTT-3′),

NHE2

and

Reverse:

(Forward:

5′5′-

CTTCTGATTCGGGAAAACA-3′ and Reverse: 5′-ATCAGGATCTCCTTGGCTTG-3′), SOX9

(Forward:

5'-CGGAACAGACTCACATCTCTCC-3

and

Reverse:

'5'-

GCTTGCACGTCGGTTTTGG-3') or GAPDH (Forward: 5’-AACGACCCCTTCATTGAC-3’ and Reverse: 5’-TCCACGACATACTCAGCAC-3’). Each PCR amplification was

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performed in duplicate wells in a StepOnePlus Real-Time PCR System (Applied Biosystems).

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Immunofluorescence

Mouse intact or regenerated stomach tissue was embedded in OCT and frozen and stored at -80°C. Serial cryosections (10 µm) were prepared by cryostat. The

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section was stained with hematoxylin & eosin. For immunostaining, sections were fixed with 4 % paraformaldehyde followed by heat activated antigen retrieval (Vector

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Laboratories, Burlingame, CA USA), then incubated with goat serum (5 %) or 3 % BSA for 60 min. Sections were then incubated with primary antibodies indicated below for 60 min in room temperature. Primary antibodies to H+/K+-ATPase alpha subunit (Mouse monoclonal, 1: 1000, Thermo Science or Rabbit polyclonal, 1: 1000, Santa Cruz), TFF2

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(Rabbit polyclonal, 1:200), GIF (Rabbit polyclonal, 1:100, Abcam), DCLK1 (Mouse monoclonal, 1:200 or Rabbit polyclonal 1:1000, Abcam), SOX9 (Rabbit polyclonal, 1:1000, Millipore), NHE2 (Rabbit polyclonal, 1:200), Ki67 (Rabbit polyclonal, 1:200,

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Abcam), PDX1 (Goat polyclonal, 1:5000, Abcam), gastrin (Rabbit polyclonal, 1: 500, Dako) or SOX2 (Mouse monoclonal, 1:200, Abcam) were used. Secondary antibodies

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(Alexa 633-labeled goat anti-mouse IgGγ1: 1:1000, and Alexa 488-labeled goat antirabbit IgG, 1:1000 or 1: 500 for H+/K+-ATPase, TFF2, GIF, DCLK1, SOX9, NHE2, Ki67, or SOX2, and Alexa 633-labeled goat anti-rabbit IgG for gastrin, and Alexa 647-labeled donkey anti-rabbit IgG 1:1000 for SOX9 and Alexa 488-labeled donkey anti-goat IgG 1:1000 for PDX1) were incubated for 60 min in room temperature. In some cases, Alexa 647-Lectin GSII (1:100, Life Technologies) and Rohdamin-UEA1 (1:100, Vector

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Laboratories) were incubated for 60 min in room temperature. Nuclear staining was performed by incubation with Hoechst 33342 (Invitrogen) at 1 µg/ml for 30 seconds.

Whole mount staining Mouse intact or regenerated stomach

tissues

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Sections were imaged with ZEISS 710 confocal microscopy.

were fixed with 4 %

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paraformaldehyde overnight at 4°C and followed by Dent’s fixative for 2 hr. Following rehydration, tissues were incubated with goat serum (4 %) for 2 hr. Tissues were then

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incubated with primary antibody to H+/K+-ATPase alpha subunit (Rabbit polyclonal 1: 100, Santa Cruz) for overnight at 4°C, followed by secondary antibodies (Alexa 633labeled goat anti-rabbit IgG: 1:400, Invitrogen) were incubated for overnight at 4°C. Nuclear staining was performed by incubation with Hoechst 33342 (Invitrogen) at 10

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µg/ml for 60 min. Then tissue was dehydrated with methanol and clearing agent was applied. (2:1=Benzyl Benzoate: Benzyl Alcohol). Whole mount images were obtained

(Bitplane).

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via z-stack using the Zeiss LSM710, and 3D images were rendered by Imaris 7.7

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Western blot analysis

Extracted proteins from mouse intact or regenerated stomach tissues were

separated on 1-15 % gradient

SDS-PAGE gel (Bio-rad, Hercules, CA USA), and

transferred to a PVDF membrane (Immobilon-P; Millipore Corp., Billerica, MA). Membranes were incubated for 1 h with Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE) and then incubated with primary antibodies to H+/K+-ATPase alpha subunit

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(Mouse monoclonal 1: 10000), GIF (Rabbit polyclonal, 1: 1000), TFF2 (Rabbit polyclonal, 1:1000), DCLK1 (Rabbit polyclonal, 1:2000), NHE2 (Rabbit polyclonal, 1:1000), SOX9 (Rabbit polyclonal, 1:2000), or GAPDH (Mouse monoclonal 1: 10000).

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Membranes were then washed and incubated for 1 h in goat anti-rabbit secondary antibody (Alex Fluor 680, 1:1000 dilution; Invitrogen) or goat anti-mouse secondary antibody (IR800 dye, 1:10000, Rockland, Gilbertsville, PA). Fluorescent blots were

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imaged and quantified using an Odyssey infrared imaging system (Li-Cor Biosciences). Immunoreactive bands were quantified using background-corrected integrated pixel

from intact tissue or WT tissue.

RNA sequence and analysis

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intensity. Results were normalized to GAPDH, then showed as fold change to value

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Stomach corpus regenerated tissue and corpus non-involved tissue were harvested after 30 days of ulceration under a dissection scope. Tissue was homogenized in TRI Reagent. Extracted total RNA was purified by the PureLink DNA

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Mini Kit (Life Technologies, Carlsbad, CA USA), followed by quantifying with Qubit Fluorometer (Life Technologies, Carlsbad, CA USA). The mRNA was poly-A selected

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and converted into a single-stranded cDNA library for Next Generation Sequencing using the Illumina IHiSeq 2500 (Illumina Inc., San Diego, CA USA) rapid sequencing system at the Cincinnati Children’s Hospital Medical Center Sequencing Core. RNA Seq was single-end 75 base pair reads with an average read depth of 30 million per sample. All analysis was performed in GeneSpring 12.6 following removal of barcodes and primers.

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Sequence data were aligned to the mouse reference genome (mm10), with reference annotations produced by the University of California, Santa Cruz (UCSC)25. Reads with multiple mappings were removed, and aligned reads subsequently filtered

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on quality, including a quality threshold ≥30 and zero ‘Ns’ allowed. Reads per kilobase per million reads (RPKMs) were computed using quantified read counts for each sample, subsequently normalized using the DESeq algorithm and thresholded to 1.

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Normalized counts were baselined to the median of control samples. The expression

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data are available through NCBI's Gene Expression Omnibus under GSE76565.

Statistical analysis

All values are reported from representative experiments as the mean ± standard error of the mean (SEM) from multiple experiments. All results were reproduced in at

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least three animals. Statistical significance was determined using unpaired Student’s Ttest, or one-way ANOVA with Dunnett’s multiple comparison post-hoc test. A p value of < 0.05 was considered significant.

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Expression values were compared between regenerated and non-involved tissue from same mouse stomach using paired-tests, with significance defined as p value
1.5 (N=1165 significant genes; Supplementary Table 1). Additionally, genes with known involvement in gastric function were assessed for significance and fold change between regenerated and non-involved tissues. Heatmaps were generated and clustered on both gene and condition, using the Euclidean distance metric and average linkage rule.

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Ontological analyses and

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biological networking were performed using GATACA (https://gataca.cchmc.org), ToppGene Suite8, ToppCluster20, and Cytoscape3. All authors had access to the study data and reviewed and approved the final

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manuscript.

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Results Regenerated epithelial appearance and gene expression

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Acetic acid-induced ulcers are a well-established rodent model that has similar features of human ulcers1. Consistent with our previous observation11, serosal application of acetic acid induced necrosis of the local gastric epithelium, covering the

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ulcerated area with dead cells. It is noted that no HK-ATPase positive cells were found in the ulcerated area, while HK-ATPase positive cells (bright red) with bright Hoechst

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nuclear staining (indication of cell death) were seen on the edge of the ulcerated area undergoing cell death at Day 2 (Figure 1A). At Day 30, the ulcerated area was visually covered by newly generated epithelium, although microscopic observation showed the regenerated epithelial appearance to be distinguished from normal gastric epithelium, lacking HK-ATPase positive cells (Figure 1B). Characteristically, the regenerated

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surface epithelium was connected to many glandular infoldings (Figure 2 and Supplementary Movie 1). Sectioned gastric regenerated tissue lacked HK-ATPase positive cells (Figure 1C), and had significantly reduced mucosal thickness versus

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normal epithelium (Figure 1B). Consistent with our previous finding11, we observed

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upregulation of TFF2 in the ulcer margin as well as the base of regenerated epithelium where HK-ATPase positive cells were lacking (Figure 1D). The changes in TFF2 and HK-ATPase were independently confirmed by Western blot (Figure 3A and 3B) and RNAseq (Figure 3C). RNAseq also showed upregulation of CXCR4, a putativeTFF2 receptor16,

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, in the regenerated epithelium (Figure 3C). Interestingly, we observed

downregulation of NHE2 in the regenerated tissue (Figure 1E and Figure 3A and 3B). The RNAseq data reported that the gastric surface marker, Muc5ac, and parietal cell 17

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marker, HK-ATPase (Atp4a), H2 receptor (Hrh2), endocrine cell marker, chromogranin A (Chga) were downregulated (Figure 3C). In addition to downregulation of parietal cell markers, we observed significant upregulation of a gastric atrophy marker27, gastrokine

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3 (Gkn3). However, we did not observe any changes of Cdx1, Cdx2, or Muc2 expression, suggesting that intestinal metaplasia does not arise in the corpus ulcer healing process (Figure 3C).

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Surface mucus marker, UEA-1(Ulex europaeus 1) and neck mucus marker, GSII

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(Griffonia simplicifolia lectin II) were expressed in their expected compartments of the gland in the intact epithelium (Figure 4A and 4B). However, both GSII and UEA1positive mucus staining also expanded into the bottom of the gland in the regenerated tissue (Figure 4A). GSII, which co-expresses with TFF2, is used as a marker of spasmolytic polypeptide expressing metaplasia (SPEM)28-30. SPEM has been

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documented in the ulcerated area 7 days after ulceration induced by acetic acid12, 31. Consistent with other findings at early time points after ulceration31, we confirmed that 30 days after ulceration TFF2 co-expressed with GSII in the deep gland of the

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regenerated epithelium (Figure 4B). The fold change values of DMP777/H. felis-induced SPEM-related genes, identified by RNAseq32, positively correlated to our RNAseq data

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which exhibited borderline significance in DMP777 and nominal significance in H. felis. Results confirmed that the Day 30 ulcer regenerated epithelium includes SPEM lineages. Interestingly, UEA1 was also found in TFF2 expressing sites, whereas it was negative in the deepest gland where TFF2 and GSII were positive (Figure 4B). Gastric intrinsic factor (GIF) and Mist1 (Bhlha15) were downregulated in the regenerated epithelium (Figure 3A-C). GIF was found at the base of the gland in the intact stomach

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(Figure 4C), but GIF positive cells were dramatically downregulated in the regenerated epithelium and were found only in the deepest gland where GSII is positive and UEA1

regenerated epithelium. Stem/progenitor zone in regenerated epithelium

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negative (Figure 4C). These data suggest that mature chief cells are not present in the

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Gastric epithelial proliferation normally occurs in the isthmus region where we observe Ki67 positive cells (Figure 5A). In contrast, Ki67 positive cells were found at the

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base of regenerated epithelium, and Ki67 immunoreactivity was notably localized in UEA1 and GSII positive cells (Figure 5A). We also observed that SOX9 was upregulated in regenerated tissue (Figure 3A, 3B, 3D and Figure 5B), especially within UEA1 and GSII positive cells deep in the gland, whereas SOX9 expression was weak in the intact gastric epithelium and co-localized with GSII (Figure 5B). The expression

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pattern of SOX9 in regenerated epithelium appeared deeper in the gland than that of Ki67. Results suggest that stem/progenitor and proliferation zone are both present at the gland base of regenerated epithelium. In the RNAseq data, we found

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downregulation of Tnfrsf19 (Troy), Bmi1 and Msi1 (Musashi1), whereas we observed

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upregulation of Prom1, SOX9, and SOX2 (Figure 3D). SOX2 was expressed in both nuclei and cytoplasmic compartments in the neck/isthmus of the mouse stomach (Figure 6), and was expressed sporadically in the entire epithelium, consistent with other findings21, 33. SOX2 positive cells are partially positive for Ki67 or SOX9 in normal epithelium. Similarly, SOX2 was partially expressed in Ki67 and SOX9 positive cells in the regenerated epithelium. However, SOX2 expression was not strong in the base of

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the gland where SOX9 was upregulated, although it was highly expressed in the above proliferation zone (Ki67) (Figure 6).

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Since SOX9 also expresses in the tuft cell, we checked the expression pattern of another tuft cell marker, doublecortin like kinase 1 (DCLK1). It has also been reported that DCLK1 expressing cells are expanded in an ulcer12 as well as in response to loss of parietal cells34. We observed that DCLK1 was significantly upregulated in the

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regenerated epithelium (Figure 3A-C and Figure 5C). Immunofluorescence showed that

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DCLK1 positive cells also expressed SOX9, whereas a large number of SOX9 positive cells did not co-express with DCLK1 (Figure 5D). However, Ki67 was not expressed in DCLK1 positive cells (Figure 5E).

Using uninjured tissue, we also observed GSII co-localized with Sox9 and Ki67 in the bottom of antral glands, and UEA1-positive mucus was observed throughout the

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pyloric gland (Figure 7). We observed upregulation of PDX1 in the regenerated epithelium, despite no expression of PDX1 in the normal corpus epithelium, (Figure 3C and 8A). PDX1 was expressed in the antral epithelium (Figure 8B). However, gastrin

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(Figure 3C) or Lgr5 (Figure 3D) expression did not change in the corpus regenerated

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tissue and gastrin was not detected in the regenerated epithelium (Figure 8C). These data suggest that regenerated corpus epithelium may share some antral features, but within the time frame evaluated there is no induction of mature antral cell types in the regenerated corpus epithelium under our experimental conditions. Role of TFF2/NHE2 in ulcer healing TFF2 is secreted predominantly by gastric neck cells and appears to have a central role in gastric injury and repair. TFFs promote gastric healing after injury through 20

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motogenic (cell migratory) and anti-apoptotic activities12,

13, 19, 35

. We previously

observed that TFF2 was upregulated even at Day 9 after ulceration11, and sustained an increase at Day 30 (Figure 1). Although ulcer healing was significantly delayed in the

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TFF2 KO mouse stomach (Figure 9A), regeneration of the epithelium was observed at Day 30 even in TFF2 KO mice, including sustained loss of HK-ATPase (Figure 9C). The expression pattern of UEA1 and GSII positive cells is similar to WT regenerated

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epithelium and we observed mucous metaplasia both at Day 9 and Day 30 (Figure 9B and 9C). In addition, NHE2 was downregulated in both intact and regenerated

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epithelium (Figure 9C), consistent with our previous observation that NHE2 expression is low in the TFF2 KO stomach16. These data suggest that TFF2 is most prominently involved in the initial closure of an ulcer, but only slows re-epithelialization. TFF-dependent repair of (at least microscopic) gastric lesions requires the NHE2

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Na/H exchanger isoform16. In normal tissue, we observed NHE2 expression in the surface epithelium and also in the deep gland, whereas we observed down-regulation of NHE2 in regenerated tissue (Figure 1E). Since we observed the down-regulation of

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NHE2 in regenerated epithelium, we considered the lack of NHE2 could contribute to TFF2 upregulation or generation of the poor appearance of regenerated epithelium. To

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test this hypothesis, we compared some outcomes in the

ulcer regenerated tissue to

the healthy NHE2 KO stomach. In the NHE2 KO corpus, HK-ATPase and GIF protein was significantly downregulated (Figure 10A and 10B) while TFF2 was markedly upregulated deep in the gland and co-localized with GSII (Figure 10A-C). UEA1 positive cells, also found deep in the gland, were co-localized with TFF2 or GSII (Figure 10C and 10D). Additionally, we found that DCLK1 was significantly upregulated in NHE2 KO

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gastric epithelium, whereas there was no difference in SOX9 expression between WT and NHE2 KO stomach (Figure 10A and 10B). SOX9 was found deep in the gland as well as DCLK1 positive cells (Figure 11A and 11B). Ki67 positive cells were also

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observed deep in the gland, and did not overlap with DCLK1 positive cells (Figure 11C). Similar to ulcer regenerated epithelium, SOX2 was partially expressed in Ki67 and SOX9 positive cells in the NHE2 KO stomach throughout the entire epithelium (Figure

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12A and 12B). On the other hand, we also observed that PDX1 was expressed throughout the NHE2 KO mouse corpus epithelium (Figure 11D). Combined, results

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suggest that NHE2 has a role in the maintenance as well as construction of a normal gastric epithelium.

Long term monitoring of regenerated epithelium

We monitored ulcer healing up to 8 months to ask how long it takes to restore a

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normal gastric epithelium after an initial ulceration. In the same experimental series, we asked if the 30 day regenerated epithelium was differentially susceptible to subsequent damage. We inoculated low amounts of Sydney strain 1 H. pylori (106 per mouse in one

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inoculum) at 30 days post ulceration (Figure 13A). We visually observed that ulcer

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regenerated epithelium was disrupted by H. pylori 14 days post inoculum (44 days after ulcer) (Figure 13B and 13C). It should be noted that no ulcer was observed in other sites of the stomach, suggesting that the regenerated epithelium defense is relatively weak. H. pylori colonization in the ulcerated site was not consistently higher than that of un-injured sites (Figure 13D). The disruption of regenerated epithelium by H. pylori did not heal by 8 months later (Figure 13B and 13C), although H. pylori colonization in the stomach tended to decrease over time (Figure 13D). 22

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At 44 days post ulceration without H. pylori, HK-ATPase was largely lacking in regenerated epithelium. Then at 4 and 8 months post ulceration without H. pylori, HKATPase expression began to slowly return. Whereas, H. pylori significantly suppressed

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the return of HK-ATPase (Figure 13E). At 4 months, TFF2 and NHE2 sustained upregulation and down-regulation in the regenerated epithelium, respectively (Figure 13E and Figure 14) with TFF2 showing strong expression in the ulcerated site with H. SOX9 was significantly upregulated in regenerated epithelium with and without

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pylori.

H. pylori (Figure 13E). SOX9, Ki67, GSII and UEA1 expression patterns were similar to

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that as in the Day 30 regenerated epithelium (Figure 14). However, distribution of SOX9 and Ki67 was similar to non-H. pylori condition (Figure 14).

At 8 months post ulceration without H. pylori, we clearly observed HK-ATPase and NHE2 expression in the regenerated epithelium and the decrease of TFF2 in the

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bottom of the gland (Figure 13E and Figure 15). Furthermore UEA1, GSII, Ki67 and SOX9 expression patterns became more similar to normal gastric epithelium although the height of epithelium was still shorter (Figure 15). Conversely, no recovery of HK-

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ATPase and NHE2 was found in the relapsed ulcer site caused by H. pylori and TFF2 expression remained high (Figure 13E). In addition, we observed a strong upregulation

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of Ki67 distribution and SOX9 in the ulcerated site as well as intact area (Figure 15), suggesting that H. pylori also affects the intact area at this late time point. However, only in the ulcerated area was UEA1 co-localized with GSII. These results suggest that ulcer regenerated epithelium takes more than 8 months to return to normal, and has a potential to progress into further damage with further insulting injury to the gastric mucosa.

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Discussion We have shown using a mouse model that ulcer regenerated epithelium is

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characterized by abnormal epithelium that is sustained for almost half a lifetime. Furthermore, repaired tissues are more susceptible to subsequent damage. Consistent with this, in humans, long term follow-up studies (up to 17 years) showed that gastric recurrence or cancer development is still observed in many ulcer patients even with

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successful H. pylori eradication5. The gastric mucosa is exposed to acid and other

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chemical hazards, including food intake, assuming that there is a high chance to receive several challenges during ulcer healing. In the acetic acid-induced ulcer rodent model, several reports showed that administration of NSAIDs or H. pylori causes an ulcer in the area where the initial ulcer had healed10, 36, 37, although why an offense-induced relapse ulcer occurs at the same or near site is still unknown. In the present study, we observed

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that H. pylori cause ulcer relapse, specifically at the site of visually healed regenerated epithelium. Additionally, H. pylori-infected regenerated epithelium sustains metaplasia and morphological abnormalities. The low inoculum of H. pylori that we used in the

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present study explains why it was unable to generate inflammation in the intact gastric epithelium, and our work suggests that the regenerated epithelium has weakened

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defenses against H. pylori. We found that metaplasia occurs in the regenerated epithelium and the

metaplastic state is sustained for at least 4 months during normal ulcer healing. Previous studies have shown that a loss of parietal cells induces metaplasia in the stomach12, 19, 24, 35. It has been reported that DCLK1 is upregulated in response to a loss of parietal cells and metaplasia, including ulcerated tissue, but its functional role 25

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remains unknown12, 34. Since DCLK1 positive cells were not positive for Ki67, they likely do not function as stem/progenitor cells. In the ulcer, UEA1 positive cells expanded deep into the gland along with GSII or TFF2 positive cells. Maloum et al. also observed

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an increase in the number of UEA1 positive cells that were found deep in the gland, along with mucous metaplasia, a loss of parietal cells, and downregulation of MUC5ac38. UEA1 binds α-L-fucose containing glycoproteins. It has been reported that

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some human TFF2 contains unusual fucosylated oligosaccharide39, but we observed UEA1 staining deep in the gland of regenerated epithelium in TFF2 KO mice.

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Fucosylation is involved in many physiological and pathological processes such as development, cell proliferation, and inflammation40. We observed an increase in the number of UEA1 positive cells, but it is unclear if this expansion of staining is due to simply more cells of a defined type, such as gastric foveolar hyperplasia, or an

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expansion of fucosylation among a diverse set of cell types.

We reported that TFF2 plays an important role in gastric epithelial restitution though

CXCR4

in

response

to

microscopic

damage16,

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.

The

surface

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epithelial/metastasis markers TFF2 and CXCR4 are upregulated and the former is confirmed to be widely distributed deep in the regenerated gastric gland. It is also

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reported that CXCR4 is upregulated at the ulcer granulation tissue and this expression is inhibited by acetylacetic acid administration, resulting in delayed ulcer healing42. In the present study we showed that ulcer healing was delayed in the TFF2 KO mouse stomach, although we observed regenerated epithelium. This suggests that TFF2 may be involved in regulation of proliferation as well. It has also been reported that TFF2 contributes to proliferation43. On the other hand, it is reported that H. pylori accelerates

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progression of gastritis to dysplasia (6 months) in the TFF2 KO mouse stomach, although development of atrophy and epithelial hyperplasia appeared 19 month after H. pylori inoculation in the wild-type mouse stomach

44

. We observed that TFF2 is

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upregulated in the ulcerated area in H. pylori infection even 8 months after an ulcer episode. Furthermore, it has been reported that TFF2 acts as a tumor suppressor in gastric carcinogenesis and TFF2 mRNA and protein are downregulated in gastric 45, 46

, these results suggest that upregulation of TFF2 contributes to

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adenomas13,

prevent cancer progression in the ulcer regenerated gastric epithelium. We did not

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detect any cancer-like morphology even 8 month after ulceration in the wild-type mouse stomach. Thus, upregulation of TFF2 is likely a necessary event in regenerated or inflamed tissue, which could prevent further developing pathological changes in the long term.

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We previously reported that NHE2 plays a novel (albeit unknown) role as an effector of TFF2 in gastric surface epithelial restitution16. We observed the downregulation of NHE2 in the regenerated epithelium where TFF2 is upregulated. This

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suggests that the regenerated epithelium has a weakened defense against insult, which may explain why a low abundance of H. pylori could selectively relapse the ulcer at the

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site of the regenerated tissue. In the present study, we observed that the number of parietal cells are decreased in NHE2 KO corpus. This is consistent with another finding that NHE2 KO stomachs have fewer parietal cells with hyperplasia/atrophy15. The same group also suggested that NHE2 is essential for parietal cell long-term viability47. It is likely that NHE2 deletion leads to a loss of parietal cells, resulting in the generation of abnormal epithelium, which is similar to ulcer-induced regenerated epithelium. While

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NHE2 may be involved in cell differentiation, further investigation will be needed to determine whether NHE2 affects the local ionic environment or directly alters the cell

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differentiation pathway through another mechanism. Stem cells in the gastric corpus epithelium have not been fully identified yet, although proliferating cells are normally detected in the isthmus. We observed strong proliferation activity in the base of regenerated epithelium where the UEA1, GSII and

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TFF2 abnormally expresses at 30 days after ulceration. In addition, the SOX9 was

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upregulated in the same region, and returned to normal expression levels during long term ulcer healing. We also observed that SOX2, which recently was reported as a gastric corpus stem cell marker21, was upregulated in the regenerated epithelium, while it does not specifically appear in the proliferative (Ki67+) compartment. SOX9 is reportedly upregulated within mucous metaplasia or gastric carcinoma22-24. Gupta et al.

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reported that upregulated SOX9 co-expressed with GSII and some SOX9 positive cells demonstrated proliferating activity22. Although SOX9 marks tuft cells and mucous metaplasia in our current experiments, some SOX9 positive cells may also be

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stem/progenitors to regenerate tissue in response to injury. This could only be resolved using SOX9 lineage tracing experiments in the regenerated epithelium. Lgr5+ cells are

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now recognized as the stem cell in antrum, small intestine and colon17, but the expression of Lgr5 in the adult corpus is limited. We did not see any changes of Lgr5 mRNA in the regenerated tissue. It is reported that Lgr5+ cells are not involved in developing metaplasia in mouse corpus19. Thus, Lgr5+ cells unlikely play any role in the ulcer regeneration in the corpus. More recently, Troy-positive cells are expressed at the corpus gland base in a subset of differentiated chief cells and demonstrate that sorted

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Troy-positive chief cells generate long-lived gastric organoids, and these cultures are differentiated toward the mucus-producing cell lineages of the neck and pit regions12, 20, Troy has been proposed as a marker of quiescent stem-like cells, but Troy mRNA did

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not change in the regenerated epithelium. However, it is possible that Lgr5+ cells or Troy positive cells give rise to progenitor cells while they do not renew themselves. Further studies will be needed to identify the stem cells that respond to regenerate the

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damaged gastric epithelium.

A central question is whether such outcomes occur in humans. In human there is

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some limited evidence that there is a longer time for recovery of parietal cells after injury. In human ulcer patients, Blom demonstrated that parietal cells did not reappear until 3 months after ulcer repair, and even by 8-9 months the number of parietal cells in the regenerated gastric mucosa never reached normal levels48. We observed that PDX1

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was increased in the regenerated epithelium. Although regenerated epithelium may have features of antral epithelium based on expression patterns of mucus, SOX9, and PDX1, we did not detect any genes of mature antral cell types, such as gastrin. Another

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investigator also found PDX1 expression in the adjacent gastric gland in atrophic corpus gastritis49. They suggested that PDX1 may play a role in the initiation of metaplasia49. It

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has also been reported that overexpression of TGFα stimulated PDX1 throughout corpus atrophic epithelium with up-regulation of TFF2 in the deep gland50. Importantly, they found that inhibition of PDX1 by anti-EGF antibody led to an increase in the number of parietal cells50. More recently, the same group found that inhibition of MAPK (downstream of EGFR signaling) regresses metaplasia in the stomach and turned on the normal gastric gland lineage, including appearance of parietal cells51. Our RNA-seq

29

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analyses revealed that TGFα and EGFR were upregulated in the regenerated epithelium. Thus, these findings could provide an explanation of why differentiation to parietal cells is slow in the regenerated epithelium in our ulcer model. Further studies

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will be needed to investigate this point.

We conclude that even after visual evidence of healing from gastric ulceration, it cannot be assumed that the tissue is normal. The gene expression profile and

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morphology of the regenerated epithelium is abnormal in mice for the long term, and

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this can contribute to further damage. This is especially important because the gastric epithelium is continually exposed to harsh environments and commonly exposed to the pathogen H. pylori. In human patients, the ulcer recurrence rate is varied among the studies as well as countries, but overall there is a concurrence regarding the beneficial effect of H. pylori eradication or anti-ulcer medicine on ulcer recurrence. Laine et al.

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observed a 20 % ulcer recurrence rate at 6 months in patients with successful H. pylori eradication and non-NSAID user52. They also reported that the ulcer recurrence rate was an increase over time (i.e. 6 month: 0%, 12 month: 7.5%, 18 month: 10 %, 24

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month: 10%) in patients in whom H. pylori was eradicated53. We speculate that the regenerated epithelium in patients may be molecularly and cellularly abnormal for years,

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and that this increases the risk for a variety of gastric disorders.

Further study is

needed in human patients, as it may be necessary to develop a more rigorous diagnostic criterion for ulcer healing as an opportunity to protect against prevention of further damage.

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Acknowledgements We are grateful to Dr. Gary E. Shull and Dr. Roger T. Worrell (University of

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Cincinnati) for providing animals from their NHE2 colony, and Dr. Timothy C. Wang (Columbia University) for providing animals from their TFF2 colony, and Dr. Karen M. Ottemann (University of California at Santa Cruz) for the kind gift of H. pylori. We thank Dr. Akihiko Asai (Cincinnati Children’s Hospital Medical Center) for sharing his tissue

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clearing protocol, and Dr. James M. Wells and his lab member, Kyle W. McCracken

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(Cincinnati Children’s Hospital Medical Center) for PDX1 and gastrin antibody and feedback. We thank Cincinnati Children’s Hospital Medical Center DNA Sequencing and Genotyping Core who ran the RNA sequence. This work was supported in part by the Office of the Provost and VP for Research at University of Cincinnati, and the National Institutes of Health Grants, P30 DK078392 (DNA Sequencing and Genotyping

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(MHM).

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Core of the Digestive Disease Research Core Center in Cincinnati) and RO1 DK54940

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Figure Legends

Figure 1. Gastric regenerated epithelium in acetic acid-induced ulcer mouse

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model. Gastric tissue was evaluated 2 days (A) or 30 days (B) after ulcer induction. Gross morphology with low resolution, high resolution of dotted rectangle on low resolution image, and whole mount staining with HK-ATPase (red) and nuclei

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(Hoechst33342: cyan). Epithelium height was measured in uninjured area (Control: C) and regenerated area (Re). Significant difference at *, p