Mechanisms of allergy and clinical immunology
Eosinophilic esophagitis: Epithelial mesenchymal transition contributes to esophageal remodeling and reverses with treatment

https://doi.org/10.1016/j.jaci.2012.03.005Get rights and content

Background

Mechanisms underlying esophageal remodeling with subepithelial fibrosis in subjects with eosinophilic esophagitis (EoE) have not been delineated.

Objectives

We sought to explore a role for epithelial mesenchymal transition (EMT) in subjects with EoE and determine whether EMT resolves with treatment.

Methods

Esophageal biopsy specimens from 60 children were immunostained for epithelial (cytokeratin) and mesenchymal (vimentin) EMT biomarkers, and EMT was quantified. Subjects studied had EoE (n = 17), indeterminate EoE (n = 15), gastroesophageal reflux disease (n = 7), or normal esophagus (n = 21). EMT was analyzed for relationships to diagnosis, eosinophil counts, and indices of subepithelial fibrosis, eosinophil peroxidase, and TGF-β immunostaining. EMT was assessed in pretreatment and posttreatment biopsy specimens from 18 subjects with EoE treated with an elemental diet, 6-food elimination diet, or topical corticosteroids (n = 6 per group).

Results

TGF-β1 treatment of esophageal epithelial cells in vitro for 24 hours induced upregulation of mesenchymal genes characteristic of EMT, including N-cadherin (3.3-fold), vimentin (2.1-fold), and fibronectin (7.5-fold). EMT in esophageal biopsy specimens was associated with EoE (or indeterminate EoE) but not gastroesophageal reflux disease or normal esophagus and was correlated to eosinophil counts (r = 0.691), eosinophil peroxidase (r = 0.738), and TGF-β (r = 0.520) immunostaining and fibrosis (r = 0.644) indices. EMT resolved with EoE treatments that induced clinicopathologic remission with reduced eosinophil counts. EMT decreased significantly after treatment by 74.1% overall in the 18 treated subjects with EoE; pretreatment versus posttreatment EMT scores were 3.17 ± 0.82 versus 0.82 ± 0.39 (P < .001), with similar decreases within treatment groups. Pretreatment/posttreatment EMT was strongly correlated with eosinophil counts for combined (r = 0.804, P < .001) and individual treatment groups.

Conclusions

EMT likely contributes to subepithelial fibrosis in subjects with EoE and resolves with treatments that decrease esophageal inflammation, and its resolution correlates with decreased numbers of esophageal eosinophils.

Section snippets

Cell culture and induction of EMT in vitro

Human esophageal epithelial HET-1A cells (American Type Culture Collection, Manassas, Va) were maintained in Bronchial Epithelial Growth Media without gentamycin-amphotericin B (Lonza/Clonetics, Walkersville, Md). For mRNA analysis, cells at confluence in 6-well plates were cultured an additional 24 or 48 hours in fresh media with or without 5 ng/mL TGF-β1 (R&D Systems, Minneapolis, Minn). Expression of mRNAs encoding adhesion and cytoskeletal proteins representative of epithelial cells

EMT is induced by TGF-β in cultured esophageal epithelial cells

To determine whether esophageal epithelium has the capacity to undergo EMT, we analyzed the ability of TGF-β1 to induce EMT in vitro in the HET-1A esophageal epithelial cell line. Culture of HET-1A cells with TGF-β1 decreased gene transcription for epithelial biomarkers, including adhesion proteins and cytoskeletal components representative of the epithelial phenotypes cytokeratin 8 (22% decrease, P < .01) and cytokeratin 14 (44% decrease, P = .27; Fig 2). Correspondingly, increased mRNA

Discussion

Because esophageal tissue can demonstrate significant epithelial basal zone hyperplasia and subepithelial fibrosis in subjects with EoE, we hypothesized that EMT might be one of the processes associated with these remodeling events. Our results identified the presence of EMT in children with active EoE and showed that treatments that resolve eosinophilic inflammation and epithelial hyperplasia reverse EMT and subepithelial fibrosis. We also showed that the degree of EMT and its resolution in

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      Citation Excerpt :

      Additionally, we found that select EAPs captured by the EST (EDN and CLC/Gal-10) correlate significantly and negatively with esophageal distensibility (ρ −0.44 and ρ −0.47, respectively (P < .01) (Table II). In the setting of chronic inflammation, epithelial cells take on characteristics of mesenchymal cells (fibroblasts, myofibroblasts), expressing collagens and vimentin and losing their epithelial cadherins (E-cadherin).1,15,21,22 These changes in the epithelium contribute to esophageal remodeling.

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    Supported in part by a translational research award from the AGA (to S.J.A. and G.T.F.), National Institutes of Health (NIH) grant R21AI079925 (to G.T.F. and S.J.A.), unrestricted gifts from the Campaign Urging Research on Eosinophilic Diseases (CURED; to S.J.A., G.T.F., and A.F.K.) and the Buckeye Foundation (to A.F.K.), and research funds from the UIC Department of Pediatrics (to A.F.K. and S.J.A.) and the Mayo Foundation and its NIH grant NCRR K26 RR0109709 (J.J.L.). B.A.R. was supported in part by a Craig Medical Student Summer Research Fellowship from the UIC COM. The CURED and Buckeye Foundations did not play any role in development of the study or in the preparation of this manuscript aside from providing research funding. This project was also supported in part by NIH/NCRR Colorado CTSI grant UL1 RR025780 (to G.T.F., S.A.W., and J.C.M.). Its contents are the authors' sole responsibility and do not necessarily represent official NIH views. S.J.A. and G.T.F. are members of the Medical Advisory Panel of the American Partnership for Eosinophilic Diseases.

    Disclosure of potential conflict of interest: A. F. Kagalwalla is on the speakers' bureau for Abbott Nutrition and has received research support from the Campaign Urging Research on Eosinophil Diseases, the Buckeye Foundation, and the University of Illinois at Chicago Department of Pediatrics. G. T. Furuta has received research support from the American Gastroenterological Association. The rest of the authors declare that they have no relevant conflicts of interest.

    These authors contributed equally to this work.

    These authors were co-senior authors.

    §

    S. A. Woodruff is currently at the University of Massachusetts Memorial Medical Center, Pediatric Gastroenterology and Nutrition, Worcester, Mass, and V. Mukkada is currently at Pediatric Gastroenterology, Nutrition, and Liver Diseases, Pediatric Food Allergy Program, Hasbro Children's Hospital, Alpert Medical School, Brown University, Providence, RI.

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