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Regulation of Esophageal Epithelial Function in Eosinophilic Esophagitis

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Regulation of esophageal epithelial function in Eosinophilic Esophagitis A dissertation submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirement of the degree of DOCTOR OF PHILOSOPHY In the Department of Pharmacology & Systems Physiology of the College of Medicine 2018 By Chang Zeng B.S. Sun Yat-sen University, 2012 Committee Chair: Anjaparavanda P. Naren, Ph.D. Mentor: Simon P. Hogan, Ph.D. Abstract Eosinophilic Esophagitis (EoE) is an allergic inflammatory disorder with increasing prevalence in the western world. Patients with EoE demonstrate symptoms including vomiting, dysphagia and food impaction which decreased the quality of life. One of the histopathological features of EoE is esophageal tissue remodeling, including dilated intercellular spaces (DIS) and basal zone hyperplasia (BZH). However, the underlying molecular s that drive these features is largely unknown. Here, we investigate the 1) involvement of sodium‐hydrogen exchanger 3 (NHE3) in esophageal epithelium remodeling and 2) the role of the transcription factors, signal transducer and activator of transcription (STAT), in the regulation of gene networks that control esophageal epithelial proliferation and histopathological features of EoE. By analyzing RNA sequencing comparing transcriptome difference in esophageal biopsies from normal control (NL) and EoE patients, we identified NHE3 as the most upregulated transmembrane transporters in patients with active EoE. We found that the expression pattern of NHE3 closely correlated with the disease severity and DIS. Functional analyses demonstrated that NHE3 activity is upregulated in IL‐13 treated primary esophageal epithelial cells derived from EoE patients, as well as in IL‐13‐induced stratified squamous epithelium generated by the air‐liquid interface (EPC2‐ALI). Pharmacological Inhibition of NHE3 activity protected from IL‐13 induced DIS in esophageal epithelium. Thus, we concluded that NHE3 plays a functional role in DIS formation and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in EoE IL‐13 has previously been shown to activate STAT proteins, particularly STAT3 and STAT6 and regulate the transcriptome changes in EoE patients. Using transcription factor binding site (TFBS) analysis, we identified STAT protein binding motif is one of the most enriched transcription factor binding site (TFBS) in the dysregulated genes in both EoE biopsies and IL‐13 treated EPC2‐ALI cultures. In particular, we ii identified the STAT3 binding site as the most enriched TFBS in IL‐13 induced upregulated genes in EPC‐ ALI. By knocking down STAT3 in EPC2 cells, we revealed a role for STAT3 in the regulation of epithelium barrier function and IL‐13‐induced proliferation. In contrast, we show that STAT6 plays a pro‐ inflammatory role regulating cytokine and chemokine production in esophageal epithelium. Together, we identified several molecular targets in the esophageal epithelium that are important in modulating the esophageal epithelium remodeling in EoE. This dissertation, for the first time, uncovers the role of transmembrane transporters in the pathogenesis of EoE. Also, we provide valuable information on the two distinct divergent pathways regulated by STAT3 and STAT6 driving IL‐13‐ mediated transcriptome changes in EoE. Further investigations into the contribution of these mechanisms in the clinical manifestations of EoE will facilitate the evolvement of new therapeutic approaches in EoE treatment. iii iv Acknowledgements First, I would like to express my utmost gratitude to my mentor Dr. Simon Hogan. I had a tough beginning for my graduate study, but it all paid off when I ended up joined Simon’s laboratory. Simon is an excellent mentor. He showed me his enthusiasm and passion for science, guided me of how to do science correctly and efficiently, and told me to explore all the possibility of life in the future. These are all valuable assets and shaped me into who I am right now. Next, I would like to thank my thesis committee members. All of my committee members, Drs. Anjaparavanda Naren, Hong‐Sheng Wang, Gary Shull, Marshall Montrose and Robert Rapoport have provided me invaluable support and advice, which are essential for my completion of the dissertation. Primarily, I want to thank Dr. Naren for the guidance on experimental techniques and preparedness to assist with completion of my thesis dissertation. Also, I am grateful to Dr. Rapoport for being supportive not only as my committee member but also my graduate program director. I would like to offer my special thanks to Dr. Ronald Millard. He helped and guided me to get through the most terrible time of my graduate study. I would not be able to join Simon’s lab and achieve what I have made now without him. I would also like to thank Nancy for her endless help during both my graduate program transition and process towards graduation. During the past four years, I have met amazing people in Hogan Lab, and I would like to express my thankfulness individually. Taeko, thank you for sharing your experiences and stupid videos with me all the time, I always enjoyed the time we spent together. Simone, thank you for your guidance on experiments and your company to different places in Cincinnati. It is sort of sad that we are not going to drink KSFM when I graduate. Amna, we have been together to go through both foundation and numerous dreadful lab meetings, I think this would be enough to keep our friendship for a long time. Lisa, thank you for always being like a mom and take care of me, I wish I could bring you more exotic v food to try. Now I’m starting to run out of spaces, but I still want to thank Jazib, Yanfen, Sunil, Varsha, Andy, Justin, Heather, Nianrong, David, Ania, and Marjan. We have shared amazing memories together, and thank you all for your friendship. I would also like to thank the great people in Division of Allergy and Immunology at Cincinnati Children’s Hospital Medical Center who offered me help and friendship during my training as a graduate student. Special thanks to Julie, Nurit and Mark Rochman, who have given me useful advice on both scientific problems and career development. Finally, I would like to thank my mom Zhi, my aunt Tun, my boyfriend Yongkun, my friend Yu and Jiuzhou, and all my other friends and families for their unconditional support and love. vi Table of Contents Regulation of esophageal epithelial function in Eosinophilic Esophagitis ..................................................... i Abstract ......................................................................................................................................................... ii Acknowledgements ....................................................................................................................................... v Table of Contents ........................................................................................................................................ vii List of Abbreviations .................................................................................................................................... xi List of Figures and Tables ............................................................................................................................xiii 1 Chapter I: Introduction ....................................................................................................................... 15 1.1 Eosinophilic Esophagitis .............................................................................................................. 15 1.1.1 Introduction ........................................................................................................................ 15 1.1.2 Pathogenesis of EoE ............................................................................................................ 15 1.1.3 Therapy ............................................................................................................................... 25 1.1.4 Summary ............................................................................................................................. 28 1.2 Interleukin‐13 (IL‐13) .................................................................................................................. 29 1.2.1 Introduction ........................................................................................................................ 29 1.2.2 Production of IL‐13 .............................................................................................................. 29 1.2.3 IL‐13 signaling pathway ....................................................................................................... 29 1.2.4 IL‐13 function under physiological and pathological conditions ........................................ 32 1.2.5 IL‐13 in EoE .......................................................................................................................... 33 1.2.6 IL‐13‐related treatment ...................................................................................................... 34 vii 1.2.7 Summary ............................................................................................................................. 34 1.3 Ion transporters .......................................................................................................................... 36 1.3.1 Introduction of Ion transporters ........................................................................................
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