The Role of LRRC31 & LRRC32 in Allergic Inflammation A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) In the Immunology Graduate Program of the College of Medicine 2015 by Rahul Joseph D’Mello B.S., The Johns Hopkins University, 2009 Advisory Committee: Marc E. Rothenberg, M.D., Ph.D. (Chair) H. Leighton Grimes, Ph.D. Andrew B. Herr, Ph.D. Simon P. Hogan, Ph.D. Ian P. Lewkowich, Ph.D. Louis J. Muglia, M.D., Ph.D. ABSTRACT Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus that is caused by both genetic and environmental factors. Herein, we investigated leucine-rich repeat– containing protein 31 (LRRC31), a protein that regulates esophageal epithelial function, and LRRC32, which was genetically associated with EoE and allergic diseases. We show that LRRC31 increased in the esophagus of patients with active EoE. LRRC31 mRNA and protein were increased in differentiated, IL-13–treated esophageal epithelial (EPC2) cells grown at the air liquid interface (ALI). LRRC31 overexpressing EPC2 cells had increased epithelial barrier function and RNA sequencing analysis identified 38 dysregulated genes, including 5 kallikrein (KLK) proteases. Indeed, KLK protein and proteolytic activity levels were decreased in LRRC31- overexpressing EPC2 cells. KLK expression was similarly dysregulated in the esophagus of EoE patients and in IL-13–treated esophageal epithelial cells. Thus, we propose that LRRC31 is induced by IL-13 and modulates epithelial barrier function, potentially by regulating KLK expression. LRRC32 is an immune-related protein that is similar in structure to LRRC31. We identified rs2155219, a single nucleotide polymorphism (SNP) associated with EoE that was an enhancer of gene transcription. The minor allele at rs2155219 decreased the risk of developing EoE and increased esophageal expression of LRRC32 mRNA in EoE patients. In addition, IL-13 induced LRRC32 mRNA expression in EPC2 cells. We propose that LRRC32 is important for disease pathogenesis and decreased esophageal expression may correlate with lower risk of EoE. In conclusion, LRRC31 and LRRC32 are both involved in two independent pathways contributing to EoE pathogenesis. A further understanding of their roles in EoE and allergic diseases may facilitate the development of alternative therapeutics to improve the quality of life for patients. ! ii! ! iii! ACKNOWLEDGEMENTS I want to thank God, my parents Gilroy and Cheryl, my sister Esther, Dusty, and all my family and friends, especially my roommate Carlton, for getting me to this point in my life. I am grateful for my mentor Dr. Marc Rothenberg, Mel Mingler, Dr. Julie Caldwell, Dr. Ben Davis, Dr. Mark Rochman, Jared Travers, Kiran KC, Dr. Nurit Azouz, Dr. Ting Wen, Dr. Joe Sherrill, Dr. Tom Lu, and everyone in the Allergy & Immunology family who invested time, energy, and resources in my training and development as a scientist and a co-worker. I am grateful to the Medical Scientist Training Program and the Immunology Graduate Program, who helped guide and support me through medical and graduate school. I want to thank the mentors on my dissertation committee, Dr. Simon Hogan, Dr. Lee Grimes, Dr. Ian Lewkowich, Dr. Andy Herr, and Dr. Louis Muglia for their support, and Dr. Jim Heubi, who continues to guide me towards success. There were many times where I struggled but your patience and belief in me inspired me to persevere forward towards completion of my PhD. I want to thank my other mentors from all levels of my training, starting from high school with Mr. Bob Berg my biology teacher, to the Cleveland Clinic with Dr. Anthony Calabro, Dr. Aniq Darr, Dr. Ediuska Laurens, and Stephon Weber, at Hopkins with Dr. Sharon Gerecht and Dr. Guoming Sun, and here at University of Cincinnati and Cincinnati Children’s. I want to thank all the patients with EoE who participate in our studies – thank you for participating and sharing your tissue with us. I also want to thank all the administrative staff, especially Terry Fettig, Dr. Rothenberg’s administrative assistant, who helped coordinate meeting times and scheduling, and everyone I know who wished me the best on this endeavor. I would like to dedicate this dissertation to the memory of my grandparents Frank, Ettie, Selina, and Reginald. Thank you and God bless all of you. ! iv! TABLE OF CONTENTS ABSTRACT…………………………………..……………………………………………………………ii ACKNOWLEDGEMENTS…………………………..…………………………………………………...iii TABLE OF CONTENTS…………………………………..…………...………………………………..iv LIST OF FIGURES……………………………..……………………………………………………..…ix LIST OF TABLES……………………………..……………………………………………...………….xi LIST OF ABBREVIATIONS……………………..…………………………………………...…….…..xii PUBLICATIONS AND CONFERENCE PROCEEDINGS ARISING FROM THIS WORK………xiii STATEMENT OF AUTHORSHIP…………………………..………………………………….……..xiv CHAPTER 1: INTRODUCTION………………………………….……..……………………………….1 1.1. Allergic Diseases………………………………………………………..……………………….1 1.1.1. Introduction……………………………………….…….…..…..……………….1 1.1.2. Epidemiology of Allergic Diseases…..………...………...…..……………….1 1.1.3. General Pathogenesis of Allergic Diseases…………...…..……….……….2 1.1.4. Summary………………………………………………..…..….……...…….….3 1.2. Eosinophilic Esophagitis (EoE)..………………………………………...….……………….…4 1.2.1. Introduction……………………………………………..…………………….…4 1.2.2. History of EoE………...……………………...…………………………………4 1.2.3. Pathogenesis of EoE……………………………………………..……………5 1.2.4. EoE Therapy……………………………...………………...…………………12 1.2.5. Summary………………………………………………………….……………15 1.3. Epithelial Cells……..……………………………………………………………………………17 1.3.1. Introduction…………………………………………………………….………17 1.3.2. Epithelial Development...…………………...……………..…………………17 1.3.3. Epithelial Differentiation…………………………..……………….........……18 ! v! 1.3.4. Epithelial Barrier Function…………………………………………………....19 1.3.5. Epithelial Immunity……………………………………………..……………..19 1.3.6. Summary……………………………..……………………..…………………21 1.4. Interleuking-13 (IL-13)………….………………………………..……………………….……23 1.4.1. Introduction…………..……………………………………..………….………23 1.4.2. IL-13 Cytokine and IL-13 Receptor Structure…………….………......……23 1.4.3. Regulation of IL-13……………………………………………………………24 1.4.4. IL-13 Function…………………………………………………………………25 1.4.5. IL-13 in EoE………………………………………………………………..….26 1.4.6. Summary………………………………………..…………..…………………27 1.5. Kallikreins (KLKs)………………………………….………….……………..…………………28 1.5.1. Introduction……………………………………………….....…………………28 1.5.2. Serine Proteases……………………….…………………..…………………28 1.5.3. KLKs………………………………….……….……………..…………………29 1.5.4. Regulation of KLKs………………………………………..…………….……29 1.5.5. Epithelial KLKs….………………………………………..………...…………30 1.5.6. Summary………………………………………..…………..…………………31 1.6. Leucine-Rich Repeat (LRR) Domains……………………………………..…………………32 1.6.1. Introduction……………..…………………………………..……...…….……32 1.6.2. LRR Structure……………...……..………………………..………….………32 1.6.3. LRR Function……………………………...………………..…………………32 1.6.4. Summary………………………………………………..…………..…………33 1.7. Leucine-Rich Repeat–Containing Protein 32…………………..…………………….……..35 1.7.1. Introduction……………………………………..…………..….………………35 1.7.2. LRRC32 Structure………...…………………………..………………………35 1.7.3. LRRC32 Function…………………………………………..…………………36 ! vi! 1.7.4. Summary…………………………..………………………..…………………37 1.8. References…...……………………………………………..………………..…………………38 CHAPTER 2: LRRC31 IS INDUCED BY IL-13 AND REGULATES KALLIKREIN EXPERSSION AND BARRIER FUNCTION IN THE ESOPHAGEAL EPITHELIUM…………………………...,…68 2.1. Abstract…...……………………………………………………………………………………..69 2.2. Introduction……..……………………………………………………………………………….70 2.3. Results….…………………………………………………………………………………….…72 2.3.1. Identification of LRRC31….……………………………………………….…72 2.3.2. LRRC31 is specifically induced in EoE……………………….………….…72 2.3.3. LRRC31 is expressed in the colon and mucosal tissue………….…….…72 2.3.4. LRRC31 mRNA parallels disease activity……………………………….…73 2.3.5. LRRC31 mRNA correlates with esophageal eosinophilia and IL13 mRNA…………………………………………………………………………..73 2.3.6. IL-13 induces LRRC31 in epithelial cells….…………………….……….…74 2.3.7. Overexpression of LRRC31 increases barrier function……………..…….76 2.3.8. LRRC31 regulates epithelial serine proteases….…………………...….…77 2.3.9. Loss of LRRC31 increases KLK expression….………………...……….…78 2.3.10. KLK expression in EoE and in response to IL-13….………..…………….79 2.4. Discussion….……………………………………………………………………...……………80 2.5. Methods…………………………………………………………………….……...……………84 2.6. Acknowledgements……..……………………………………………….……...……..………88 2.7. References…...…………………………………………………………….……...……………89 2.8. Figures…………………………………………………………………….……...……….…….95 ! vii! CHAPTER 3: ESOPHAGEAL LRRC32 EXPRESSION IS REGULATED BY ALLELIC VARIATION AT rs2155219……………………………………..……………………...……….……112 3.1. Abstract…..……………………………………………………………….……...……………113 3.2. Introduction……..…………………………………………..……………….……...…………114 3.3. Results……………………….……………………………………….……...………………...117 3.3.1. EoE shares genetic associations with other allergic diseases……….…117 3.3.2. Chromosome 11q13 has SNPs associated with allergic diseases…….117 3.3.3. Epigenetic and transcriptional landscape at rs2155219……...…………118 3.3.4. Expression of LRRC32, C11ORF30, and CAPN5 in the esophagus ....119 3.3.5. Expression of LRRC32, C11ORF30, and CAPN5 in epithelial cells.…..120 3.4. Discussion….…………………………………………………………...…...………………..122 3.5. Methods…………………………………...………………………….……...………………..127 3.6. Acknowledgments…..……...……………………….……………….……...………………..131 3.7. References…..……………………………………………………….……...………………..132 3.8. Figures....…………………………………………………………….……...…………………139 CHAPTER 4: GENERAL DISCUSSION