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Zepp Thesis Merged Document with References THE REGULATORY ROLES OF TRAF4 IN IL-17 RECEPTOR MEDIATED SIGNALING AND DISEASE PATHOLOGY BY JAROD A. ZEPP Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Xiaoxia Li Department of Molecular Medicine CASE WESTERN RESERVE UNIVERSITY August 2014 1 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jarod A. Zepp candidate for the degree of DOCTOR OF PHILOSPHY (signed) Committee Chair Timothy Nilsen, Ph.D. Committee Member, Advisor Xiaoxia Li, Ph.D. Committee Member Serpil Erzurum, M.D. Committee Member Paul Fox, Ph.D. Committee Member Olga Stenina-Adognravi, Ph.D. Date of Defense May 30, 2014 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Copyright © 2014. Jarod A. Zepp Copyright © 2012. The American Association of Immunologists, Inc. All rights reserved 3 Dedication To my courageous, compassionate and devoted mother. To my grandparents Arthur & Ann Thompson. To my grandfather Robert Zepp. 4 Table of Contents: ♦ Title page...................................................................................................................1 ♦ Committee signature sheet.................................................................................. 2 ♦ Dedication page...................................................................................................... 3 ♦ Table of Contents................................................................................................... 4 ♦ List of Figures.......................................................................................................... 7 ♦ Acknowledgements................................................................................................ 9 ♦ Abstract ♦ Chapter 1: Introduction to the Interleukin-17 cytokine family................13 I. Interleukin-17 cytokine family.........................................................................14 II. ACT1; A critical adaptor protein for IL-17R signaling............................... 16 a. Discovery of ACT1...................................................................................... 16 b. The STIR domain superfamily................................................................... 18 c. ACT1 is a U-box type E3 ubiquitin ligase................................................ 19 d. ACT1, the double-edged sword................................................................. 22 III. IL-17 and IL-25-associated disease pathology............................................. 26 a. Pathogenic Th17 cells and Autoimmune Diseases................................26 b. Th17 and IL-17 in EAE, a murine model for the immunopathogenesis of multiple sclerosis............................................. 27 c. IL-17-induced ACT1-mediated signaling in CNS resident cells is necessary for Th17-induced CNS inflammation and EAE..................32 d. IL-17 and IL-25 mediate allergic airway inflammation and airway hyperresponsiveness................................................................................... 34 IV. IL-17 and IL-25 signaling................................................................................ 38 a. TNF-receptor associated factor (TRAF) protein family.......................38 b. IL-17 and IL-25 signaling, a current overview........................................41 ♦ Chapter 2: TNF Receptor-associated factor 4 restricts IL-17-mediated pathology and signaling processes................................... 44 I. Abstract............................................................................................................... 45 II. Introduction....................................................................................................... 46 III. Materials and Methods..................................................................................... 48 IV. Results a. IL-17–induced signaling and gene expression are increased in TRAF4- deficient cells................................................................................................52 b. TRAF4 deficiency exacerbates EAE severity......................................... 56 c. ACT1-TRAF4 complex is distinct from other ACT1-TRAF complexes..................................................................................................... 60 d. TRAF4 competes with TRAF6 for ACT1 TRAF binding sites.......... 62 V. Discussion.......................................................................................................... 68 5 ♦ Chapter 3: TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation............................... 71 I. Abstract...............................................................................................................72 II. Introduction.......................................................................................................73 III. Materials and Methods..................................................................................... 76 IV. Results a. Differential requirements for TRAF proteins in the IL-25 response.83 b. TRAF4 is required for IL-25-dependent airway inflammation........... 86 c. TRAF4 is required for IL-25 responses in T-cells and airway epithelial cells................................................................................................................ 9 4 d. TRAF4 and SMURF2 cooperate to mediate the IL-25 response..... 102 e. TRAF4 and SMURF2 are required for IL-25-induced degradation of the inhibitory molecule DAZAP2..........................................................108 f. IL-25R recruits DAZAP2 through p-Tyr 355 residing within the SEFIR domain...........................................................................................117 g. IL-25R Y355F mutant mediates IL-25 response in the absence of TRAF4........................................................................................................ 122 V. Discussion.........................................................................................................124 ♦ Chapter 4: Conclusions and future study.....................................................129 I. Introduction......................................................................................................130 II. TRAF4 in the IL-17 signaling pathway........................................................ 131 III. TRAF4/SMURF2 in the IL-25 pathway......................................................132 IV. Future study...................................................................................................... 134 a. TRAF4 as a therapeutic target in inflammatory diseases.................... 134 b. IL-25R-dependent STAT activation.......................................................135 c. IL-25R as a therapeutic target and biomarker in respiratory Disorders.....................................................................................................137 V. Final Remarks................................................................................................... 138 ♦ Appendix 1.............................................................................................................140 ♦ Appendix 2............................................................................................................. 141 ♦ Appendix 3............................................................................................................. 142 ♦ Bibliography..........................................................................................................144 6 List of Figures Figure 1-1 IL-17 cytokine and receptor family............................................15 Figure 1-2 Act1 structure and IL-17 signaling cascade..............................18 Figure 1-3 Th17 cells in the Initiation and effector stages of EAE.........31 Figure 1-4 IL-17 and IL-25 in airway inflammation.................................. 37 Figure 1-5 TNF-receptor Associated Factor (TRAF) Family.................. 40 Figure 1-6 IL-17 and IL-25 signaling....................................................43 Figure 2-1 A-B, IL-17 induced signaling and gene expression in TRAF4-deficient primary cells...................................................54 Figure 2-1 C-E, IL-17 induced signaling and gene expression in TRAF4-deficient primary cells...................................................55 Figure 2-2 A, TRAF4 deficiency exacerbates Th17 mediated EAE....... 57 Figure 2-2 B, TRAF4 deficiency exacerbates Th17 mediated EAE....... 58 Figure 2-2 C-E, TRAF4 deficiency exacerbates Th17 mediated EAE.. 59 Figure 2-3 Distinct Act1-TRAF interactions form following IL-17 stimulation.....................................................................................61 Figure 2-4 A-B, TRAF4 restricts Act1/TRAF6 interaction.................... 64 Figure 2-4 C-D, TRAF4 restricts Act1/TRAF6 interaction................... 65 Figure 2-4 E, TRAF4 restricts Act1/TRAF6 interaction........................ 66 Figure 2-5 Working model of IL-17 signaling cascade............................. 67 Figure 3-1 Screening for TRAF involvement in the IL-25R-dependent response.......................................................85 7 Figure 3-2 A-B, TRAF4 regulates IL-25 responses in vivo........................87 Figure 3-2 C, TRAF4 regulates IL-25 responses in vivo............................ 88 Figure 3-2 D-E, TRAF4 regulates IL-25 responses in vivo....................... 89 Figure 3-3 Enhanced IL-17A-induced
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