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Interrogating Raf-1 Kinase Inhibitor Protein (RKIP) As a Novel Therapeutic Target For

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Interrogating Raf-1 Kinase Inhibitor Protein (RKIP) As a Novel Therapeutic Target For Interrogating Raf-1 Kinase Inhibitor Protein (RKIP) as a Novel Therapeutic Target for Modulating Inflammatory Responses Kyle T. Wright, M.D., Ph.D. University of Connecticut, 2016 The studies presented here were designed to test if Raf-1 kinase inhibitor protein (RKIP) plays a functionally significant role in immunity, and to interrogate its possibility of providing a novel therapeutic target for modulating inflammatory responses. Based on previous studies from other laboratories that attributed negative regulatory functions to RKIP in the context of MAPK and NF-κB signaling in cell lines, we tested the hypothesis that its function was to suppress the production of pro-inflammatory cytokines, proliferation, and cell survival. However, after extensive investigation, our data clearly demonstrate that RKIP is actually necessary for the production of certain cytokines, namely Type-I and Type-II interferons, but had less robust effects on cell survivability and proliferation. Specifically, this work shows that RKIP is integrated in the signaling pathway downstream of TCR triggering in CD8+ T cells and TLR ligation in APCs. Finally, these studies highlight RKIP as a druggable protein, and through its targeted inhibition, cytokine responses can be significantly diminished. Thus, this provides elementary rationale for its potential clinical applicability in therapeutic interventions for inflammatory diseases, especially those associated with dysregulated IFN responses. Through this current work, we have provided a solid foundation for future studies that seek to investigate further the molecular mechanisms of RKIP function within the immune system, as well as its advancement into clinically relevant inflammatory disease models. i Interrogating Raf-1 Kinase Inhibitor Protein (RKIP) as a Novel Therapeutic Target for Modulating Inflammatory Responses Kyle T. Wright B.S., University of Illinois Urbana-Champaign, 2008 A Dissertation Submitted in Partial Fulfillment of the Doctor of Philosophy at the University of Connecticut 2016 ii Copyright by Kyle T. Wright 2016 iii APPROVAL PAGE Doctor of Philosophy Dissertation Interrogating Raf-1 Kinase Inhibitor Protein (RKIP) as a Novel Therapeutic Target for Modulating Inflammatory Responses Presented by: Kyle T. Wright Major Advisor_____________________________________________________ Anthony T. Vella, Ph.D. Associate Advsior__________________________________________________ Robert B. Clark, M.D. Associate Advsior__________________________________________________ Lynn Puddington, Ph.D. Associate Advsior__________________________________________________ Thiruchandurai V. Rajan, M.D., Ph.D. University of Connecticut- 2016 iv ACKNOWLEDGEMENTS First and foremost, I would like to thank Tony for his invaluable guidance and support throughout the development of this thesis project, as well as challenging me both intellectually and personally during my time in his laboratory. Not only did he bolster my training of how to think about and conduct scientific research, but he also taught me how to be a scientist. This is perhaps his greatest gift to me, and for this I will always be grateful. I would also like to thank my thesis committee members Drs. Robert Clark, Lynn Puddington, and T.V. Rajan for their support and ever poignant feedback during the course of our scientific discussions. I am also grateful to the members of the Vella Lab group which have helped guide and strengthen not only my project through intelligent discussions and (occasionally pointed) feedback, but also my desire to learn, teach, and advocate science for the long-term. I’ve learned so much from them: Drs. Adam Adler, Francisco Sylvester, Antoine Ménoret, Soo-Mun Ngoi, Sanjeev Kumar, Andrew Draghi, Raghunath Ramanarasimhaiah, Naomi Tsurutani, Wenhai Liu, and all other past and present lab members. Most importantly, I would like to thank my mom for instilling in me at an early age a desire to always strive for greatness in everything I do, but never at the cost of doing it the right way; and also to my wife, Bo, for providing unwavering emotional and intellectual support throughout this process. I can only ever hope to be half as good at what I do, as what she does. Finally, I want to acknowledge all of my family and friends that are constant reminders of the fact that although having a strong devotion to your craft is important, it’s equally as important to, every once in a while, sit on the couch with a bottle of wine and watch a good movie. v TABLE OF CONTENTS LIST OF TABLES ------------------------------------------------------------------------------------ viii LIST OF FIGURES ------------------------------------------------------------------------------------ ix CHAPTER 1 INFLAMMATION IN HEALTH AND DISEASE: HOW UNDERSTANDING DIFFERENT INFLAMMOMES MAY LEAD TO ENHANCED THERAPUETIC INNOVATION AND CLINICAL IMPLEMENTATION -------------------------------------------------- 1 I. Introduction ----------------------------------------------------------- 2 II. The Major Inflammomes ----------------------------------------- 4 A. Innate (TNF Dominant) ----------------------------------------- 4 B. Innate (IFN Dominant) ----------------------------------------- 6 C. Innate (Inflammasome Dominant) -------------------------------- 7 D. Adaptive (T cell Centric) ----------------------------------------- 9 E. Adaptive (B cell Centric) ----------------------------------------- 13 F. Reverse-Phase Immunity ----------------------------------------- 13 III. RKIP: A New Therapeutic Target? -------------------------------- 14 IV. Conclusions ----------------------------------------------------------- 16 V. Figures ----------------------------------------------------------- 18 CHAPTER 2 MATERIALS AND METHODS ----------------------------------------- 22 CHAPTER 3 RKIP CONTRIBUTES TO IFNγ SYNTHESIS BY CD8+ T CELLS AFTER SERIAL TCR TRIGGERING IN SYSTEMIC INFLAMMATORY RESPONSE SYNDROME ---------------------- 34 I. Abstract ----------------------------------------------------------- 35 II. Introduction ----------------------------------------------------------- 36 III. Results ----------------------------------------------------------- 39 IV. Discussion ----------------------------------------------------------- 45 V. Figures and Tables -------------------------------------------------- 53 vi CHAPTER 4 RKIP IS INVOLVED IN DOWNSTREAM SIGNALING AFTER PATTERN RECOGNITION RECEPTOR ENGAGEMENT, IMPACTING CYTOKINE PRODUCTION ----------------------- 72 I. Abstract ----------------------------------------------------------- 73 II. Introduction ----------------------------------------------------------- 74 III. Results ----------------------------------------------------------- 76 IV. Discussion ----------------------------------------------------------- 83 V. Figures and Tables -------------------------------------------------- 90 CHAPTER 5 RKIP IS NECESSARY FOR THE GENERATION OF OPTIMAL TYPE I INTERFERON RESPONSES DURING B CELL ACTIVATION BY TLR LIGANDS ------------------------------ 101 I. Abstract --------------------------------------------------------- 102 II. Introduction --------------------------------------------------------- 103 III. Results --------------------------------------------------------- 105 IV. Discussion --------------------------------------------------------- 114 V. Figures and Tables ------------------------------------------------ 120 CHAPTER 6 DISCUSSION: RKIP IS AN IMPORTANT, CO-EVOLVED REGULATOR OF INTERFERON RESPONSES AND MAY PROVIDE A NOVEL TARGET FOR INTERVENTIONAL CYTOKINE-BASED THERAPY --------------------------------------- 143 I. Introduction --------------------------------------------------------- 144 II. RKIP drives Type-I interferon responses after nucleic acid sensing: A working model ------------------------------------------------ 145 III. Establishing RKIP’s role within the immune response --- 147 IV. The great arms race: viral evasion, interferons, and RKIP co- evolution --------------------------------------------------------- 150 V. Future Directions ------------------------------------------------ 152 VI. Locostatin: A new cytokine-based therapeutic intervention? --- 154 VII. Conclusions & Figures --------------------------------------- 156 REFERENCES ------------------------------------------------------------------------------------ 166 vii LIST OF TABLES TABLE I Normal clonal expansion, contraction, and survival in RKIP-/- mice after SEA-LPS Immunization -------------------------------------------------- 71 TABLE II Bioinformatic Analysis of RKIP in Mus musculus --------------------- 100 TABLE III Ingenuity Analysis of Pathways Enriched in RKIP-/- B Cells --- 136 TABLE IV Altered Genes Important for Mitochondrial Function in RKIP-/- B Cells ------------------------------------------------------------------------------------ 138 TABLE V Altered Genes Important for TLR-Type I IFN Signaling in Naïve RKIP-/- B Cells ------------------------------------------------------------------ 139 TABLE VI Normalization of TLR-Type I IFN Genes after TLR Stimulation in RKIP-/- B Cells ------------------------------------------------------------------ 142 viii LIST OF FIGURES Figure 1-1 An inflammome-based disease taxonomy -------------------------------- 18 Figure 1-2 RKIP: A regulator of kinase-mediated signaling cascades -------------- 20 Figure 3-1 RKIP is necessary for optimal production of IFNγ by splenocytes during SIRS ----------------------------------------------------------------------------- 51 Figure 3-2 RKIP-/-
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