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ABSTRACT TOKARZ, DEBRA ANN. Tripartite Motif Containing 9 As A

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ABSTRACT TOKARZ, DEBRA ANN. Tripartite Motif Containing 9 As A ABSTRACT TOKARZ, DEBRA ANN. Tripartite Motif Containing 9 as a Novel Innate Immune Response Gene and Mediator of Macrophage Migration. (Under the direction of Dr. Jeffrey Yoder). Innate immunity provides the first line of defense against invading pathogens. Pathogens are sensed by pattern recognition receptors (PRRs) present on cells throughout the body, which recognize diverse pathogen associated molecular patterns (PAMPs). Activation of PRRs by PAMPs turns on the expression of numerous genes that regulate innate immune responses. A critical component of this response is the recruitment and activation of macrophages and neutrophils to sites of infection and inflammation. These phagocytic cells are highly adept at engulfing and killing pathogens, as well as recruiting additional inflammatory cells. However, excessive or prolonged infiltration of tissues by neutrophils and macrophages is implicated in the pathogenesis of a variety of inflammatory disorders. Therefore, understanding how migration of these leukocytes is regulated will facilitate the design of novel therapeutic strategies for modulating inflammation. Recently, we identified tripartite motif containing 9 (trim9) as a gene with increased transcript levels in larval zebrafish following exposure to different PAMPs. TRIM9 is a member of the tripartite motif family of RING E3 ubiquitin ligases. TRIM9 is highly expressed in neurons and, until recently, was considered to be brain specific. We find that TRIM9 is expressed in larval zebrafish neutrophils and macrophages and in human myeloid cell lines, and responds transcriptionally to PAMP stimulation in these cells. In neurons, TRIM9 serves as an intracellular mediator of axon migration in response to the chemoattractant Netrin, a role conserved in invertebrates and mammals. We hypothesized that TRIM9 may act as a mediator of migration in neutrophils and macrophages during the immune response. Using mosaic transgenic zebrafish larvae we show that disrupting Trim9 ubiquitin ligase activity in macrophages alters cell morphology and significantly reduces in vivo macrophage migration. Our findings support a role for TRIM9 in mediating migration in cell types other than neurons, and suggest that ubiquitination of TRIM9 substrates may be critical for this function in macrophages. Identifying the TRIM9 protein interaction network in macrophages will further elucidate the mechanisms of TRIM9 function within these cells and reveal new potential targets for modulating leukocyte migration and inflammation. © Copyright 2016 Debra Ann Tokarz All Rights Reserved Tripartite Motif Containing 9 as a Novel Innate Immune Response Gene and Mediator of Macrophage Migration by Debra Ann Tokarz A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Comparative Biomedical Sciences Raleigh, North Carolina 2016 APPROVED BY: _______________________________ _______________________________ Dr. Jeffrey A. Yoder Dr. Samuel Jones Committee Chair _______________________________ _______________________________ Dr. Matthew Koci Dr. Barbara Sherry DEDICATION To my parents, for their faith in me and To my husband, for his patience ii BIOGRAPHY Debra Tokarz was raised in southeast Michigan by her parents, Ray and Sandi, along with her younger brother J.R. and her older half-siblings Rebecca, Bryant, Andrew, Matt, Kristopher, and James. Having been preceded in the world by her hell-raising eldest brothers who brought notoriety to the Tokarz name in her home town of St. Clair Shores, Debra was forced to become an exceptionally well-behaved child. This she accomplished by spending most of her time with her nose in books, a habit that lead to extremely thick glasses and early aspirations of becoming a writer. However, she fell in love with biology in junior high, and hasn’t looked back since. Debra attended college at the University of Michigan in Ann Arbor, MI, where she earned a B.S. in Cell and Molecular Biology. While there she worked as a research assistant in the laboratory of Dr. John Fink, where she helped analyze genes for mutations associated with inherited neurologic disorders. So began her interest in biomedical research, which continues to this day. Forced to come to grips with choosing an actual career path as college came to an end, she fell back on her love of animals and chose to attend veterinary school at Michigan State University. Through her courses at vet school and through summer research projects, Debra was introduced to veterinary pathology and chose to specialize in this area. She completed a residency in anatomic veterinary pathology at the Veterinary Medical Teaching Hospital at the University of California-Davis and was boarded in September 2011. iii During her pathology residency, Debra learned about host-pathogen interactions and the immune response. She decided she wanted to learn more and chose to do her PhD training at North Carolina State University in the lab of Dr. Jeff Yoder. In the Yoder lab, Debra has enjoyed doing great research with great people and a great model organism, the zebrafish. Upon completing her PhD training, she is very excited to be staying on at NC State as a comparative veterinary pathologist. iv ACKNOWLEDGMENTS I am incredibly grateful for the outstanding mentorship and guidance provided by my advisor, Dr. Jeff Yoder. I am also grateful for the help and insight provided by my committee members: Dr. Sam Jones, Dr. Barbara Sherry, and Dr. Matthew Koci. I am also thankful for funding support provided by the NCSU Comparative Medicine and Translational Research Training Program (NIH T32 OD011130) and the American Association of Immunologists Careers in Immunology Fellowship. To all the members of the Yoder lab – Ivan, Hayley, Jess, Dustin, Amanda, Amyn, Ashley – you made it a joy to come to lab each day! Many others shared their technical expertise with me throughout this work and I am grateful for their help, especially Shannon Chiera, Dr. Katie Sheats, Dr. Stephanie Gupton, and Dr. Eva Johannes. I would never be where I am today without the love and support of my family, especially my parents, Ray and Sandi Tokarz, who have stood by me 100% in all of my endeavors. Finally, I cannot express enough how thankful I am for my husband, Kip. His love, his patience, and his humor have all been a source of strength for me through all of my training. He truly deserves a degree for each one he has seen me through! v TABLE OF CONTENTS LIST OF TABLES ............................................................................................................... ix LIST OF FIGURES ............................................................................................................. x INTRODUCTION................................................................................................................ 1 CHAPTER 1: LITERATURE REVIEW .......................................................................... 4 The Innate Immune Response ...................................................................................... 4 Pattern Recognition Receptor Signalling .................................................................. 4 Toll-like Receptors (TLRs) .................................................................................. 5 Retinoic Acid-Inducible Gene I-Like Receptors (RLRs) .................................... 8 Nucleotide Binding Domain and Leucine Rich Repeat Containing Receptors (NLRs) .......................................................................................... 10 The inflammatory response ........................................................................................ 14 Macrophage and Neutrophil Migration..................................................................... 16 Chemotaxis: a Primer ........................................................................................... 17 Extravasation........................................................................................................ 18 Intracellular Signaling and Cytoskeletal Dynamics of Chemotaxis .................... 23 The Zebrafish as a Model of Innate Immunity ........................................................... 26 Tripartite Motif (TRIM) Protein Family ..................................................................... 28 Ubiquitination ............................................................................................................ 29 TRIMs and Innate Immunity ...................................................................................... 31 Tripartite Motif Containing 9 (TRIM9) ..................................................................... 33 vi References ....................................................................................................................... 43 CHAPTER 2: DISRUPTION OF TRIM9 FUNCTION ABROGATES MACROPHAGE MOTILITY IN VIVO .......................................................................................................... 71 Abstract ........................................................................................................................... 73 Introduction .................................................................................................................... 74 Materials and Methods .................................................................................................. 77 Results ............................................................................................................................
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