TPL2 (MAP3K8) REGULATES THE MIGRATION, DIFFERENTIATION, AND FUNCTION OF CRITICAL INNATE IMMUNE CELLS DURING THE INFLAMMATORY RESPONSE by SEAN MICHAEL ROWLEY (Under the Direction of Wendy Watford) ABSTRACT The protein kinase Tpl2 (MAP3K8) regulates innate inflammatory responses and is being actively pursued for therapeutic inhibition during chronic autoimmunity. Herein, we addressed the contribution of Tpl2 to pro-inflammatory responses of NK cells and macrophages. Despite the Tpl2-dependent regulation of IFN secretion by CD4+ T-cells, NK cell IFNγ production, STAT4 expression, and expression of cytotoxic machinery occurred independently of Tpl2. In contrast, tpl2-/- macrophages were functionally defective, as they displayed impaired chemokine and chemokine receptor expression following LPS stimulation and were defective in migrating in vivo to inflamed tissues. Tpl2-/- macrophages were also impaired in their differentiation towards a pro-inflammatory phenotype (M1) while conversely displaying an enhanced anti-inflammatory phenotype (M2). Overall, this work provides additional support for targeting Tpl2, through its effects on macrophage recruitment and differentiation, for the treatment of autoimmunity. INDEX WORDS: autoimmunity, inflammation, cytokines, chemokines, cellular trafficking, NK cells, macrophage classical activation, macrophage alternative activation TPL2 (MAP3K8) REGULATES THE MIGRATION, DIFFERENTIATION, AND FUNCTION OF CRITICAL INNATE IMMUNE CELLS DURING THE INFLAMMATORY RESPONSE by SEAN MICHAEL ROWLEY B.S., University of Georgia, 2009 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2012 © 2012 Sean Michael Rowley All Rights Reserved TPL2 (MAP3K8) REGULATES THE MIGRATION, DIFFERENTIATION, AND FUNCTION OF CRITICAL INNATE IMMUNE CELLS DURING THE INFLAMMATORY RESPONSE by SEAN MICHAEL ROWLEY Major Professor: Wendy Watford Committee: Liliana Jaso-Friedmann Donald Harn Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2012 v ACKNOWLEDGEMENTS I would like to thank the many people that have helped me along the way in completing this project. First, and foremost, I would like to thank Dr. Wendy Watford for her guidance and oversight on this project. Her advice has been invaluable and she has truly been an amazing mentor to study under. I would also like to thank Dr. Liliana Jaso-Friedmann for her advice and guidance on the project as well as Dr. Donald Harn for his guidance and for his gracious supply of experimental materials related to the work involving Schistosoma mansoni. I also would like to thank Smanla Tundup in the Harn Laboratory for his extensive help with the S. mansoni infection experiments. I would like to thank those in my lab including Teneema Kuriakose, Rebecca Kirkland, Nicole Acuff, and Xin Li for their help with day to day assistance in the laboratory. Additionally, I would like to thank the Animal Resources staff at both the Paul D. Coverdell Center for Biomedical Sciences and the College of Veterinary Medicine here at the University of Georgia for their help in maintaining our mice colonies. I also want to thank those in the CVM Cytometry Core Facility, particularly Barbara Reaves and James Barber, for their assistance related to use of the flow cytometer for experiments. Finally, I would like to thank my family including my parents Mike and Becky and my brother Chris for their ongoing support during my graduate studies. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION .........................................................................................................1 2 LITERATURE REVIEW ..............................................................................................2 Tpl2 Overview .........................................................................................................2 The natural killer (NK) cell and innate immunity ...................................................8 Innate cell trafficking during inflammation ...........................................................11 Macrophage differentiation: The M1 vs. M2 subtype ...........................................17 3 MATERIALS AND METHODS .................................................................................34 Mice .......................................................................................................................34 Media and Buffers..................................................................................................34 Cell Culture and Purification .................................................................................35 Cell stimulation ......................................................................................................38 Infection with X31 Influenza virus ........................................................................38 Schistosoma mansoni egg-induced inflammation ..................................................39 Cell counting ..........................................................................................................40 Cytokine measurement by ELISA .........................................................................40 vii RNA Isolation ........................................................................................................40 cDNA Reverse Transcription .................................................................................41 Quantitative Real-Time PCR .................................................................................41 Western Blotting ....................................................................................................43 Flow Cytometry .....................................................................................................43 Statistical Analysis .................................................................................................44 4 TPL2 IS DISPENSABLE FOR NK CELL DEVELOPMENT AND FUNCTION ...46 Introduction ............................................................................................................46 Results ....................................................................................................................47 Discussion ..............................................................................................................54 5 TPL2 PROMOTES MACROPHAGE CHEMOKINE/CHEMOKINE RECEPTOR EXPRESSION AND MACROPHAGE MIGRATION DURING INFLAMMATION ................................................................................................................................66 Introduction ............................................................................................................66 Results ....................................................................................................................67 Discussion ..............................................................................................................70 6 TPL2 REGULATES MACROPHAGE DIFFERENTIATION BETWEEN CLASSICAL (M1) AND ALTERNATIVE (M2) PHENOTYPES ............................80 Introduction ............................................................................................................80 Results ....................................................................................................................81 Discussion ..............................................................................................................85 7 CONCLUSIONS AND FUTURE DIRECTIONS .......................................................94 REFERENCES ..............................................................................................................................97 viii LIST OF TABLES Page Table 3.1: Antibodies used for FACS staining .............................................................................45 ix LIST OF FIGURES Page Figure 2.1: TLR4 signaling pathway .............................................................................................22 Figure 2.2: NK cell effector functions ...........................................................................................24 Figure 2.3: IL-12/IL-18/STAT4 signaling pathways leading to IFNγ production ........................26 Figure 2.4: Macrophage polarization during the inflammatory response ......................................28 Figure 2.5: Schistosoma mansoni life cycle ...................................................................................30 Figure 2.6: Immune responses to S. mansoni life cycle stages in the vertebrate host ...................32 Figure 4.1: Tpl2/MAP3K8 is expressed in lymphocytes including NK cells ...............................57 Figure 4.2: NK cell numbers are similar in the spleens of WT and tpl2-/- mice ............................58 Figure 4.3: Proportions of IFNγ+ NK cells are similar in WT and tpl2-/- mice..............................59 Figure 4.4: NK cell IFNγ secretion is not dependent on Tpl2 .......................................................60 Figure 4.5: STAT4 is differentially regulated by CD4+ T