The Role of Irgm1 in Mitochondrial Dynamics and Metabolism by Elyse Schmidt Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Gregory Taylor, Supervisor ___________________________ Jörn Coers ___________________________ Tso-Pang Yao ___________________________ Nancie MacIver ___________________________ David Pickup Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2017 i v ABSTRACT The Role of Irgm1 in Mitochondrial Dynamics and Metabolism by Elyse Schmidt Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Gregory Taylor, Supervisor ___________________________ Jörn Coers ___________________________ Tso-Pang Yao ___________________________ Nancie MacIver ___________________________ David Pickup An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2017 i v Copyright by Elyse Schmidt 2017 Abstract The Immunity-Related GTPases (IRG) are a family of proteins that are induced by interferon (IFN)-γ and play pivotal roles in immune and inflammatory responses. IRGs ostensibly function as dynamin-like proteins that bind to intracellular membranes, and promote remodeling and trafficking of those membranes. Prior studies have shown that loss of Irgm1 in mice leads to increased lethality to bacterial infections, as well as enhanced inflammation to non-infectious stimuli; however, the mechanisms underlying these phenotypes are unclear. In this dissertation, I studied the role of Irgm1 in mitochondrial biology and immunometabolism. Past studies of Irgm1’s human orthologue, IRGM, reported that IRGM localized to mitochondria and induced mitochondrial fragmentation. Further, absence of IRGM inhibited the cell’s ability to undergo IFN-γ and starvation induced autophagy and promoted the formation of elongated mitochondrial networks. In the first chapter, I confirmed that mouse Irgm1 shares IRGM’s mitochondrial localization, induced mitochondrial fragmentation, and that its absence promoted a mitochondrial hyperfused state. The structural determinants required for Irgm1’s mitochondrial localization and Irgm1-mediated mitochondrial fragmentation were also identified. In chapter two, I studied the metabolism of Irgm1-deficient embryonic fibroblasts and macrophages through a series of bioenergetic and metabolomic assays, and found a iv number of metabolic phenotypes in Irgm1-deficient cells suggesting enhanced proinflammatory activation. In chapter 3, I describe the increased pro-inflammatory cytokine production observed in our Irgm1-deficient macrophages. A series of metabolic studies indicated that the enhanced cytokine production was associated with marked metabolic changes in the Irgm1-deficient macrophages, including increased glycolysis and an accumulation of long chain acylcarnitines, whereas Irgm1-deficient macrophages exposed to the glycolytic inhibitor, 2-deoxyglucose, or fatty acid synthase inhibitors resulted in dampening of the excessive cytokine production. Finally, Irgm1-deficient mice displayed high levels of serum cytokines typifying profound auto-inflammation. Taken together, these results suggest that Irgm1-deficiency drives metabolic dysfunction in macrophages in a manner that is cell autonomous and independent of infectious triggers. This may be a significant contributor to excessive inflammation seen in Irgm1- deficient mice. v Dedication This dissertation is dedicated first to my grandfather, who has weathered more challenges, and accomplished more in his life by the age of thirty with an eighth-grade education that I have up to this point in my life. This is for you Opa. This manuscript is dedicated to my parents and my husband. Without their hard work and encouragement I would not be here. vi Contents Abstract ......................................................................................................................................... iv List of Figures .............................................................................................................................. xii 1. Introduction ............................................................................................................................... 1 1.1 Interferon Signaling in Innate Immunity ...................................................................... 1 1.1.1 Introduction to Interferon .......................................................................................... 1 1.1.2 Interferon Signaling .................................................................................................... 2 1.1.2.1 Type I Interferon Signaling Pathway ................................................................ 2 1.1.2.2 Type II Interferon Signaling Pathway ............................................................... 4 1.1.3 Interferons in Immunity ............................................................................................. 5 1.2 Immunity related GTPases .............................................................................................. 6 1.2.1 Features of the IFN-Inducible GTPases .................................................................... 6 1.2.2 The IRG Protein Family .............................................................................................. 8 1.2.2.1 The Proteomic and Structural Features of the IRG Family ............................ 8 1.2.2.2 IRG Subfamilies .................................................................................................... 9 1.2.3 The Murine IRGs ....................................................................................................... 11 1.2.3.1 Murine IRGs Interactions in Lipid and Membrane Binding ........................ 12 1.2.3.2 Murine IRGs in Intracellular Pathogen Defense ............................................ 13 1.2.3.3 Mechanisms of IRG-Mediated Pathogen Defense ......................................... 14 1.2.4 Functions of Murine Irgm1 ...................................................................................... 16 1.2.5 The Human IRGs ....................................................................................................... 20 vii 1.2.5.1 Parity of Function between Mouse Irgm1 and Human IRGM .................... 23 1.3 Immunometabolism of Proinflammatory Macrophages .......................................... 25 1.3.1 Modulation of Immunometabolism in Proinflammatory Macrophages. .......... 26 1.3.2 Glycolytic Metabolism drives M1 Mitochondrial Polarization .......................... 28 1.3.3 The Pentose Phosphate Pathway Acts as a Hub to Sustain Carbohydrate Metabolism and the Redox Demands of M1 Macrophages ......................................... 32 1.3.4 Fatty Acid Synthesis and Oxidation in Macrophage Polarization ..................... 34 1.3.5 The Tricarboxylic Acid Cycle is Broken in Two Places During M1 Macrophage Polarization ......................................................................................................................... 37 1.4 Mitochondrial Dynamics and Their Functions .......................................................... 39 1.4.1 Mechanisms of mitochondrial fission and fusion ................................................. 40 1.4.1.1 Mitochondrial Fission ........................................................................................ 40 1.4.1.2 Mitochondrial Fusion ........................................................................................ 42 1.4.2 Mitochondrial Dynamics in Response to Stress .................................................... 43 1.4.3 Mitochondrial Dynamics in Metabolism ............................................................... 45 1.5 Motivation for this Work ............................................................................................... 47 2. Irgm1-Deficiency in MEFs Causes Changes in Mitochondrial Morphology ................. 50 2.1 Introduction ..................................................................................................................... 50 2.2 Results .............................................................................................................................. 53 2.2.1 Evidence of Altered Mitochondrial Morphology in Irgm1-Deficient Enterocytes .......................................................................................................................... 53 2.2.2 A Subset of Irgm1 Localizes to the Mitochondria and Induces Mitochondrial Fragmentation ..................................................................................................................... 54 viii 2.2.3 Characterization of the Structural Elements Required for Irgm1’s Mitochondrial Localization and Irgm1-Dependent Mitochondrial Fragmentation . 57 2.2.4 Irgm1-Dependent Mitochondrial Fragmentation is Dependent on Drp1 Activity................................................................................................................................. 61 2.2.5 Mitochondrial Function in Irgm1-Deficient MEFs ............................................... 63 2.3 Discussion .......................................................................................................................