Sickness Behavior and the Metabolic Demand of Immunity: Insight from a Live Bacterial Infection Model. by Robert Michael Johnson A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 8, 2020 Keywords: Immunometabolism, Life-history Theory, Listeria monocytogenes Sickness Behavior, Trade-offs Copyright 2020 by Robert Michael Johnson Approved by Elizabeth Hiltbold Schwartz, Chair, Associate Professor of Biology Michael Greene, Associate Professor of Nutrition Robert Judd, Associate Professor of Pharmacology Kate Buckley, Assistant Professor of Biology Abstract Life-history theory states that animals have access to a finite amount of resources over their lifespan. These resources are allocated between growth, reproduction, and maintenance, and how these resources are allocated will affect the overall fitness of an organism. A fundamental assumption of this theory is that once a resource is utilized by a trait, it cannot be used by another trait. Thus, when the demand for resources for one trait increases, it will come at the cost of the other traits. The host’s immune system is responsible for survival from pathogenic invasions. Therefore, it is a critical part of maintenance. Studies have examined the trade-offs induced by an immune response. The current knowledge on trade-offs incurred during an immune response come from the use of pathogen-associated molecular patterns (PAMPs), non-pathogenic antigens such as sheep red blood cells (SRBCs), or keyhole limpet hemocyanin (KLH). These studies have observed trade-offs with metabolism and weight. Additionally, some of these studies have observed decreased activity, increased fatigue, loss of appetite, and fever. Collectively, these symptoms are knowns as sickness behavior. In recent years, the field of science investigating the cellular metabolism of immune cells termed immunometabolism has observed a rapid growth. However, the knowledge gained from these studies relies on PAMPs or non-specific activation of cells of the adaptive immune system. Thus, the understanding of trade-offs and immunometabolism to a pathogen remains poorly characterized. Our present study aims to longitudinally characterize the trade-off to life-history traits, induced sickness behavior, and immunometabolism to a well-characterized model Listeria monocytogenes. During a primary immune response, we observed trade-offs and 2 sickness behavior that corresponded to the timing of the innate immune response. Additionally, during this time, we observed a shift in cellular metabolism towards aerobic glycolysis in cells of the innate immune system. During the time of maximal cost of adaptive immunity, clonal expansion, we observed the resolution trade-offs and sickness behavior. Additionally, the cell’s immunometabolism resembled cells from control. Thus, during a primary immune response, the innate immune system likely is the driver of trade-offs. During a secondary response, we observed trade-offs that coincided with the timing of reactivation of cells of the adaptive immune system. Thus, reactivation of the adaptive immune response is likely to cause trade-offs. 3 Acknowledgments My time at Auburn University has allowed me the opportunity to interact with amazing people, and without their unwavering support, I would not have been able to complete my Ph.D. I will be forever thankful for them and that our paths crossed. The first person I want to thank is my major advisor Dr. Elizabeth Schwartz. She provided me with countless advice, guidance, and support, which I appreciate. Additionally, she has been a second mother to me while away from my mom, and I will never be able to express how grateful I am for that. I want to thank my graduate advisory members. To Dr. Michael Greene, thank you for all insight into metabolism and allowing me to house infected mice in the metabolic cages for 14 consecutive days. Without this, I would not have been able to achieve my dissertation work. Dr. Robert Judd, thank you for your kind words of support and smiling face during my committee meetings. Dr. Kate Buckley has only recently joined my committee, but I am thankful for her contribution to my research and lab meeting discussions. My time in The Schwartz’s lab was not spent in isolation. I had five wonderful lab mates that made the time there rememberable. Dr. Peter Rogers, I want to thank you for the scientific guidance you provided me as the senior lab member. I want to thank Dr. Adesola Olatunda for her assistance with my experiments and for being the mother of the lab to keep us all in line. Dr. Keah Higgins provided me with emotional guidance, which I am forever grateful for since emotions do not come easily to me. I am thankful for Haley Hallowell for providing me suggestions for flow cytometric analysis and ensuring I was always caffeinated, which the lab greatly appreciated. 4 To the Department of Biological Sciences, thank you for providing me to the opportunity to be a graduate teaching assistant, which sparked my interest in teaching and forged my path towards academia. The faculty members of Biological Sciences have always been there for the graduate students. I wish to say thank you for standing behind your graduate students and for providing me with laughter when I was down. To the fantastic Department of Biological Sciences staff, I want to say thank you for always having a smiling face and going above and beyond to help me with my requests. Dr. Sang-Jin Suh, I want to thank you for the time you spend on my committee while at Auburn University. You allowed me to guest lecture the class Microbial Physiology. After you departed from Auburn, you provided me with insight when I taught Microbial Physiology for which I am forever grateful. I would be remised if I did not say thank you to my dear friend Simon Taylor. Through all my trials and tribulations of graduate school, he has always checked in on me physically and emotionally to ensure I was able to achieve my dream of getting a Ph.D. Thank you for always being there. I would never have been able to attend college if it were not for my late maternal grandmother setting up a college trust fund. She allowed me to be the first-generation college student for which I am forever grateful. My mother has been the most nurturing and caring mother a son could ask for. She was always willing to help keep me afloat when I had no idea what I was doing, and I am incredibly thankful to have her as a mother. 5 Table of Contents Abstract ......................................................................................................................................... 2 Acknowledgments......................................................................................................................... 4 List of Tables ................................................................................................................................ 8 List of Figures ............................................................................................................................... 9 List of Abbreviations .................................................................................................................. 10 Chapter 1. Literature Review ...................................................................................................... 12 Life-history Theory and Sickness Behavior ......................................................................... 12 Primary Immune Response ................................................................................................... 14 Secondary Immune Response ............................................................................................... 16 Listeria monocytogenes ........................................................................................................ 17 Immunometabolism .............................................................................................................. 19 Hypothesis and Goals ........................................................................................................... 22 References ............................................................................................................................ 23 Chapter 2. The Systemic and Cellular Metabolic Phenotype of Infection and Immune Response to Listeria monocytogenes .................................................................................... 34 Abstract ................................................................................................................................. 34 Introduction ........................................................................................................................... 36 Materials and Methods .......................................................................................................... 38 Results ................................................................................................................................... 42 Metabolic Phenotype ...................................................................................................... 42 Metabolic Rate and activity ............................................................................................ 49 Kinetics of bacterial colonization and T cell response ................................................... 51 6 Changes in cellular metabolic status upon infection ......................................................