Changing the Fate of Histoplasma Capsulatum-Infected Cells with Small
Total Page：16
File Type：pdf, Size：1020Kb
Changing the fate of Histoplasma capsulatum-infected cells with small molecules: investigation of zinc modifying agents and the antioxidant Ferrostatin-1 A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Immunobiology of the College of Medicine 2017 by MICHAEL HORWATH B.S. University of Dayton, 2009 Committee Chair: George S. Deepe, Jr., MD i Thesis abstract The dimorphic fungal pathogen Histoplasma capsulatum causes significant morbidity and thousands of deaths each year in endemic regions including North America, South America, and Africa. In its pathogenic yeast form, H. capsulatum has a complex relationship with macrophages (MPs) and dendritic cells (DCs) of the host mononuclear phagocyte system. The yeast is a facultative intracellular pathogen, and multiplies within MPs, eventually resulting in MP death. Control of the infection requires activation of MPs by cytokines and upregulation of antimicrobial mechanisms, including sequestration of intracellular zinc. DCs are capable of killing H. capsulatum yeast and presenting antigen to T-helper cells; this provides a crucial link to protective cytokine production by the adaptive immune system. However, the mechanisms involved in DC activation and antigen presentation in response to H. capsulatum remain only partially understood. This report describes two experimental investigations of the interactions between H. capsulatum yeast and mononuclear phagocytes. The first study focuses on the role of zinc in DCs. We hypothesized that, in response to H. capsulatum infection, sequestration of free cytoplasmic zinc by DCs may promote DC activation and induction of a protective T-helper adaptive response. Using small molecule zinc chelators and ionophores, we demonstrated that intracellular zinc has an inverse relationship with DC activation in the context of H. capsulatum exposure. Although DCs stimulated by the yeast upregulated metallothionein and metal transporter genes involved in zinc trafficking, genetic manipulation of these genes had only minor impact on DC phenotype. This may reflect redundancy of multiple mechanisms maintaining optimal zinc levels in DCs during activation. ii The second study focused on the lipophilic antioxidant Ferrrostatin-1 (Fer-1), and its ability to prevent death of H. capsulatum-infected MPs by unexpected antifungal activity. Fer-1 was the most potent inhibitor of MP cell death in our screen of small molecule death modifying agents. Although we initially hypothesized that Fer-1 was preventing ferroptosis of infected MPs, we discovered that Fer-1 is directly fungistatic toward H. capsulatum as well as several related dimorphic fungal pathogens. We further demonstrated that Fer-1 functions as an inhibitor of fungal sterol synthesis, and is capable of reducing ergosterol content in H. capsulatum yeast. Investigation with analog ferrostatins and other small molecule lipophilic antioxidants revealed that the antifungal activity of these compounds is distinct from the antioxidant activity, and is highly sensitive to changes in molecular structure. In conclusion, zinc modifying agents and ferrostatins each represent novel small molecule tools for changing the outcome of mononuclear phagocyte interaction with H. capsulatum. iii iv Acknowledgements Reflecting on my years of PhD training and research, I am very grateful for the support, mentorship, and encouragement I have received along the way. I would first like to thank my thesis advisor, Dr. George Deepe. Dr. Deepe’s guidance in scientific thought and writing, and his enthusiasm for immunology and microbiology, have been central to my training as a scientist. His door was always open, and I am grateful for his patience and support. I also thank and acknowledge the members of my dissertation committee—Drs. Dave Hildeman, Edith Janssen, Michael Jordan, and Edward Merino. Each of these individuals shared their insights and expertise on numerous occasions, and helped me progress as a scientist. Dr. Joseph Caruso, an original member of my committee, sadly passed away during my training—I am also thankful for his guidance. I would also like to thank the members of the Deepe laboratory who have shared insights, experimental techniques, and sometimes frustrations along the way—including William Buesing, Victor Lescano, Mariam George, Jamie Tweedle, Roger Fecher, Kavitha Subramanian-Vignesh, Akash Verma, Ye Xiong, Chelsea Bueter, and Daniel Kroetz. I especially have good memories of many hours spent collaborating at the bench with Roger and Victor. Beyond the Deepe laboratory, I had the opportunity to learn from and collaborate with many talented scientists during my PhD training. I appreciated learning from many researchers at CCHMC and UC who shared our interest in infectious mycology, included Simon Newman, Karthik Krishnan, Juwen DuBois, Jessica Dade, and Kris Orsborne. I further appreciate the help and collaboration I received from multiple researchers in the UC Chemistry Department, especially Larry Sallans, Julio Landero, and Anna Donnell. The collaborative environment at CCHMC and UC has been a central part of my graduate school experience--the times that I benefited from advice and help from other students, post-docs, faculty members, and core facility scientists are countless. I would also like to thank the Immunobiology Graduate Program and the Medical Scientist Training Program at the University of Cincinnati. Immunobiology graduate program director Dave Hildeman, MSTP Director Neeru Hershey, and the rest of the program leadership have given the students a tremendous amount of support—as have the program coordinators, including Isabel Castro, Laurie Mayleben, and Andrea DeSantis. I am also thankful for the other students in these programs, many of whom became friends or role models (or both). I was lucky to have a wonderful set of classmates in both my MSTP and Immunology starting classes—Jared, Dan, Vivian, and Kun-Po; Rahul, Steve, Jed, Julie, and Martine. v Finally, I am very grateful to my family for their love and encouragement. My parents John and Maureen instilled in me an early love of learning, science, and the natural world, and I owe so much to the upbringing an education they gave me. My siblings Tom, Mary, and Elizabeth are always there for me and have (usually) tolerated my nerdy jokes. Most importantly, I am deeply thankful for my wife Tiffany and our son Oliver. Tiffany is a talented chemist, a loving wife, and a great friend. Her patience and support have helped me through the tough times, while her humor, wit, and enthusiasm make the good times great. Oliver arrived about midway through my PHD training, and he really brought a wonderful extra dimension to our life. Ollie’s constant curiosity (and frequent stubbornness) are a source of inspiration to me as a father and scientist. I can’t wait to see the places you’ll go! Thanks again all, Mike Horwath vi Table of contents Thesis abstract ii Acknowledgments v Table of contents vii List of figures and tables ix Common abbreviations xii Chapter 1: Introduction 1 I. Immune response to the fungal pathogen Histoplasma capsulatum 1 1. Biology, virulence, and health impact of H. capsulatum 1 2. The mononuclear phagocyte system 4 3. The innate immune response to H. capsulatum 19 4. Adaptive immune response to H. capsulatum 25 5. Translation from mouse to human 29 6. Antifungal therapy and H. capsulatum 31 II. Zinc: An essential metal in cell function and immune response 35 1. Zinc, an essential nutrient 35 2. Zinc toxicity: too much of a good thing 38 3. Cellular zinc trafficking and homeostasis 41 4. Zinc and Immunity 52 III. Regulated cell death, H. capsulatum infection, and ferroptosis 59 1. Host cell death during intracellular infection 59 2. Ferroptosis: a novel form of regulated cell death 60 3. Ferroptosis, inflammation, and immunity 67 IV. Statement of Aims 73 References 74 Chapter 2: Zinc Suppresses Dendritic Cell Activation 95 Title and Abstract 95 Introduction 96 Methods 98 vii Results 101 1. Regulation of Cellular Zinc During H. capsulatum Exposure 101 2. Intracellular zinc suppresses DC activation 107 Discussion 113 References 118 Figure legends 122 Figures 126 Supplemental figure legends 135 Supplemental figures 136 Chapter 3: Antifungal activity of the lipophilic antioxidant Ferrostatin-1 140 Title and Abstract 140 Introduction 141 Results 141 1. Discovery of Novel Antifungal Activity of Ferrostatin-1 141 2. Investigation of Fer-1 antifungal mechanism and 145 structure-activity relationship Discussion 148 Methods 150 Acknowledgments 151 References 151 Image for table of contents 154 Supplemental figures 155 Supplemental methods 168 Chapter 4: Discussion 173 1. Zinc and dendritic cell phenotypes: Implications of findings 173 2. Ferrostatin-1: Implications of findings 180 3. Connections between zinc, reactive oxygen, and ferroptosis 183 4. Future directions for the study of Zn trafficking in DC activation 185 5. Future directions for the analysis of the antifungal mechanisms of Fer-1 186 References 196 viii Figures and Tables Chapter 1: Introduction (Pages 1-94) Figure 1. Major mammalian zinc trafficking proteins 41 Figure 2. Generation and propagation of free radicals, reactive oxygen species (ROS), 62 and reactive nitrogen species (RNS) Figure 3. Oxidation and radical propagation of polyunsaturated fatty acids 63 Figure 4. Mechanism, inducers, and inhibitors