Investigations into the role of exogenous estrogenic endocrine disrupting chemicals on immune dysregulation in autoimmune disease Michael R. Edwards Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biomedical and Veterinary Sciences S. Ansar Ahmed Thomas E. Cecere Liwu Li Xin M. Luo Lijuan Yuan May 8, 2019 Blacksburg, VA Keywords: Autoantibody, nephritis, immune complex, dietary This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA. Investigations into the role of exogenous estrogenic endocrine disrupting chemicals on immune dysregulation in autoimmune disease Michael Edwards ABSTRACT Estrogenic endocrine disrupting chemicals (EEDCs) are defined as chemicals that bind to estrogen receptors (ERs) and augment estrogenic functions, either through promoting or blocking estrogen receptor signaling. Recent reports highlight the growing concern surrounding environmental exposure to EEDCs and immune system modulation. A commonly prescribed EEDC, 17α-ethinyl estradiol, is a synthetic analog of 17β-estradiol (E2), and is also found in many environmental reservoirs of human and animal exposure. Little is known regarding the immunomodulatory effects of this EEDC. Autoimmune diseases, such as systemic lupus erythematosus (SLE), are characterized by a dysregulated immune system that has lost tolerance to self-antigens. The pathogenesis of SLE is still poorly understood. However, it is likely that genetics, epigenetics, hormones, and environmental factors, such as EEDC exposure, contribute to the pathogenesis and severity of SLE. The work presented in this dissertation focused on investigating the immunomodulatory effects of exogenous estrogens in mouse models of SLE. Chapter 1 describes an overview of environmental endocrine disruptors and autoimmune disease, with a particular emphasis on estrogens. Chapter 2 represents a review of the current and pertinent literature surrounding the contributions of sex differences, hormones, and EDCs to the induction of autoantibodies and development of autoimmunity, as well as the contributions of anti- microbial responses to SLE. We explored the contribution of dietary components to SLE disease severity. Mice fed a diet devoid of exogenous phytoestrogens developed significantly reduced glomerulonephritis and glomerular immune complex deposition compared to mice fed a diet containing soy isoflavones. Diet also influenced cytokine production and epigenetics of LPS-stimulated splenic leukocytes. We identified similar effects of E2 and EE implantation with regards to innate immunity, and distinct cellular subset, cytokine production profiles, gene expression, and epigenetic responses between E2 and EE treated NZB/WF1 mice. Oral exposure to a very low human relevant dose of EE promoted glomerulonephritis and augmented responses to viral and bacterial mimics in MRL/lpr mice. Overall, our findings suggest that chronic exposure to environmental EEDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals and have greatly expanded the current body of knowledge surrounding 17α-ethinyl estradiol. ii Investigations into the role of exogenous estrogenic endocrine disrupting chemicals on immune dysregulation in autoimmune disease Michael Edwards GENERAL AUDIENCE ABSTRACT Chemicals that can disrupt the normal effects of hormones are termed endocrine disrupting chemicals (EDCs). Estrogenic EDCs promote or suppress the ability of estrogen receptors to carry out normal functions within the body. Normal immune system functions require a fine balance of inflammatory and anti-inflammatory cellular responses. This delicate balance is a prime target for dysregulation by EDC exposure. Autoimmune diseases, such as systemic lupus erythematosus, are characterized by a loss of immune tolerance to ones’ own cells and tissues. There is a lack of knowledge surrounding the immunomodulatory effects of a commonly prescribed EDC, 17α-ethinyl estradiol, especially as it pertains to autoimmune disease patients. The aim of this dissertation work is to investigate the immunomodulatory effects of exogenous EDC exposure in mouse models of SLE. We found that MRL/lpr mice fed a diet devoid of phytoestrogens had reduced kidney disease and immune-complex deposition and had augmented cytokine response and epigenetics in LPS-stimulated splenic leukocytes compared to mice fed a diet high in isoflavones. We next compared the immunomodulatory effects of chronic pharmacologic dose exposure to 17β-estradiol or EE, and found that while both estrogens have similar effects on innate immune cellular responses, EE has distinct effects on T cell population subsets, cytokine production, gene response and epigenetic alterations in female NZB/WF1 mice. Finally, chronic low-dose oral exposure to EE exacerbated clinical signs of kidney disease and suppressed the normal response of toll-like receptor 9 in MRL/lpr mice. Overall, we have found that chronic exposure to environmental estrogenic EDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals. iii Acknowledgements Many people deserve my gratitude and have been instrumental in guiding and supporting my success. I would like to acknowledge the many people who contributed to the following works and to my graduate career as a whole, including my graduate committee Dr. Ahmed, Dr. Dr. Thomas Cecere, Dr. Liwu Li, Dr. Xin Luo, and Dr. Lijuan Yuan. Thank you especially to my mentor Dr. S Ansar Ahmed who has facilitated my growth as a student and researcher. I also would like to acknowledge and thank Dr. Divaker Choubey. Dr. Choubey has agreed to travel the long distance to VA from the University of Cincinnati in order to serve as an external examiner. The many researchers who have either directly contributed to the following works, or indirectly through support and mentorship include Dr. Qinghui Mu, Dr. Xiaofeng Liao, and Melissa Makris. Additionally, I would like to acknowledge and thank my lab mates, Dr. Rujuan Dai, Dr. Catharine Cowan, Zhuang Wang, and Bettina Heid. Without their input, tutelage, and continued support, none of this would have been possible. Thank you for the times you got me back on track, patiently taught me a technique, and discussed science in a positive, engaging manner. The TRACSS staff and BMVS administration were integral to helping maintain the animal colony and health. Thank you to my parents, siblings, and in-laws for all the encouragement and support. Finally, I would certainly like to thank my beloved wife, Dr. Virginia Edwards, and my two boys Gibson and Noah, for all their support throughout this academic training journey. Without the emotional and intellectual support of my family, I would not be where I am today. iv Table of Contents Page # Abstract……………………………………………………………………………………………ii General Audience Abstract……………………………………………………………………….iii Acknowledgements……………………………………………………………………………….iv Table of Contents………………………………………………………………………………….v List of Figures…………………………………………………………………………………....vii List of Tables……………………………………………………………………………………..ix Attribution……………….…………………………………………………………………….......x Chapter 1: Introduction: Autoimmune dysregulation and estrogenic EDC exposure .…………...1 Chapter 2: Literature Review: Our environment shapes us: The importance of environment and sex differences in regulation of autoantibody production …………………………………….....22 2.1 Abstract………………………………………………………………………………22 2.2 Introduction………………………………………………………………..….….…..24 2.3 Sex Differences in Genetics and autoimmunity ……………………………….….…26 2.4 Sex Hormones and Environmental Endocrine Disrupting Chemical Regulation of Immunity and Autoimmunity ………………………………………….………..27 2.5 Sex Differences in Stress Response of Autoimmunity …………………………...…35 2.6 Sex Differences in Epigenetic Regulation and Autoimmunity …………………..….37 2.7 Autoimmunity and Microbial Agents ……………………………………………….42 2.8 Conclusions and Future Directions ………………………………………………….48 Chapter 3: Commercial rodent diets differentially regulate autoimmune glomerulonephritis, epigenetics, and microbiota in MRL/lpr mice …………………………………………………. 91 3.1 Abstract………………………………………………………………………………91 3.2 Introduction…………………………………………………………………………..93 3.3 Materials and Methods…………………………………………………………….…94 3.4 Results………………………………………………………………………………102 3.5 Discussion…………………………………………………………………………..110 Chapter 4: 17-β estradiol and 17α-ethinyl estradiol exhibit immunologic and epigenetic regulatory effects in NZB/WF1 female mice ….…………………………………………….....147 4.1 Abstract……………………………………………………………………………..147 4.2 Introduction…………………………………………………………………………148 4.3 Materials and Methods……………………………………………………………...151 4.4 Results………………………………………………………………………………157 4.5 Discussion…………………………………………………………………………..165 v Chapter 5: Environmental exposure to 17α-ethinyl estradiol augments kidney disease and TLR7/9 signaling in female autoimmune-prone MRL/lpr mice ......……………………...……224 5.1Abstract…………………………………………………………………………….. 224 5.2 Introduction…………………………………………………………………………226 5.3 Materials and Methods………………………………………………………...……229 5.4 Results…………………………………………………………………………...….235 5.5 Discussion………………………………………………………………………..…241 Chapter 6: Conclusions and Future Directions……………………………………………...….279