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UNIVERSITY of CALIFORNIA RIVERSIDE Impact of Alcohol on Intestinal Homeostasis a Dissertation Submitted in Partial Satisfaction UNIVERSITY OF CALIFORNIA RIVERSIDE Impact of Alcohol on Intestinal Homeostasis A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences by Tasha Marie Barr June 2018 Dissertation Committee: Dr. Ilhem Messaoudi, Co-Chairperson Dr. Declan McCole, Co-Chairperson Dr. James Borneman Copyright by Tasha Marie Barr 2018 The Dissertation of Tasha Marie Barr is approved: Committee Co-Chairperson Committee Co-Chairperson University of California, Riverside ACKNOWLEDGEMENTS To Dr. Ilhem Messaoudi: Thank you for never giving up on me and for the countless talks and words of encouragement. You are such an inspiration and your incredibly strong work ethic is my motivation. You have shown me what it takes to be a successful scientist and I will be forever grateful for you taking a chance with me. To the Messsaoudi Lab: Thank you all for the support you have given me over these years. I was fortunate to have amazing lab mates that are truly my best friends. Special thanks to Andrea – I am so grateful I had you by my side throughout this adventure. To Dr. Declan McCole and Dr. James Borneman: Thank you for agreeing to be on my committee. I truly do appreciate the both of you taking the time out of your busy days to give me advice and forcing me to think critically like a biomedical scientist. The text of this dissertation, in part or in full, is a reprint of the material as it appears in Progress in Neuro-Psychopharmacology and Biological Psychiatry 2016 (Chapter 1); Journal of Immunology 2016 (Chapter 2); and Gut Microbes 2018 (Chapter 3). The co- author Ilhem Messaoudi listed in those publications directed and supervised the research that forms the basis for this dissertation. This work was funded by the National Institute on Alcohol Abuse and Alcoholism (R21AA021947, R21AA024981, U01AA013510, R24 AA109431, F31AA025278-01). iv DEDICATION This dissertation is dedicated to my Grammy and Poppop. I love you both so much! Thank you for the emotional, spiritual, and financial support throughout my graduate school years. I couldn’t have done it without you. v ABSTRACT OF THE DISSERTATION Impact of Alcohol on Intestinal Homeostasis by Tasha Marie Barr Doctor of Philosophy, Graduate Program in Biomedical Sciences University of California, Riverside, June 2018 Dr. Ilhem Messaoudi, Co-Chairperson Dr. Declan McCole, Co-Chairperson Chronic heavy alcohol consumption results in intestinal injury characterized by increased permeability, dysbiosis, nutrient malabsorption, increased risk of colorectal cancer, and functional alterations in mucosal immune cells. Studies have implicated dysregulation of tight junction proteins as well as quantitative and qualitative changes in gut microbiota as key contributors to gut injury. Recently, we showed using a macaque model of voluntary ethanol self-administration that moderate consumption resulted in a more robust T-cell and antibody vaccine response to Modified Vaccinia Ankara (MVA), while heavy drinkers generated blunted T-cell and antibody response compared to controls. First, we show that chronic alcohol consumption modulates host defense and response to booster vaccination by altering gene expression in circulating leukocytes. Using this model, we vi previously examined the effect of chronic ethanol consumption on cytokine production using intracellular cytokine staining, and found that colonic T cells exhibited a disruption of inflammatory cytokine production. Next, we show the modulation of transcriptome and microbiota profiles by chronic alcohol consumption within the different segments of the gastrointestinal tract. Lastly, we report alterations in the transcriptional profiles of intestinal lamina propria lymphocytes following chronic alcohol consumption. Since our animals lack overt organ damage, the alterations in gene expression and microbial composition precede clinical disease and could potentially serve as early biomarkers before detrimental effects occur. These findings provide novel insight into the mechanisms of ethanol-induced mucosal damage and lay the foundation for follow up studies to develop new interventions to reverse mucosal damage and improve immunity in heavy alcohol abusers. vii TABLE OF CONTENTS Acknowledgements iv Dedication v Abstract vi List of Figures xi List of Tables xiv Chapter 1: Introduction and review of literature 1 1.1 Alcohol use disorder 1 1.2 Alcohol metabolism 3 1.3 Animal models used in alcohol research 5 1.4 Overview of the immune system 7 1.5 Opposing effects of alcohol on the peripheral immune system 8 1.5.1 Modulation of innate immunity by alcohol 8 1.5.2 Modulation of adaptive immunity by alcohol 15 1.5.3 Impact of alcohol on infection and vaccination 18 1.5.4 Impact of alcohol on circulating factors 20 1.5.5 Mechanisms of dose-dependent modulation of immunity by alcohol 22 1.6 Impact of alcohol on the gastrointestinal tract 25 1.6.1 Gastrointestinal mucosal immune system 26 1.6.2 Alcohol-induced barrier dysfunction 27 1.6.3 Impact of alcohol on intestinal immunity 30 1.6.4 Association between alcohol consumption and colorectal cancer 31 viii 1.6.5 Alcohol and dysbiosis 32 1.7 Thesis overview and aims 34 Chapter 2: Alcohol consumption modulates host defense in rhesus macaques by altering gene expression in circulating leukocytes 35 Abstract 36 Introduction 37 Materials and Methods 40 Results 45 Discussion 65 Chapter 3: Concurrent gut transcriptome and microbiota profiling following chronic ethanol consumption in nonhuman primates 74 Abstract 75 Introduction 76 Materials and Methods 80 Results 87 Discussion 112 Chapter 4: Transcriptional profiling reveals alterations in intestinal lamina propria lymphocytes following chronic ethanol consumption 120 Abstract 121 Introduction 122 Materials and Methods 124 Results 130 Discussion 162 Chapter 5: Discussion and future directions 169 ix 5.1.1 Modulation of peripheral immunity by alcohol 169 5.1.2 Chronic alcohol consumption alters intestinal immunity 170 5.2 Final thoughts 174 References 175 x List of Figures Figure 1.1 Pathways of ethanol metabolism 4 Figure 1.2 Alcohol-induced barrier dysfunction 29 Figure 2.1 Peripheral blood mononuclear cell (PBMC) gene expression 46 Figure 2.2 Chronic heavy alcohol consumption downregulates genes that promote wound healing and contribute to obstructive lung diseases compared to controls and moderate drinkers 49 Figure 2.3 Alcohol abuse uniquely downregulates additional genes that promote wound healing and contribute to obstructive lung diseases 52 Figure 2.4 Heavy alcohol consumption downregulates genes associated with regulation of the immune system 54 Figure 2.5 Alcohol abuse upregulates genes that interfere with wound healing and contribute to cancer 57 Figure 2.6 Moderate ethanol consumption modulates genes associated with immune response 59 Figure 2.7 Heavy ethanol consumption changes expression of several microRNA 62 Figure 3.1 Characteristics of macaques used in these studies 86 Figure 3.2 Chronic ethanol consumption modulates jejunum mucosal gene expression 88 Figure 3.3 Chronic heavy ethanol consumption modulates expression of genes important for immune system processes in the ileum 91 Figure 3.4 In silico analysis shows most gene expression changes are driven by B cells 93 Figure 3.5 Chronic heavy ethanol consumption modulates expression of genes associated with cancer development and immune function in the colon 96 Figure 3.6 Dynamic gene expression changes mediated by ethanol consumption 101 Figure 3.7 STEM gene expression patterns 102 xi Figure 3.8 Dose-dependent increase in intestinal permeability 103 Figure 3.9 Alpha diversity along the gastrointestinal tract 104 Figure 3.10 Beta diversity along the gastrointestinal tract 106 Figure 3.11 Chronic ethanol intake results in minor changes in mucosal microbial communities 107 Figure 3.12 Inter-individual family differences 108 Figure 3.13 Chronic ethanol consumption alters bacterial communities and subsequently critical metabolic pathways in a dose-dependent manner 111 Figure 4.1 Flow cytometric analysis reveals LPLs are primarily CD4+ and CD8+ T cells 131 Figure 4.2 Chronic ethanol consumption modulates gene expression in resting LPLs in a dose-dependent manner 134 Figure 4.3 Chronic ethanol consumption modulates the expression of genes associated with metabolic processes in duodenal LPLs 137 Figure 4.4 Chronic ethanol consumption alters gene expression in LPLs isolated from the jejunum 140 Figure 4.5 Resting ileal LPLs isolated from drinkers are in a hyper-inflammatory state before response to stimulus 142 Figure 4.6 Gene expression changes in colonic LPLs 145 Figure 4.7 Moderate consumption uniquely modulates gene expression of small intestinal LPLs 149 Figure 4.8 Alterations in protein secretion by LPLs with ethanol consumption 152 Figure 4.9 Cytokine, chemokine, and growth factor production by duodenal LPLs in response to PMA/ionomycin 154 Figure 4.10 Cytokine, chemokine, and growth factor production by jejunal LPLs in response to PMA/ionomycin 157 xii Figure 4.11 Cytokine, chemokine, and growth factor production by ileal LPLs in response to PMA/ionomycin 160 Figure 5.1 Ethanol-mediated disruptions in intestinal homeostasis characterized in our macaque model of voluntary ethanol self-administration 172 xiii List of Tables Table 1.1 Opposing effects of alcohol consumption on immune cells 14 Table 2.1 mRNA-miRNA pairs 64 Table 4.1 Characteristics of macaques used in LPL studies 125 xiv A version of this chapter is published in: Tasha Barr, Christa
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