The Anti-Inflammatory Effects of the Dietary Dipeptide, Gamma- Glutamylvaline in a Mouse Model of LPS-Induced Sepsis by MacKenzie Chee A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Food Science Guelph, Ontario, Canada © MacKenzie Chee, April, 2016 ABSTRACT THE ANTI-INFLAMMATORY EFFECTS OF THE DIETARY DIPEPTIDE, GAMMA- GLUTAMYLVALINE IN A MOUSE MODEL OF LPS-INDUCED SEPSIS MacKenzie Chee Advisor: University of Guelph, 2016 Professor Y. Mine This study endeavored to identify the anti-inflammatory activity and signaling mechanism of the bioactive dietary dipeptide gamma-L-glutamyl-L-valine (γ-EV) via calcium- sensing receptor (CaSR) activation in a lipopolysaccharide (LPS)-induced mouse model of sepsis. Sepsis occurs when the otherwise tightly-regulated immune and inflammatory response that eliminates invading pathogens and repairs injured tissues, switches to an uncontrolled response that causes an acute life-threatening inflammatory syndrome. γ-EV had anti- inflammatory activity in BALB/c mice with LPS-induced sepsis as measured by the reduction of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in plasma and small intestine tissue. There was a γ-EV-mediated decrease in the phosphorylation of the downstream signaling proteins JNK and IκBα in small intestine tissue, which indicates γ-EV likely has an effect on an upstream signaling protein common to both the AP-1 and NF-κB pathways. The study proposes that a direct interaction of CaSR-recruited β-arrestin2 with TRAF6, TAB1, and IκBα constitutes γ-EV’s anti-inflammatory activity in LPS-induced inflammation. This study provides evidence that γ-EV has health-promoting activity. ii ACKNOWLEDGEMENTS I would like to express my gratitude to my entire support system during my Graduate studies. My advisor, Dr. Yoshinori Mine, provided me with guidance in academia and research, and encouraged me to lead a balanced ‘bushido’ life. I would like to thank my lab peers Dr. Toshihiko Fukuda, Dr. Fang Geng, Dr. Hua Zhang, and Dr. Kaustav Majumder for educating and training me in the lab. I would not have the lab skills set that I have today without these fine scientists teaching me along the way. I would also like to express my appreciation for Dr. Rong Cao who provided me with guidance as a member of my Advisory Committee. Thank you to my partner in life – Zachary Munroe, who has stood by me through my journey as a Graduate student. Thank you for always believing in me, and encouraging me to be the best person I can possibly be. Your support has been paramount during my Masters studies. I would like to thank the Advanced Foods and Materials Network (AFMNet) and the Natural Sciences and Engineering Research Council of Canada (NSERC) for the financial support for this project. As well, I would like to express my utmost gratitude for the OAC Class of 1950, Mary Frances Hucks and family, Kenneth W. Knox and family, as well as Martha Robb and family for their tremendous generosity. iii TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………... vii LIST OF FIGURES…………………………………………………………………….. viii ABBREVIATIONS…………………………………………………………………….. ix 1. LITERATURE REVIEW 1.1 Gut Health, Intestinal Inflammation and Chronic Inflammatory Diseases of the Gut………………………………………………………. 1 1.2 Sepsis…………………………………………………………………….. 3 1.2.1 Definition of Sepsis……………………………………….. 3 1.2.2 Causative Pathogens and Models of Sepsis………………. 7 1.2.3 Pathogen-Associated Molecular Patterns and Their Receptors…………………………………………… 8 1.2.4 Pathogenesis………………………………………………. 12 1.2.5 Diagnostic Biomarkers of Sepsis…………………………. 14 1.2.6 Clinical Diagnostic Criteria……………………………….. 16 1.2.7 Treatment of Sepsis……………………………………….. 17 1.2.8 Vulnerable Populations…………………………………… 19 1.2.9 Prevalence and Healthcare Costs………………………….. 19 1.3 Immunology of Sepsis…………………………………….……………… 20 1.4 Bioactive Peptides……………………………………..…………………. 21 1.5 Gamma-glutamylvaline..……………….………………………………… 23 1.6 Calcium-Sensing Receptor (CaSR) ……………………………………… 28 1.6.1 Background…………………………………….………….. 28 1.6.2 Structure and Function of CaSR…………………………... 28 1.6.3 Agonists and Antagonists…………………………………. 30 1.6.4 Binding Site for L-Amino Acids…………………………... 31 1.6.5 CaSR Signaling…………………………..………………... 32 2. RATIONALE, HYPOTHESIS AND OBJECTIVES………………………………. 35 2.1. Rationale………………………….……………………………………… 35 2.2. Hypothesis………………………….……………………………………. 35 2.3. Objectives………………………….…………………………………….. 35 3. MATERIALS AND METHODS………………………….………………………… 36 3.1. Reagents………………………….………………………….…………... 36 3.2. Animal Study………………………….………………………….……… 36 3.2.1. Animals………………………….………………………… 36 iv 3.2.2. Treatment………………………….……………………... 37 3.2.3. Sepsis Induction………………………….……………..... 37 3.2.4. Tissue Collection………………………….…………..….. 38 3.2.5. Tissue Protein Extraction and Protein Concentration…..... 38 3.3. ELISA………………………….………………………….……….…… 39 3.4. Western blot………………………….…………………………..…….. 40 3.5. Histological Analysis………………………….……………………….. 41 3.6. Data Analysis………………………….…………………...….……….. 42 4. RESULTS 4.1. Effects of γ-EV on Acute Inflammatory Cytokine Secretion in Plasma and Small Intestine……………………….…...………………. 43 4.1.1. Effects of γ-EV on Acute Inflammatory Cytokine Secretion in Plasma………..…………………………….. 43 4.1.2. Effects of γ-EV on Acute Inflammatory Cytokine Secretion in Small Intestine……………………………... 46 4.2. Effects of γ-EV on Phosphorylation and Expression of Signaling Proteins………………………………………………….….. 48 4.2.1. p-IκBα/IκBα……………...……….…………………….. 48 4.2.2. p-JNK/JNK…………...………….………………………. 48 4.2.3. TRAF6/ β-actin……………………….………………….. 48 4.2.4. Ubiquitin/ β-actin……………………….………………... 50 4.3. Small Intestine Histology………………………….…………………… 53 5. DISCUSSION 5.1. γ-EV Suppressed Pro-Inflammatory Cytokine Secretion in Plasma…….. 55 5.2. γ-EV Suppressed Pro-Inflammatory Cytokine Secretion in Small Intestine………………………….………………………………… 56 5.3. γ-EV Inhibited Phosphorylation of IκBα………………………………… 57 5.4. γ-EV Inhibited Phosphorylation of JNK………………………………… 58 5.5. γ-EV Had an Effect on Background Ubiquitin Protein Levels that was Unrelated to TRAF6………………………….…………………………. 59 5.6. Protein Concentration of Total TRAF6 Decreased with LPS Stimulation………………………….………………………….….. 60 5.7. Cellular Signaling Crosstalk Mechanism………………………………. 60 5.8. Applications to Sepsis Prevention.……………………….…………….. 67 6. CONCLUSION AND FUTURE WORK………………………….………………. 69 v 7. REFERENCES………………………….………………………….……………… 70 vi LIST OF TABLES Table 1: Diagnostic criteria for sepsis………………………….……………………. 5 Table 2: The Sepsis-related organ failure assessment (SOFA) ……………………... 6 Table 3: Diagnostic biomarkers of sepsis….………………………………………… 15 Table 4: Treatments for sepsis……………….………………………………………. 18 Table 5: Bioactive food-derived peptides and their anti-inflammatory activities.….... 24 vii LIST OF FIGURES Figure 1: The structure of the gut barrier integrity……………….…………………… 2 Figure 2: The structure of lipopolysaccharide (LPS) ……………….……………….. 9 Figure 3: The Toll-like receptor 4 (TLR4) structure and its associated TIR signaling domain……………….……………………………………………………… 11 Figure 4: The three phases of sepsis……………….…………………………………. 13 Figure 5: The structure of γ-EV……………….……………………………………… 27 Figure 6: Calcium-sensing receptor (CaSR): the structure of the receptor and its intracellular signaling domain……….…………………………………….. 33 Figure 7: The TNF-α, IL-6 and IL-1β protein expression in the plasma…………….. 45 Figure 8: The TNF-α, IL-6, IL-1β, and IL-10 protein expression in the small intestine…..………….………………………………………………. 47 Figure 9: The p-JNK/JNK and p-IκBα/ IκBα protein expression in the small intestine…..………….………………………………………………. 49 Figure 10: The TRAF6/β-actin and Ubiquitin/β-actin protein expression in the small intestine……………….…………………………………………….. 52 Figure 11: Small intestine histological slides stained with H&E…………………...… 54 Figure 12: Proposed signaling crosstalk interaction between β−arrestin2 and TRAF6.. 64 Figure 13: Proposed signaling crosstalk interaction between β−arrestin2 and TAB1… 65 Figure 14: Proposed signaling crosstalk interaction between β−arrestin2 and IκBα..... 66 viii LIST OF ABBREVIATIONS 1,25(OH)2D3 1,25-dihydroxyvitamin D3 AP-1 activator protein 1 APACHE acute physiology and chronic health evaluation AS ankylosing spondylitis APC antigen-presenting cell BSA bovine serum albumin JNK c-Jun N-terminal kinase CRP C-reactive protein CT calcitonin CaSR calcium-sensing receptor CD14 cluster of differentiation 14 Co-IP co-immunoprecipitation CD Crohn’s disease DAMP damage-associated molecular pattern DC dendritic cells ELISA enzyme-linked immunosorbent assay ECD extracellular domain γ-EV gamma-glutamylvaline GI gastrointestinal (tract) H&E hematoxylin and eosin HLA-DR human leukocyte antigen – antigen D related HUVEC human umbilical vein endothelial cell ix IKK IκB kinase IRAK IL-1R-associated kinase iNOS inducible nitric oxide synthase IBD inflammatory bowel disease ICU intensive care unit ICAM-1 intercellular adhesion molecule-1 IFN interferon IL interleukin IEC intestinal epithelial cell IP intraperitoneal LP lamina propria LPS lipopolysaccharide LBP lipopolysaccharide-binding protein LTA lipoteichoic acid MAPK mitogen-activated protein kinase MCP-1 monocyte chemoattractant protein-1 Mal MyD88-adaptor-like protein MD-2 myeloid differentiation-2 NICU neonatal intensive care unit NO nitric oxide NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells IκBα nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha PTH parathyroid hormone x PAMP pathogen-associated