THE EFFECT OF SULFUR-CONTAINING COMPOUNDS AND HYDROGEN SULFIDE IN THE MODULATION OF THE IMMUNE RESPONSE By CAMILA RODRIGUES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Camila Rodrigues To my husband (Andre) and my daughter (Luiza) ACKNOWLEDGMENTS I would like to express my deepest appreciation to my committee chair Dr. Susan S. Percival, who gave me the great opportunity to develop my doctoral research in the field of nutrition in her laboratory. She is a very knowledgeable person and an inspired professional with a very kind big heart. It was a pleasure to have Dr. Percival as a mentor and as an inspiration model. I also express my gratitude to my other committee members, Dr. Bobbi Langkamp-Henken, Dr. Jesse F. Gregory and Dr. Alexander J. Grenning for their invaluable suggestions and contributions to this project. I would like to thank Dr. Robin da Silva for welcoming on his lab and for all the knowledge and support shared during these last two years. I am also very grateful to my current and former colleagues, Dr. Cheryl Rowe, Dr. Xiaoshuang Dai, Dr. Rasha Mansouri, Dr. Changjie Xu, Alison O’Donoughue, Brandon Eudy, Sawsan Ahmed, Caitlin McDermott and others for their uncountable assistance, suggestions, and good times. My profound gratitude goes to my amazing husband (Andre) and daughter (Luiza) for all their love, and tremendous support on making this happening. I would not be able to achieve this degree and to be who I am without both of you. Thank you for making me more than I am. Lastly, I would like to acknowledge my wonderful parents (Joao and Terezinha), who raised me to be who I am today and always believe that I could achieve anything in life. Thank you for all the love and support. 4 TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................... 10 ABSTRACT ........................................................................................................................ 17 CHAPTER 1 BACKGROUND .......................................................................................................... 19 Introduction ................................................................................................................. 19 Immune System .......................................................................................................... 20 Inflammation ......................................................................................................... 22 NF-κB Pathway .................................................................................................... 23 Cellular Redox State and Oxidative Stress ................................................................ 25 Glutathione .................................................................................................................. 28 GSH and Aging .................................................................................................... 31 GSH and Immunity ............................................................................................... 33 Garlic (Allium sativum L.) ............................................................................................ 36 Aged Garlic Extract (AGE) ................................................................................... 37 AGE and Immunity ............................................................................................... 38 Hydrogen Sulfide ........................................................................................................ 40 Hydrogen Sulfide as a Signaling Molecule .......................................................... 42 Hydrogen Sulfide and Inflammation .................................................................... 44 2 SIGNIFICANCE AND OBJECTIVES .......................................................................... 57 Research Gaps and Significance ............................................................................... 57 Research Implications ................................................................................................ 58 Hypothesis................................................................................................................... 59 Objective and Specific Aims ....................................................................................... 59 3 ORAL SUPPLEMENTATION OF GLUTATHIONE AND THE EFFECT IN THE IMMUNE RESPONSE IN HEALTHY OLDER INDIVIDUALS.................................... 60 Introduction ................................................................................................................. 60 Material and Methods ................................................................................................. 61 Blood Collection, PBMC Isolation and Autologous Serum ................................. 61 Cell Culture ........................................................................................................... 62 Flow Cytometry .................................................................................................... 62 5 Respiratory Burst Activity (RBA) in Neutrophils .................................................. 63 Cytokine Determination ........................................................................................ 63 IgG and IgM Levels in serum ............................................................................... 63 Glutathione Levels in PBMC ................................................................................ 63 Thiol Determination in Serum .............................................................................. 64 Statistical Analysis................................................................................................ 64 Results ........................................................................................................................ 65 Systolic Blood Pressure and Immunological Parameters ................................... 65 Thiol Determination .............................................................................................. 66 Discussion ................................................................................................................... 66 4 THE ROLE OF GLUTATHIONE AND AGED GARLIC EXTRACT ON HYDROGEN SULFIDE PRODUCTION IN THP-1 MACROPHAGES ...................... 79 Introduction ................................................................................................................. 79 Material and Methods ................................................................................................. 80 Cell Culture ........................................................................................................... 80 Direct Measurement of Endogenous H2S Using a Fluorescent Probe ............... 80 Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT- PCR) .................................................................................................................. 81 Statistical Analysis................................................................................................ 81 Results ........................................................................................................................ 82 Effect of GSH and AGE on H2S Concentration ................................................... 82 Effect of GSH and AGE on H2S-Generating Enzymes ....................................... 83 Discussion ................................................................................................................... 83 5 THE EFFECT OF GLUTATHIONE, AGED GARLIC EXTRACT AND HYDROGEN SULFIDE DONORS IN THE REGULATION OF NF-B ACTIVATION IN THP-1 MACROPHAGES ................................................................ 90 Introduction ................................................................................................................. 90 Material and Methods ................................................................................................. 91 THP-1 Cell Culture and Treatments .................................................................... 91 Western Blot ......................................................................................................... 92 Statistical Analysis................................................................................................ 94 Results ........................................................................................................................ 94 Effect of Two Different Concentrations of H2S Donors on NF-B Activation ..... 94 Effect of GSH, AGE and H2S Donors on NF-B Activation ................................ 95 Effect of GSH, AGE and H2S Donors on NF-B Activation in CSE-inhibited cells ................................................................................................................... 95 Discussion ................................................................................................................... 96 6 CONCLUSIONS AND FUTURE DIRECTIONS .......................................................108