MUCOSAL TOLERANCE STRATEGIES FOR TREATING TYPE 1 DIABETES IN NON- OBESE DIABETIC MICE By ANDREW SCOTT NELSON 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 2018 © 2018 Andrew Scott Nelson To my parents, my brother, and my wife Lisa ACKNOWLEDGMENTS I would like to thank my parents for their love and support. I would also like to thank my brother, Adam, whose standards I constantly strive to meet. To the friends I have made in graduate school, thank you for your support and help. And a big thank you to my wife, Lisa Lundgren, for working with me to not only understand my science, but to communicate it as well. I sincerely thank my advisor, Dr. David Pascual for providing me with the opportunity to study and train in his lab, and also for his patience and guidance. Thank you David for establishing a high standard for my research, how I present my work, and how I present myself. Thank you as well to Dr. Brusko, Dr. Nguyen, Dr. Zhou, and Dr. Abbott – my dissertation committee – for their support and guidance, without which these projects would not have been completed. 4 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Historical Context .................................................................................................... 18 Classification as an Autoimmune Disease........................................................ 19 Modern Diabetes .............................................................................................. 20 Genetics of Type 1 Diabetes ................................................................................... 21 Environmental factors ............................................................................................. 22 The NOD mouse ..................................................................................................... 25 Disease Etiology ..................................................................................................... 26 Viruses as Disease Initiators ............................................................................ 26 β Cells as Disease Initiators ............................................................................. 27 Disease Pathogenesis ............................................................................................ 28 Mechanisms of Pathogenesis ................................................................................. 30 Oral Tolerance ........................................................................................................ 33 Regulatory T cells ............................................................................................. 34 T1D and Tolerance ........................................................................................... 35 Reovirus Protein Sigma-1 ....................................................................................... 36 Colonization factor antigen 1 .................................................................................. 38 Purpose of the Study .............................................................................................. 40 2 ORAL LACTOCOCCAL IMMUNOTHERAPY FOR THE TREATMENT OF T1D .... 43 Background ............................................................................................................. 43 Methods .................................................................................................................. 46 NOD Mouse Husbandry ................................................................................... 46 Monitoring blood glucose levels ................................................................. 47 Grading Insulitis ......................................................................................... 47 Growing L. lactis for Oral Administration .......................................................... 47 Neutralization of stomach acid ................................................................... 48 Oral gavage of bacteria .............................................................................. 49 Tissue Collections ............................................................................................ 49 Splenocyte isolation ................................................................................... 50 5 Lymphocyte isolation ................................................................................. 51 Counting cells ............................................................................................ 51 Cell Culture ....................................................................................................... 51 Flow Cytometry ................................................................................................ 52 Staining with tetramers ..................................................................................... 53 Cytokine ELISA ................................................................................................ 53 Statistics ........................................................................................................... 55 Results .................................................................................................................... 55 Oral Treatment with LL-CFA/I Ameliorates T1D in NOD Mice .......................... 55 LL-CFA/I induced CD25+ Tregs are suppressive in vitro .................................. 57 Optimization of LL-CFA/I therapy ..................................................................... 59 Phenotyping Tregs at 11 weeks ....................................................................... 60 LL-CFA/I does not induce Tr1 cells at 11 weeks .............................................. 63 LL-CFA/I induced Tregs are stable out to 17 weeks ......................................... 65 Optimized Therapy Reduces Incidence of T1D in NOD mice ........................... 66 Discussion .............................................................................................................. 66 3 ESTABLISHMENT OF A REGULATORY MICROENVIRONMENT BY LACTOCOCCUS EXPRESSION COLONIZATION FACTOR ANTIGEN I .............. 96 Background ............................................................................................................. 96 Methods .................................................................................................................. 98 NOD Mouse Husbandry ................................................................................... 98 Growing L. lactis for Oral Administration .......................................................... 99 Neutralization of stomach acid ................................................................... 99 Oral gavage of bacteria ............................................................................ 100 Tissue Collections .......................................................................................... 100 Splenocyte isolation ................................................................................. 100 Lymphocyte isolation ............................................................................... 101 Counting cells .......................................................................................... 101 Cell Culture ..................................................................................................... 101 Infecting BMDCs ...................................................................................... 102 Co-culture of CD4+ T cells with APCs ...................................................... 103 Flow Cytometry .............................................................................................. 104 Cytokine ELISA .............................................................................................. 104 Statistics ......................................................................................................... 105 Results .................................................................................................................. 105 LL-CFA/I induces regulatory function in BMDCs ...................................... 105 Kinetics of LL-CFA/I induced regulatory DCs and Tregs .......................... 106 LL-CFA/I reduces inflammatory potential of cDCs ................................... 108 LL-CFA/I infected BMDCs do not induce Foxp3+CD4+ Tregs .................. 108 Discussion ............................................................................................................ 109 4 MUCOSAL TOLERANCE INDUCTION FACILITATED BY REOVIRUS PROTEIN SIGMA-1 AMELIORATES T1D IN NOD MICE ..................................... 129 Background ........................................................................................................... 129 6 Methods