Linköping University Medical Dissertations No. 1410. STUDIES OF MUCOSAL IMMUNE REGULATION IN CELIAC DISEASE AND TYPE 1 DIABETES ANNE LAHDENPERÄ Division of Paediatrics Department of Clinical and Experimental Medicine Faculty of Health Sciences, Linköping University, Sweden Linköping 2014 © Anne Lahdenperä, 2014 ISBN: 978-91-7519-286-4. ISSN: 0345-0082. Paper I, II and III has been printed with permission from the publisher. During the course of the research underlying this thesis, Anne Lahdenperä was enrolled in Forum Scientium, a multidisciplinary doctoral programme at Linköping University, Sweden. Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2014. II To my family III SUPERVISORS Johnny Ludvigsson, Professor Emeritus Division of Paediatrics, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. Outi Vaarala, Professor Institute of Clinical Medicine, University of Helsinki, Helsinki, Finland. Translational Science, Innovative Medicine/Respiratory, Inflammatory and Autoimmune Diseases, AstraZeneca. Rosaura Casas, Associate Professor Division of Paediatrics, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. OPPONENT Eva Sverremark-Ekström, Professor The Wenner-Gren Institute, Department of Molecular Biosciences, Stockholm University, Stockholm, Sweden. COMMITTEE BOARD Marie-Louise Hammarström, Professor Immunology, Department of Clinical Microbiology, Umeå University, Umeå, Sweden. Jonas Wetterö, Associate Professor Autoimmunity and Immune Regulation Unit (AIR)/Rheumatology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. Torbjörn Ledin, Professor Oto-Rhino-Laryngology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. IV CONTENTS LIST OF ORIGINAL PAPERS ................................................................................................. 3 ABSTRACT ............................................................................................................................... 5 SAMMANFATTNING .............................................................................................................. 7 ABBREVIATIONS .................................................................................................................... 9 INTRODUCTION .................................................................................................................... 11 The immune system ............................................................................................................. 11 The innate immune system ............................................................................................... 11 The adaptive immune system ........................................................................................... 13 The mucosal/gut immune system ..................................................................................... 18 Oral tolerance ................................................................................................................... 20 Probiotics .......................................................................................................................... 21 Infant immunity ................................................................................................................ 22 Celiac disease (CD) .............................................................................................................. 23 History .............................................................................................................................. 23 Genetic factors .................................................................................................................. 23 Environmental factors ...................................................................................................... 24 Symptoms ......................................................................................................................... 24 Diagnosis .......................................................................................................................... 27 Histopathology ................................................................................................................. 27 Serologic markers ............................................................................................................. 29 HLA typing ...................................................................................................................... 30 Epidemiology ................................................................................................................... 30 Development of CD/(Molecular) Pathogenesis ............................................................... 31 Treatment ......................................................................................................................... 35 Associated diseases .......................................................................................................... 36 Mortality & malignancy ................................................................................................... 36 Type 1 diabetes (T1D) .......................................................................................................... 37 History .............................................................................................................................. 37 Definition & Symptoms ................................................................................................... 38 Diagnosis .......................................................................................................................... 38 Genetic factors .................................................................................................................. 38 Environmental factors ...................................................................................................... 39 Serologic markers ............................................................................................................. 42 Epidemiology ................................................................................................................... 43 Development of T1D/(Molecular) Pathogenesis .............................................................. 44 Treatment ......................................................................................................................... 45 Associated diseases .......................................................................................................... 45 Mortality & Complications .............................................................................................. 46 Intervention & prevention studies in T1D ........................................................................ 46 AIMS OF THE THESIS .......................................................................................................... 49 MATERIAL & METHODS ..................................................................................................... 51 Study subjects ....................................................................................................................... 51 Paper I .............................................................................................................................. 51 Paper II ............................................................................................................................. 51 Paper III ............................................................................................................................ 52 Paper IV ............................................................................................................................ 53 Laboratory methods .............................................................................................................. 54 1 Sample collection (paper I-IV) ......................................................................................... 54 RNA isolation (Paper I-IV) .............................................................................................. 55 Real-time polymerase chain reaction (PCR)-array analyses (Paper I, III and IV) ........... 56 Quantitative real time reverse transcription (RT)-PCR analyses (Paper II and III) ......... 59 Immunohistochemical analyses (Paper II) ....................................................................... 60 Cytokine assessment in in vitro cultured biopsies (Paper II) ........................................... 61 Analyses of apoptotic markers in IL-17 treated Caco-2 cells (Paper II) .......................... 61 Analyses of T1D related autoantibodies (Paper IV) ........................................................ 61 Whole blood cultures in vitro stimulated with LPS or LTA (Paper IV) .......................... 62 Flow cytometric analyses (Paper IV) ............................................................................... 62 Analyses of C-reactive protein (CRP) (Paper IV) ............................................................ 62 Statistical methods ................................................................................................................ 62 PCA .................................................................................................................................. 63 Clustering ........................................................................................................................