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Inflammatory bowel diseases (IBD), with Crohn’s disease (CD) and ulcerative colitis (UC) as the two most prevalent phenotypes, span a disease spectrum of lifelong, idiopathic conditions characterized by chronic inflammation of the gut with diarrhea and abdominal pain. Although its exact pathophysiology remains elusive, IBD is believed to be caused by an aberrant mucosal immune responses towards intestinal microorganisms in genetically predisposed individuals. During the last years, new pathophysiologic pathways have been uncovered and since there still is an unmet therapeutic need in IBD, this knowledge is currently being applied by pharmaceutical companies to develop compounds that target these pathways. Therefore, in the first part of this project we aimed to contribute to the therapeutic development for IBD patients by performing a preclinical efficacy experiment in mice for a new experimental JAK1-specific inhibitor (GLPG0634/filgotinib) that has been developed by Galapagos NV. Since the efficacy of filgotinib was already shown in a chemically induced murine colitis model (DSS), we performed these experiments in the T cell transfer model of colitis which is an adaptive/T cell-driven model. Daily oral administration of filgotinib resulted in a significant reduction in body weight loss and histological inflammation scores. Our data, together with the data obtained from the chronic DSS model, provide convincing preclinical evidence for the application of filgotinib in IBD and meanwhile a large phase 3 program is ongoing. Other new therapeutic strategies, are in the research pipeline or reaching registration. This means that, in the future, the range of therapies will drastically expand. Furthermore, a well-known characteristic of IBD is the large inter-patient variability in disease course and severity, which has consequences for the type, the dose and frequency of treatment. We believe that the IBD treatment paradigm should change towards a more personalized approach that is based on targeting the underlying pathophysiologic pathways that drive the disease in a given patient. The second part of this PhD project investigated the contribution of three IBD-associated pathways (ER stress, autophagy and inflammasomes) in specific patient-subgroups at different levels. First of all, we developed a novel cell culture system that allows short term expansion of patient/biopsy-derived intestinal epithelial monolayers. We confirmed that these monolayers had an epithelial character and a normal apical-basolateral polarization. In this model we showed that the number of risk alleles in ER stress and/or autophagy leads to an increased BIP induction after thapsigargin treatment indicating functional alterations in the epithelial ER stress response. These readouts might in the future be used as an indication for ER stress reducing therapies such as TUDCA. Furthermore, we believe that this patient-derived intestinal epithelial cell culture approach has more potential applications such as co-culturing with other cell types and/or investigating patient-specific responses to pharmaceuticals or even microbes. Finally, we investigated the inflammasome, ER stress and autophagy at a transcriptional level in the mucosa of patients and healthy controls who were included in two well-characterized whole genome gene expression microarray cohorts. We found a strong upregulation of two dsDNA-responsive inflammasome sensors AIM2 and IFI16 in patients with active disease when compared to patients with inactive disease or healthy controls. We are one of the first groups to identify the potential involvement of both inflammasome sensors which are now subject of investigation as a disease activity marker (IFI16) and a key player in intestinal homeostasis (AIM2). We also found a strong upregulation of several ER stress genes and a downregulation of autophagy genes that was associated with increased disease activity. When disease was controlled normalization was observed for most of these genes, yet some (KDELR3, XBP1, MAP1LC3A and CHMP4B) remained dysregulated, indicating that these pathways are not entirely restored to their normal levels upon remission. Finally, we found a gene expression signature in these pathways that could aid in predicting the response to anti-TNF treatment caused by an altered ER stress or autophagy status. Although preliminary, these data hint towards the application of ER stress reducing or autophagy stimulating therapy in patients who show, already at baseline, dysregulated gene expression patterns. In conclusion, the promising filgotinib data demonstrate that we live in a time of increasing therapeutic diversity for IBD. In this PhD thesis we furthermore set the first steps towards a personalized pathway-based approach for treating this multifactorial disease spectrum. LIST OF ABBREVIATIONS 8 CHAPTER 1 INTRODUCTION 13 1. The paradigm of patient management in IBD 15 2. Toward innovative management of IBD 17 2.1. Anti-IL-12/23 17 2.2. Janus kinases inhibition 18 2.3. Leukocyte trafficking 20 3. Targeting early events in IBD pathogenesis to restore intestinal homeostasis 21 3.1. Anti-Smad7 (TGF-β/BMP signaling) and other antisense oligonucleotides 21 3.2. Cell-based therapies 24 4. Future prospects in IBD management 26 4.1. A mechanism-based approach 26 4.2. Toward personalized IBD management 31 5. Conclusion 33 6. References 35 CHAPTER 2 RESEARCH OBJECTIVES 43 PART I IN VIVO TARGETING OF THE JAK-STAT PATHWAY IN IBD 47 CHAPTER 3 SELECTIVE INHIBITION OF JANUS KINASE 1 (JAK1) WITH FILGOTINIB REVERSES PATHOGENIC PROCESSES IN PRECLINICAL MODELS FOR IBD 49 1. Introduction 52 2. Materials and Methods 54 2.1. Reagents 54 2.2. Mice 54 2.3. Induction of chronic colitis and Experimental setup 54 2.4. Monitoring of disease 54 2.5. Statistical analysis 56 3. Results 57 3.1. Pilot experiments 57 3.2. Filgotinib reduces body weight loss and inflammatory scores 59 4. Discussion 61 5. References 64 PART II FUNCTIONAL TRANSLATION OF IDENTIFIED PATHWAYS IN IBD 67 CHAPTER 4 BIOPSY-DERIVED INTESTINAL EPITHELIAL CELL CULTURES FOR PATHWAY BASED STRATIFICATION OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE 69 1. Abstract 71 2. Introduction 72 3. Materials and methods 74 3.1. Patients and ethical statement 74 3.2. Isolation and culturing of IECs 75 3.3. Immunocytochemistry 76 3.4. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis 77 3.5. ER stress induction 78 3.6. Binding immunoglobulin protein (BiP)/Glucose-regulated protein 78 (GRP78) ELISA 78 3.7. Statistical analysis 78 4. Results 79 4.1. IECs and epithelial characterization 79 4.2. Genetic risk in ER stress and autophagy genes and the epithelial ER stress response 82 5. Discussion 86 6. Supplementary material 90 7. References 92 CHAPTER 5 STRONG UPREGULATION OF AIM2 AND IFI16 INFLAMMASOMES IN THE MUCOSA OF PATIENTS WITH ACTIVE INFLAMMATORY BOWEL DISEASE 95 1. Abstract 97 2. Introduction 98 3. Materials and methods 100 3.1. Patients and biopsy specimens 100 3.2. Mucosal gene expression analysis 100 3.3. RNA isolation 100 3.4. Whole-genome gene expression analysis 101 3.5. qRT-PCR analysis 101 3.6. Immunohistochemistry 102 3.7. Western blot analysis 102 3.8. Ethical considerations 102 4. Results 103 4.1. Gene expression of different inflammasome subtypes in IBD colonic mucosa 103 4.2. Validation of AIM2 and IFI16 expression at the protein level 107 4.3. Immunohistochemistry 108 5. Discussion 111 6. Supplementary material 115 6.1. Protocol details 115 6.2. Supplementary tables 117 6.3. Supplementary figures 118 7. References 120 CHAPTER 6 MUCOSAL EXPRESSION OF AUTOPHAGY AND ER STRESS GENES IN INFLAMMATORY BOWEL DISEASES 125 1. Introduction and aim 127 2. Material and methods 128 2.1. Selection of ER stress and autophagy associated genes 128 3. Results 129 3.1. ER stress genes 129 3.2. Autophagy genes 134 4. Discussion 137 5. Supplementary material 142 5.1. Filtering of ER stress genes 142 5.2. Filtering of autophagy genes 142 5.3. Supplementary tables 143 6. References 144 CHAPTER 7 CONCLUDING DISCUSSION 149 1. General discussion 151 2. Future perspectives 159 3. References 162 SUMMARIES 167 1. English summary 167 2. Nederlandstalige samenvatting 169 3. Popular summary 171 4. Popular summary (Dutch) 173 SCIENTIFIC ACKNOWLEDGEMENTS 175 PERSONAL CONTRIBUTION 176 CONFLICTS OF INTEREST STATEMENT 177 ACKNOWLEDGEMENTS - DANKWOORD 178 CURRICULUM VITAE 190 SCIENTIFIC COMMUNICATIONS 192 nrpages: 194 status: published