An investigation into the role of optineurin in macrophage-mediated acute antibacterial response in inflammatory bowel disease By Thean Soon Chew A thesis submitted to UCL for the degree of Doctor of Philosophy Division of Medicine 2015 1 I, Thean Soon Chew confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 ex nihilo nihil fit 3 Abstract Crohn’s disease (CD) is now recognised to be due to defective host responses to bacteria in genetically susceptible individuals. Others in our research group previously found defective neutrophil recruitment, bacterial clearance and monocyte-derived macrophage (MDM) proinflammatory cytokine secretion in CD. To investigate the defective cytokine secretion, we performed transcriptomic analysis and identified reduced MDM optineurin (OPTN) expression in 10% CD patients. Here, I show that macrophages stimulated with E. coli, bacterial Toll-like and NOD-like receptor ligands upregulate OPTN, a Golgi complex protein that regulates vesicle transport. In vitro, Optn-/- bone marrow-derived macrophages (BMDM) stimulated with E. coli secrete significantly lower levels of proinflammatory TNF and IL6 cytokines, which is normalised to wildtype levels on addition of lysosomal function inhibitors. In vivo, Optn-/- mice develop a more severe Citrobacter colitis with greater mortality, an early defective neutrophil recruitment to the bowel and lower levels of serum proinflammatory cytokines. The Optn-/- mice also demonstrated a similar phenotype of defective neutrophil recruitment and lower serum proinflammatory cytokines in an E. coli-induced peritonitis. These results indicate that the low OPTN expression originally identified in CD macrophages has likely contributed to an attenuated antibacterial response, which potentially led to the development of bowel inflammation. 4 Table of Contents Table of Contents ......................................................................................... 5 Table of Figures ......................................................................................... 12 Table of Tables ........................................................................................... 21 Acknowledgements ................................................................................... 22 Statement of Collaborative Work .............................................................. 23 List of Abbreviations ................................................................................. 24 Chapter 1 Introduction........................................................................... 27 1.1 Diagnosis and natural history of inflammatory bowel disease 27 1.1.1 Natural history of Crohn’s disease ................................................. 27 1.1.2 Natural history of ulcerative colitis ................................................. 30 1.2 Epidemiology and burden of inflammatory bowel disease ...... 31 1.2.1 Healthcare cost of inflammatory bowel disease ............................. 31 1.2.2 Rising incidence and prevalence of IBD ........................................ 32 1.3 Environmental effects on IBD ..................................................... 33 1.3.1 Smoking ......................................................................................... 33 1.3.2 Diet ................................................................................................ 33 1.4 Treatment of inflammatory bowel disease ................................ 34 1.4.1 5-Aminosalicylates ......................................................................... 34 1.4.2 Corticosteroids ............................................................................... 35 1.4.3 Thiopurine analogues .................................................................... 35 1.4.4 Anti-TNF therapy ........................................................................... 36 1.5 Pathogenesis of Crohn’s disease .............................................. 36 1.5.1 Infectious aetiology ........................................................................ 36 1.5.2 Dysbiosis ....................................................................................... 38 1.6 Genetic factors in Crohn’s disease ............................................ 38 1.6.1 NOD2 ............................................................................................. 39 5 1.6.2 ATG16L1 ....................................................................................... 40 1.6.3 The missing heritability in CD ........................................................ 44 1.7 Innate immunity in the gastrointestinal tract ............................ 44 1.7.1 Macrophages and neutrophils in gut mucosa ................................ 45 1.7.2 Cytokine trafficking in macrophages .............................................. 48 1.7.3 The innate immune system and Crohn’s disease .......................... 51 1.8 Defective innate immunity in Crohn’s disease ......................... 51 1.9 Optineurin .................................................................................... 55 1.9.1 Optineurin structure and function ................................................... 55 1.9.2 Optineurin in vesicle trafficking ...................................................... 57 1.9.3 Optineurin and autophagy ............................................................. 59 1.9.4 Optineurin and the type I interferon response ................................ 60 1.9.5 Optineurin and disease .................................................................. 61 1.10 Outline of thesis........................................................................... 62 1.10.1 Summary of background information ............................................. 62 1.10.2 Summary of investigations and hypothesis .................................... 63 Chapter 2 Materials and Methods ......................................................... 65 2.1 CD patient and healthy control recruitment .............................. 65 2.2 gDNA extraction, OPTN sequencing and SNP genotyping. ..... 66 2.3 Optn-/- mouse generation and verification ................................. 66 2.3.1 Optn-/- mouse generation ............................................................... 66 2.3.2 Mouse husbandry .......................................................................... 66 2.3.3 Mouse genotyping ......................................................................... 66 2.3.4 Optn-/- mouse verification ............................................................... 67 2.3.5 Investigating the naïve Optn-/- phenotype ...................................... 68 2.4 Optn-/-Nod2-/- mouse generation ................................................. 71 2.4.1 Nod2-/- mice ................................................................................... 71 2.4.2 Optn-/-Nod2-/- mice ......................................................................... 71 6 2.5 Antibodies and stains ................................................................. 71 2.5.1 Antibodies used in immunoblots .................................................... 71 2.5.2 Antibodies used in confocal microscopy ........................................ 72 2.5.3 Antibodies used in flow cytometry ................................................. 72 2.5.4 Antibodies used in immunohistochemistry ..................................... 72 2.6 Reagents ...................................................................................... 73 2.6.1 Heat-killed E. coli (HkEc) stock ...................................................... 73 2.6.2 MTT cell viability assay reagents ................................................... 73 2.6.3 Buffers ........................................................................................... 74 2.6.4 Cell culture media .......................................................................... 75 2.6.5 Bacterial culture broth .................................................................... 76 2.6.6 Histology and microscopy reagents ............................................... 76 2.7 Cell culture and stimulation ........................................................ 78 2.7.1 Monocyte-derived macrophages (MDM) ........................................ 78 2.7.2 THP-1 cells .................................................................................... 79 2.7.3 Bone marrow-derived macrophages (BMDM) ............................... 80 2.7.4 Thioglycollate induced peritoneal macrophages (TiPM) ................ 81 2.8 Macrophage Expression Microarray .......................................... 82 2.9 Quantitative reverse transcription PCR (qRT-PCR) .................. 83 2.9.1 Cell preparation and conversion into cDNA ................................... 83 2.9.2 qRT-PCR protocol ......................................................................... 84 2.10 Immunoblot .................................................................................. 85 2.10.1 Whole cell lysate preparation ......................................................... 85 2.10.2 Semi-dry transfer ........................................................................... 86 2.11 Immunoprecipitation ................................................................... 87 2.12 Mass spectrometry ...................................................................... 87 2.13 Subcellular fractionation ............................................................