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An Investigation of Molecular Defects Underlying Impaired Acute Inflammation in Crohn's Disease

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An Investigation of Molecular Defects Underlying Impaired Acute Inflammation in Crohn's Disease AN INVESTIGATION OF MOLECULAR DEFECTS UNDERLYING IMPAIRED ACUTE INFLAMMATION IN CROHN’S DISEASE By Gavin William Sewell A thesis submitted to UCL for the degree of Doctor of Philosophy Division of Medicine 2011 1 I, Gavin William Sewell 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 Abstract Mounting evidence suggests the pathogenesis of Crohn’s disease (CD) involves an impaired acute inflammatory response. Monocyte-derived macrophages from CD patients release deficient levels of pro-inflammatory cytokines in response to Escherichia coli, arising from post-translational trafficking abnormalities. In this thesis, the macrophage responses to microbial stimuli were further characterised, and the underlying molecular lesions investigated. Macrophage TNF release was attenuated in response to Escherichia coli, Candida albicans and Toll-like receptor (TLR) stimulation in CD. This deficit was unrelated to use of medication, age, gender and smoking status. Patients with stricturing and colonic disease demonstrated the most profound defects in response to TLR2 and TLR4 stimulation respectively. Genotyping for 34 known CD susceptibility polymorphisms revealed no detectable association between any individual variant and impaired TNF release. However in CD, TNF secretion in response to Escherichia coli was weakly correlated with overall genetic risk score. Macrophage sphingolipid and phospholipid compositions were subsequently investigated, given the prominent role of these molecules in vesicle trafficking. Mass spectrometric analysis revealed no gross abnormalities in CD macrophages, although a reduced percentage of phosphatidylinositol 16:0/18:1 was synthesised over 3 hours; the same species was also present at reduced levels in ileal biopsies. Given the heterogeneity of CD, a microarray outlier analysis strategy was developed to identify specific gene expression abnormalities in individual patients. A subset of patients had deficient expression of optineurin, a molecule implicated in vesicle trafficking and autophagy. These individuals shared a number of allelic variants, 3 which were associated with optineurin expression levels. Macrophages from these patients had attenuated TNF secretion downstream of TLR2. Concordantly, depletion of optineurin in THP-1 cells impaired TNF release after TLR2 activation. This study supports a role for immune deficiency in the pathogenesis of CD and identifies abnormal optineurin expression as a relevant molecular abnormality in a subset of patients. 4 Table of contents Acknowledgements........................................................................................................ 14 Statement of Collaborative Work ................................................................................ 16 List of abbreviations ..................................................................................................... 18 Chapter 1: Introduction ............................................................................................... 23 1.1 Clinical and epidemiological aspects of Inflammatory Bowel Disease ................ 23 1.1.1 Clinical presentation and management of Crohn’s disease ................................ 23 1.1.2 Ulcerative colitis and other inflammatory bowel diseases ............................. 28 1.1.3 Inflammatory bowel disease is associated with genetic and environmental factors ...................................................................................................................... 29 1.2 Theories of Crohn’s disease pathogenesis ............................................................. 30 1.2.1 Abnormal mucosal barrier function in Crohn’s disease ................................ 31 1.2.2. Infectious aetiology, ‘Dysbiosis’ and Crohn’s disease pathogenesis ............ 34 1.2.3 Autoimmunity .................................................................................................. 42 1.2.4 Immunodeficiency ........................................................................................... 43 1.2.5 Integrated models of Crohn’s disease pathogenesis....................................... 47 1.3 Impaired acute inflammation in Crohn’s disease .................................................. 48 1.3.1 Acute inflammation and the role of macrophages .......................................... 48 1.3.2 Mechanism of impaired neutrophil recruitment in Crohn’s disease .............. 52 1.3.3 Mechanism of defective macrophage cytokine secretion in Crohn’s disease . 54 1.3.4 Pathways of cytokine secretion and relevance to CD ..................................... 55 1.3.5 Other macrophage defects in Crohn’s disease ............................................... 59 1.3.6 Macrophage function in ulcerative colitis ...................................................... 60 1.4 Genetic factors in Crohn’s disease and their relationship to impaired acute inflammation ............................................................................................................... 61 1.4.1 NOD2 .............................................................................................................. 61 1.4.2 IBD5 ................................................................................................................ 63 1.4.3 ATG16L1, IRGM and autophagy .................................................................... 64 1.4.4 Other CD-susceptibility loci ........................................................................... 67 1.4.5 Limitations of GWAS and ‘Missing heritability’ ............................................ 69 5 1.4.6 The missing heritability of CD remains unexplained ..................................... 70 1.5 Outline of Thesis ................................................................................................... 71 1.5.1 Summary of background information ............................................................. 71 1.5.2 Summary of investigations conducted and hypotheses ................................... 73 Chapter 2: Materials and Methods ............................................................................. 75 2.1 Subject recruitment and selection .......................................................................... 75 2.2 Cell culture and assays .......................................................................................... 76 2.2.1 Preparation of stimuli for cell culture assays ................................................. 76 2.1.2 Primary macrophage isolation, culture and stimulation ................................ 77 2.1.3 THP-1 cell culture and stimulation ................................................................ 78 2.1.4 THP-1 cell transfection and siRNA knockdown ............................................. 78 2.1.5 TNF Bioassay .................................................................................................. 79 2.1.6 Multiplex cytokine measurement .................................................................... 80 2.1.7 MTT cell viability assay to estimate cell number ........................................... 81 2.1.8 Propidium iodide staining and flow cytometry ............................................... 82 2.1.9 SDS-PAGE and Western blotting ................................................................... 82 2.3 Genomic DNA preparation and analysis ............................................................... 84 2.3.1 Genomic DNA extraction ................................................................................ 84 2.3.2 Genotyping of Crohn’s disease associated polymorphisms ........................... 84 2.3.3 Calculation of genetic risk scores ................................................................... 86 2.3.4 Sequencing of OPTN region ........................................................................... 87 2.4 Lipid extraction and analysis ................................................................................. 89 2.4.1 Sphingolipid analysis of cultured macrophages ............................................. 89 2.4.2 BCA assay ....................................................................................................... 90 2.4.3 Preparation of samples for phospholipid analysis and stable isotope incubation ................................................................................................................ 91 2.4.4 Phospholipid extraction and analysis by electrospray ionisation mass spectrometry............................................................................................................. 92 2.5 RNA preparation and analysis ............................................................................... 94 2.5.1 RNA extraction from HC, CD and UC macrophages ..................................... 94 2.5.2 Microarray hybridisation ............................................................................... 95 6 2.5.3 Microarray data analysis................................................................................ 95 2.5.4 cDNA conversion and Quantitative PCR........................................................ 98 2.6 Statistical analysis ................................................................................................. 99 2.6.1 Analysis of cytokine secretion measurements, and relationships to phenotypes and genotypes (chapter 3).......................................................................................
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