An investigation into the role of optineurin in macrophage-mediated acute antibacterial response in

241 Pages·2015·11.14 MB·English

by  Thean Chew

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

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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

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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.
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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 D iagnosis 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

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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

1.1 0 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 C D 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

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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

2.1 0 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 ............................................................. 88
2.13.1 Preparation of sucrose gradients ................................................... 88

2.13.2 Subcellular fractionation of THP-1 cells ......................................... 89

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2.14 Lysosomal inhibition and TNF production ................................ 89
2.15 Intraperitoneal E. coli infection .................................................. 90
2.15.1 Live E. coli infection ....................................................................... 90

2.15.2 Killed E. coli infection ..................................................................... 90

2.16 Zebrafish Study ........................................................................... 91
2.16.1 Optn in situ hybridisation ............................................................... 91

2.16.2 Salmonella immersion and injection infection ................................ 91

2.16.3 Optn morpholino knockdown ......................................................... 92

2.17 Dextran sodium sulphate (DSS) colitis ...................................... 92
2.17.1 DSS colitis protocol........................................................................ 92

2.17.2 DSS colitis clinical score ................................................................ 92

2.18 Citrobacter rodentium colitis ...................................................... 93
2.18.1 Preparation of C. rodentium ........................................................... 93

2.18.2 Induction of C. rodentium colitis ..................................................... 94

2.18.3 Sample preparation for C. rodentium culture ................................. 94

2.19 Lamina propria cell isolation ...................................................... 95

2.20 Histology and immunohistochemistry. ...................................... 95
2.20.1 Haematoxylin and eosin staining ................................................... 95

2.20.2 OPTN immunohistochemistry ........................................................ 96

2.20.3 Myeloperoxidase immunohistochemistry ....................................... 97

2.20.4 Steiner Silver stain ......................................................................... 98

2.21 Killing assay ................................................................................. 99

2.22 Phagocytosis assay .................................................................... 99
2.22.1 Preparation of FITC-labelled HkEc ................................................ 99

2.22.2 Phagocytosis of FITC-HkEc ......................................................... 100

2.23 Autophagy assay ....................................................................... 100
2.24 Endoplasmic reticulum stress assays ..................................... 100

2.25 Subcutaneous HkEc injection .................................................. 101
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2.26 Confocal immunofluorescence microscopy ........................... 101
2.26.1 Specimen preparation and staining ............................................. 101

2.26.2 Colocalisation analysis ................................................................ 102

2.27 Electron microscopy ................................................................. 102

2.28 Cell Viability Assay .................................................................... 103
2.28.1 MTT Assay .................................................................................. 103

2.29 Flow cytometry .......................................................................... 104
2.29.1 Cell preparation and staining ....................................................... 104

2.29.2 Compensation particle preparation and flow cytometry ............... 104

2.30 Cytokine assay .......................................................................... 105
2.30.1 ELISA and MSD® immunoassay .................................................. 105

2.30.2 Proteome Profiler™ Array ............................................................ 105

2.31 Statistical analysis..................................................................... 105
2.32 Ethics .......................................................................................... 106

Chapter 3 Optineurin in macrophages of Crohn’s disease patients 107
3.1 In troduction ................................................................................ 107

3.2 Results ........................................................................................ 108
3.2.1 Low OPTN expression is replicated in 10% of CD patient ........... 108

3.2.2 Low OPTN expression in CD shows a potential male sex bias ... 111

3.2.3 OPTN expression is associated with inheritance of a minor SNP 117

3.2.4 OPTN is expressed in monocytes, lymphocytes and macrophages

but not neutrophils ..................................................................................... 119

3.3 Discussion ................................................................................. 121
Chap ter 4 Intracellular location and function of optineurin in
macrophages ............................................................................................ 126

4.1 Introduction ................................................................................ 126
4.2 Results ........................................................................................ 127

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4.2.1 Intracellular location of OPTN in human monocytes and
macrophages ............................................................................................. 127

4.2.2 OPTN and TNF in human monocytes and macrophages ............ 131

4.2.3 OPTN is upregulated on bacterial stimulation .............................. 136

4.2.4 Optn-/- mouse generation and charaterisation.............................. 139

4.2.5 Thioglycollate-induced peritoneal macrophages .......................... 144

4.2.6 Bone marrow-derived macrophages ............................................ 148

4.3 Discussion ................................................................................. 163

Chapter 5 Animal Models of Inflammation ......................................... 171
5.1 In troduction ................................................................................ 171

5.2 Results ........................................................................................ 172
5.2.1 Zebrafish model of Salmonella infection ...................................... 172

5.2.2 Mouse model of subcutaneous E. coli infection ........................... 175

5.2.3 Mouse model of intraperitoneal E. coli infection .......................... 178

5.2.4 Mouse model of Citrobacter rodentium colitis .............................. 181

5.2.5 Mouse model of acute DSS colitis ............................................... 190

5.2.6 Mouse model of chronic DSS colitis ............................................ 195

5.3 Discussion ................................................................................. 197

Chapter 6 General Discussion ............................................................ 201
6.1 S ummary of investigations conducted and novel findings ... 201

6.2 Discussion of findings, implications and study limitations ... 201
6.3 Future directions ....................................................................... 206

6.4 Conclusion ................................................................................. 207
Refer ences................................................................................................ 209
Image References .................................................................................... 232

Appendix 1 Primers, probes and morpholinos used ............................ 234

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