The roles of interferon regulatory factors in the murine models of colitis and sepsis [Elektronische Ressource] / by Jingling Yu

The roles of interferon regulatory factors in
the murine models of colitis and sepsis


A Dissertation submitted to

The School of Medicine and Department of Biology

Johannes Gutenburg-University of Mainz


In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

by

Jingling Yu
Born in Beijing, V.R. China

Mainz, December 2009INDEX i


ABSTRACT ............................................................................................ 1
1 INTRODUCTION .............................................................................. 3
1.1 The intestinal immune system ...................................... 3
1.1.1 Gut-associated lymphoid tissues (GALT) .................. 3
1.1.2 The lamina propria and epithelium of the intestinal mucosa ...................... 4
1.1.3 Induction of intestinal immune responses .................................................. 5
1.1.4 A balance between protective immunity and homeostasis to a large
number of different foreign antigens ........................................ 6
1.1.4.1 Tolerance to food proteins and commensals ................................................................ 7
1.1.4.2 Protective immunity caused by pathogens.... 7
1.2 Inflammatory bowel diseases ........................................................................ 8
1.2.1 Genetics .................................... 9
1.2.2 Environmental factors ................ 9
1.2.3 Immunobiology ........................................................ 10
1.2.3.1 Epithelial barrier .......................................................................... 10
1.2.3.2 Dendritic cells .............. 11
1.2.3.3 Innate immune cells .... 12
1.2.3.4 Effector T cells ............................................................................................................. 12
1.2.3.5 Apoptosis of T cells ..... 13
1.2.3.6 Balance of regulatory T cells and effector T cells ....................................................... 14
1.3 Sepsis ............................................................................ 15
1.4 Immunopathogenesis of sepsis .................................. 16
1.4.1 Microbial pathogenesis ............ 18
1.4.1.1 Causative microorgarnisms ......................................................................................... 18
1.4.1.2 Microbial components ................................. 18
1.4.2 Host recognition of microbial component ................. 19
1.4.2.1 Pathogen recognition by innate immunity ................................... 20
1.4.2.2 Proinflammatory cytokines .......................................................... 22
1.4.3 Coagulation and anticoagulation ............................. 24
1.4.4 Immune suppression and apoptosis ........................................................ 24 INDEX ii


1.5 Interferon-regulatory factors ....................................................................... 25
1.6 Aims of the study.......................................................................................... 28
2 MATERIALS AND METHODS ....................... 30
2.1 Materials ........................................................................................................ 30
2.1.1 Chemicals 30
2.1.2 Equipments ............................................................................................. 30
2.2 Software ........................................ 31
2.3 Methods ......................................................................... 32
2.3.1 Human colon biopsy samples .................................. 32
2.3.2 Mice ......... 32
2.3.3 Induction of colitis .................................................................................... 33
2.3.3.1 TNBS-induced colitis ................................... 33
2.3.3.2 DSS-induced colitis ..... 34
2.3.3.3 Naive T cell-transfer colitis .......................................................................................... 34
2.3.4 Histopathology and mouse endoscopy .................... 35
2.3.5 Cell isolation and cultivation .................................................................... 37
2.3.5.1 Splenocytes isolation ... 37
2.3.5.2 Naive CD4+CD25- T cell isolation............... 38
2.3.5.3 Lamina propria mononuclear cells (LPMC) isolation .................................................. 38
2.3.5.4 Isolation of colonic lamina propria fibroblasts (CLPFs) ............... 39
2.3.6 Cytokine measurement by using ELISA .................................................. 39
2.3.7 Immunofluorescent staining ..................................... 39
2.3.8 Detection of apoptosis ............. 40
2.3.9 RNA isolation, cDNA synthesis and Real-time PCR ................................ 41
2.3.10 Mapping of putative IRF4 binding sites on murine promoters .................. 43
2.3.11 Chromatin Immunoprecipitation ............................................................... 43
2.3.12 Electrophoretic mobility shift assay (EMSA) ............................................ 45
2.3.13 Retroviral transduction ............................................. 47
2.3.14 Induction of sepsis by CLP ...... 48
2.3.15 Leukocyte counts ..................................................... 49
2.3.16 Determination of CFU .............................................. 49 INDEX iii


2.3.17 Fluorescence Activated Cell Sorting (FACS) ........................................... 49
2.3.17.1 Detection of cell surface markers ................................................ 49
2.3.17.2 Intracellular staining .................................................................... 50
2.3.17.3 Phagotest .................................................... 50
2.3.18 Determination of myeloperoxidase activity (MPO) ... 50
2.3.19 Depletion of neutrophils ........................................................................... 51
2.3.20 Statistical Analysis ................... 51
3 RESULTS ...................................................................................... 52
3.1 IRF4 expression in human IBD patients ..................... 52
3.1.1 IRF4 protein expression in colon of IBD patients ..................................... 52
3.1.2 IRF4 expressing cells .............................................. 53
3.1.3 Expression of potentially IRF4 regulated cytokines in IBD patients ......... 54
3.1.4 Correlation between IRF4 and IL-6, IRF4 and IL-17, IRF4 and IL-22 ...... 55
3.1.5 IRF4, IL-17, IL-22 expressions in inflamed mucosal tissue ..................... 56
3.2 IRF 4 in T cell dependent experimental colitis models .............................. 57
3.3 IRF4 in DSS colitis model ............................................................................ 58
3.4 Analysis of proinflammatory cytokine pattern in murine colitis models . 60
3.4.1 Expression and production of cytokines in TNBS colitis .......................... 60
3.4.1.1 IL-6 and IL-17 productions in colon specimen ............................................................ 60
3.4.1.2 Production of IL-6 in absence of IRF4 ......................................... 61
3.4.2 Expression and production of cytokines in transfer colitis model ............. 62
3.4.3 Expression of cytokines and related transcription factors in DSS colitis
model......................................................................................................................64
3.5 Detection of apoptosis in colitis models .................................................... 65
3.6 Regulatory fa