INDEX
SUMMARY 4
INTRODUCTION 7
1. Mycobacterium genus 7
1.1 General characteristics 7
1.2 Cell wall 7
1.3 Pathogenesis of TB 13
1.4 Epidemiology and control measures 16
1.5 Mtb secretion systems 18
1.5.1 Type VII secretion systems 20
1.5.2 ESX-5 secretion system 22
2. Host innate immune response to the pathogens 24 2.1 Pathogen recognition by innate immune system 24
2.2 Macrophages and DCs 28
2.3 IL-1 family cytokines 30
2.4 The inflammasome 32
2.5 Caspase-1: inflammatory caspase 38
3. Innate immune response to Mtb 39
3.1 Mtb and inflammasome modulation 43
AIM OF THE STUDY 44
MATERIALS AND METHODS 46
1. Bacterial strains and growth conditions 46
2. Cell populations 47
3. Infection 48
4. Immunofluorescence staining for surface markers 48 5. Determination of cytokines in culture supernatants 49
6. mRNA purification and Real Time PCR 49
7. SDS page and Western blot analysis 51
8. FLICA staining 52
9. Determination of apoptotic and dead cells 52
10. Statistical analysis 53
RESULTS 54
1. Characterization of macrophages and DCs 54 1.1 Morphologic and phenotypic analysis of the cell
populations 54
1.2 Functional analysis of the cell populations 56 2. Maturation marker expression and cytokine profiles 58 2.1 MoDc maturation marker expression and cytokine
profiles 58
2.2 Cytokine production by macrophage populations 61 2.3 Cytokine secretion upon infection of macrophages with
live or heat-killed bacteria 64
3. Construction of the complemented strain 66 4. Growth of Mtbrv1794ko strain in macrophage
subsets 67
5. Induction of cell death and apoptosis 68
6. Analysis of IL-1 pathway 70
6.1 Quantitative measurement of pro-IL-1 mRNA levels by 70
Real Time PCR
6.2 Analysis of pro- and active-IL-1 protein levels by immunoblotting
72
6.3 Caspase-1 inhibition 73
6.4 Quantitative measurement of mRNA levels of
inflammasome components by Real Time PCR 75
6.5 Caspase-1 activity 77
7 The inflammasome activity 79
7.1 Cytokine production via induction of the K+ efflux 79 7.2 Cytokine production via release of cathepsin B 81
DISCUSSION 83
CONCLUSION 92
BIBLIOGRAPHY 93
ACKNOWLEDGEMENTS 126
