The role of the granuloma in bacterial expansion and dissemination during early tuberculosis Open Access

Davis III, James Muse (2009)

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Despite decades of research, tuberculosis (TB) remains a major human health threat, responsible for approximately 1.5 million deaths each year. An effective vaccine remains elusive despite continuous efforts, and the existing vaccine, BCG, is of questionable efficacy. An additional grave concern is the increased prevalence of multiple-drug resistant strains of
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Most TB research has focused on later, adaptive immune aspects, while less attention has been paid to the early, innate immune reactions. In early infection, infected macrophages
aggregate to form immune structures called granulomas, hallmark lesions of tuberculosis. Just prior to graduate school, I established the embryonic zebrafish to model tuberculosis in real-time using fluorescence and Differential Interference Contrast (DIC) microscopy. With this model we found that granuloma formation can be initiated in the context of innate immunity alone. The research detailed here centers on the mechanisms and steps of granuloma formation, which have been difficult to study in other model systems. By comparing mycobacterial pathogenesis in thto embryonic zebrafish infections with other bacterial pathogens, we have confirmed that the host-pathogen interactions observed in this model are each specific to the infecting organism, and quite similar to corresponding human infections with similar pathogens.

Turning to the question of how granulomas form and what consequence they have to early infection, we used
quantitative intravital microscopy to reveal the distinct steps of granuloma formation and assess their consequence for infection. Intracellular mycobacteria use the ESX-1/RD1 virulence locus to induce recruitment of new macrophages to, and their rapid movement within, nascent granulomas. This motility enables multiple arriving macrophages to efficiently find and phagocytose infected macrophages undergoing apoptosis, leading to rapid, iterative expansion of infected macrophages and thereby bacterial numbers. Thus, although the cellular responses which make up early granuloma formation--cell migration, chemotaxis, phagocytosis, and apoptosis--might individually restrict bacterial growth, together they are induced by mycobacteria to provide an excellent growth
environment. The primary granuloma then seeds secondary granulomas via egress of infected macrophages. Our direct observations provide mechanisms whereby pathogenic mycobacteria exploit the granuloma during the innate immune phase for local expansion and systemic dissemination.

Table of Contents


Chapter 1: Introduction to tuberculosis
1.1 Pathogenesis of tuberculosis

1.2 The macrophage in tuberculosis

1.3 Granuloma Formation
1.3.1 The transition from innate to adaptive immunity to Mtb
1.3.2 The role of cytokines and chemokines
1.3.3 The role of regulated cell-cell adhesion

1.4 The embryonic zebrafish model of tuberculosis

1.5 M. marinum Infection of Zebrafish Embryos

1.6 Questions in granuloma formation, maintenance, and spread

1.7 In vivo imaging of tuberculosis pathogenesis

Chapter 2: Introduction to the zebrafish as a model of
host-pathogen interaction

2.1 Why zebrafish? Strengths and weaknesses as a model of host-pathogen interactions

2.2 Overview of the immune system of the zebrafish

2.3 Phagocytes of the zebrafish
2.3.1 Macrophages
Molecular markers
Effector pathways
2.3.2 Melanomacrophages
2.3.3 Neutrophils
Molecular markers
Effector pathways
2.3.4 Dendritic cells

2.4 Zebrafish models of infection
2.4.1 Mycobacteria
2.4.2 Salmonella
2.4.3 Streptococcus
2.4.4 Edwardsiella
2.4.5 Other microbial pathogens
2.5 Summary
2.6 Figures

Chapter 3: Specificity of infection phenotype in zebrafish embryos
3.1 Introduction
3.2 Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos
3.2.1 Introduction
3.2.2 Materials and methods
3.2.3 Results
3.2.4 Discussion
3.2.5 Figures
3.2.6 Supplementary Information

3.3 Leptospria interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues.
3.3.1 Introduction
3.3.2 Materials and methods
3.3.3 Results
3.3.4 Discussion
3.3.5 Figures
3.3.6 Supplementary information
3.4 Summary

Chapter 4: Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish
4.1 Introduction
4.2 Results
4.3 Discussion
4.4 Experimental procedures
4.5 Figures
4.6 Supplemental information

Chapter 5: The role of the granuloma in expansion and dissemination of early tuberculous infection
5.1 Introduction
5.2 Results
5.3 Discussion
5.4 Experimental Procedures
5.5 Figures
5.6 Supplementary information

Chapter 6: Discussion


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