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Leukocyte behaviour during infection - new insights from zebrafish models
thesis
posted on 2017-02-20, 00:26 authored by Vahid PazhakhZebrafish have proven
to be an excellent model for studying host-pathogen interactions. They share
highly conserved genetic and physiological aspects with mammals, and offer
technical advantages like small size, high fecundity, availability of several
genomic tools and the feasibility of large-scale phenotype screening.
I have used reverse genetic approaches to exploit the zebrafish embryo model to study the molecular basis of severe congenital neutropenia (SCN), a primary immunodeficiency, which is associated with various severe lethal infections in early childhood. Using exome sequencing on known families of SCN combined with linkage analysis and mapping approaches, a list of candidate genes for SCN was provided by Christoph Klein, (Ludwig-Maximilians University, Munich, Germany) that were potentially responsible for neutropenia in SCN patients. A morpholino knockdown strategy was chosen as the initial reverse genetic approach to see if loss of function of the smarcd2, a gene prioritized for study by Dr Klein, could replicate the neutropenia in a zebrafish model. The smarcd2 morphant embryos replicated the neutropenia phenotype. A knockout model of Smarcd2 was pursued to further evaluate the role of this gene in granulopoiesis, which provided a stable mutant line with a mild neutrophil-deficiency phenotype. These studies confirmed a genetic requirement for smarcd2 for neutrophil development in zebrafish in vivo.
Secondly, to understand the mechanisms by which the immune system protects the host against pathogens, I have exploited zebrafish as an infection model to see the interactions of immune elements with the fungal opportunistic pathogen Penicillium marneffei. In addition to characterizing macrophage dynamics during the course of infection in this model, I collected multiple additional examples of shuttling of phagocytosed spores from neutrophil to macrophage. This novel phenomenon had been previously demonstrated in Lieschke lab, but only by four examples. With multiple examples, I was able to quantify some aspects of this previously undescribed phenomenon. By demonstrating shuttling of Aspergillus fumigatus spores, another opportunistic fungi, I showed that shuttling is not a P. marneffei-specific phenomenon. Having multiple examples of shuttling events with different pathogens enabled this phenomenon to be characterised in more detail and also provided more clues regarding a possible mechanism of spore shuttling. I showed that polystyrene beads mimicking fungal spores are not shuttled. However, zymosan particles, which are fungal cell wall derivatives, are shuttled. These results provide a strong clue that zymosan contains the triggering factor for shuttling and that it is a structural components in fungal cell wall, most likely β-glucan.
Finally I evaluated the potential of this zebrafish embryo fungal infection model to test novel therapeutic strategies against fungal infection. I have provided proof-of-principle that it can be used as a tool for antifungal therapeutic studies. I showed that the biology of macrophages could be exploited to deliver antifungal drugs by showing that spore-laden macrophages are capable of engulfing nanoparticles avidly. This makes it possible to deliver antifungal agents (drugs, myeloperoxidase, etc.) inside macrophages, which is where the P. marneffei spores hide themselves and survive other effective immune elements.
The studies described in this thesis therefore provide new discoveries about: (1) a new genetic cause of severe congenital neutropenia; (2) neutrophil-to-macrophage pathogen shuttling, a novel host/pathogen interaction; (3) the utility of zebrafish fungal infection models for exploring antifungal therapeutic strategies.
I have used reverse genetic approaches to exploit the zebrafish embryo model to study the molecular basis of severe congenital neutropenia (SCN), a primary immunodeficiency, which is associated with various severe lethal infections in early childhood. Using exome sequencing on known families of SCN combined with linkage analysis and mapping approaches, a list of candidate genes for SCN was provided by Christoph Klein, (Ludwig-Maximilians University, Munich, Germany) that were potentially responsible for neutropenia in SCN patients. A morpholino knockdown strategy was chosen as the initial reverse genetic approach to see if loss of function of the smarcd2, a gene prioritized for study by Dr Klein, could replicate the neutropenia in a zebrafish model. The smarcd2 morphant embryos replicated the neutropenia phenotype. A knockout model of Smarcd2 was pursued to further evaluate the role of this gene in granulopoiesis, which provided a stable mutant line with a mild neutrophil-deficiency phenotype. These studies confirmed a genetic requirement for smarcd2 for neutrophil development in zebrafish in vivo.
Secondly, to understand the mechanisms by which the immune system protects the host against pathogens, I have exploited zebrafish as an infection model to see the interactions of immune elements with the fungal opportunistic pathogen Penicillium marneffei. In addition to characterizing macrophage dynamics during the course of infection in this model, I collected multiple additional examples of shuttling of phagocytosed spores from neutrophil to macrophage. This novel phenomenon had been previously demonstrated in Lieschke lab, but only by four examples. With multiple examples, I was able to quantify some aspects of this previously undescribed phenomenon. By demonstrating shuttling of Aspergillus fumigatus spores, another opportunistic fungi, I showed that shuttling is not a P. marneffei-specific phenomenon. Having multiple examples of shuttling events with different pathogens enabled this phenomenon to be characterised in more detail and also provided more clues regarding a possible mechanism of spore shuttling. I showed that polystyrene beads mimicking fungal spores are not shuttled. However, zymosan particles, which are fungal cell wall derivatives, are shuttled. These results provide a strong clue that zymosan contains the triggering factor for shuttling and that it is a structural components in fungal cell wall, most likely β-glucan.
Finally I evaluated the potential of this zebrafish embryo fungal infection model to test novel therapeutic strategies against fungal infection. I have provided proof-of-principle that it can be used as a tool for antifungal therapeutic studies. I showed that the biology of macrophages could be exploited to deliver antifungal drugs by showing that spore-laden macrophages are capable of engulfing nanoparticles avidly. This makes it possible to deliver antifungal agents (drugs, myeloperoxidase, etc.) inside macrophages, which is where the P. marneffei spores hide themselves and survive other effective immune elements.
The studies described in this thesis therefore provide new discoveries about: (1) a new genetic cause of severe congenital neutropenia; (2) neutrophil-to-macrophage pathogen shuttling, a novel host/pathogen interaction; (3) the utility of zebrafish fungal infection models for exploring antifungal therapeutic strategies.
