User:Kinkreet/Zebrafish and Vertebrate Immunity

Zebrafish was first identified as a investigative tool 30 years ago; at that time, it was used to elucidate early development in vertebrates. Nowadays, it is used to investigate organogenesis, and also reponses to diseases, infections and cancer.

More studies in recent years have been geared towards the immune system of zebrafish; it is used as a model because it has many common aspects with the vertebrate immune system, furthermore, its transparent embryos allow for monitoring cell movements and organogenesis in real-time and in 3D. A strain of zebrafish (named casper) has been developed which have transparent bodies even in adulthood.

Most of the research into immunity and inflammatory responses have been based on studies on rodent models and primary immune cell cultures, but there are many limitations in their use. One such limitation arise because the immune system have evolved to become quite complex, and thus making it hard to study. The zebrafish's immune response have common features with vertebrates but is much simpler. Thus, zebrafish is becoming the model of choice in the first step in identifying the basic principles or mechanisms, and only using rodent models or higher vertebrate models once the principle is established.

Transgenic Lines for Tracking Immune Cells
Cell type-specific transgenic lines have been created to allow researchers to follow each cell type in zebrafish (in vivo) during an immune response. A transgenic line gata2 has been generated to discriminate eosinophils from other cells. Cells with similar histochemical and biochemical properties to mast cells have been identified in zebrafish, but a transgenic has yet to be developed.

The development of these lines in zebrafish have been important because some of these cells, such as eosinophils, are very hard to track in mammals.

Two macrophage-specific transgenic lines have been developed: mpeg:Gal4 and fms:Gal4. Both uses the upstream activating sequence (UAS) to drive expression. From following its movement in vivo, it was found that macrophages prefers to migrate along the walls of the lumen instead of in the middle of the lumen.

Elucidating the Main Phagocyte
There has been much debate whether neutrophils or macrophages are the main phagocyte. It is found that in human, mammal and zebrafish models that when the cells are in a fluid environment, macrophages are the main phagocytes; on a tissue surface, however, neutrophils are the main phagocyte.

PI3K and F-actin
The Pleckstrin homology (PH) domain binds to phosphatidylinositol-(3,4,5)-triphosphate (PIP3), which are produced from phosphorylation of other phosphatidylinositol (PI) lipids by PI 3-kinase (PI3K).

Using a PH domain tagged with a GFP domain, we are able to track the localisation of PI3K in cells. When done on neutrophils, it was found that PI3K is concentrated at the leading edge during both forward and reverse migration, suggesting PI3K regulate the polymerisation of globular actin (G-actin) into filamentous actin (F-actin), which is the basis of cell membrane extrusion and cell migration. Other factors also regulate F-actin formation, such as RAC.

Mutagenic screens
Mutagensis can be induced by using ENU or retroviral insertions. The mutants are then checked for changes in phenotype. One such mutant has a defect in LTA4 hydrolase, and this cause a build-up of LTA4 and an absence of leukotriene B4 (the LTA4 hydrolase product). The build up of LTA4 is compensated by its converstion to lipoxin A4, which is pro-resolution, causing neutrophils to migrate from the inflammatory site. When this mutant is infected with Mycobacterium marinum, it failed to mount a response because neutrophils are not recruited to the inflammation site. This has implementations in designing new immunosuppressant drugs which targets the pro-resolution pathways rather than the co-stimulatory pathway. For example, lipoxin is already used as an anti-inflammatory mediator.

Ablation study in Macrophages
Ablation studies involves selectively destroying cells, based on the location (in space and time) and cell type. Ablation studies have already been used to study cell lineage relationships, elucidating the roles of different cells during embryogenesis, and studying the mechanism of tissue regeneration (i.e. how the organism regenerate the ablated cells).

To create specificity, a gene encoding for bacterial nitroreductase (NTR) is transformed with a promoter into the genome of specific cell types. NTR catalyse the reaction that convert a harmless prodrug metronidazole (Mtz) into a cytotoxic product. So if we develop a transgenic line of zebrafish with that produce NTR+ macrophages, we can add Mtz to the medium and this would kill off all the NTR+ cells, namely the macrophages. This allows us to see the change in phenotype by the absence of macrophages.

Macrophage interaction with pathogens
It has recently come to light that macrophages may aid in the replication of some pathogens. Burkholderia cepacia is a respiratory pathogen that might have an intra-phagocytic life cycle that make it evasive to the immune response.

Mycobacteria also have an intra-phagocytic life cycle that allows it to disseminate and to form granulomas. Previously, it was thought that granulomas are formed to protect against mycobacteria dissemination, but now it looks more likely to aid in the process rather than prevent it. Mycobacteria can also gain tolerance to both the host defense and also some antibiotics. When mycobacteria is inside the macrophage, it expresses a bacterial efflux pump, that is part of its tolerance. This is supported by the fact that introduction of verapamil, an inhibitor for the pump, shortened the time required for antibiotic treatments.

Autophagy
Autophagy is involved in the degradation of the yolk proteins during embryonic development, and because zebrafish have a rapid development, it is a good model to study autophagy. Two homologs of the Saccharomyces cerevisiae Autophagy-related protein 8 (Atg8) were identified in zebrafish - lc3 and gabarap. Congcong He and colleagues generated transgenic zebrafish which express GFP-tagged lc3 and gabarap, both of which was observed to undergo post-translational modifications from the pharyngula stage. Autophagy can be upregulated in zebrafish by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. It was found that during an autophagic response, the Atg8 homologs accumulated inside lysosomes, and so GFP-gabarap or GFP-lc3 transgenics can be used to visually indicate the level of autophagy in zebrafish.