It is well established that diets rich in cholesterol are associated with a number of serious health disorders such as metabolic and cardiovascular disease and also contribute to obesity which the World Health Organisation now classifies as a global epidemic. Clinical studies and experimental animal models have shown that prolonged ingestion of a high cholesterol diet (HCD) can lead to the formation of atherosclerotic plaques which contain lipid deposits and a range of inflammatory cells of which innate immune cells play a key role. By contrast, little is known of the early host responses to an HCD but now we have demostrated, using Danio rerio (zebrafish) larvae, that ingestion of an HCD for a short time induces a localised acute inflammatory response in the intestine. Further, prolonged feeding with an HCD leads to the deposition of fatty streaks in the vasculature around which myleoid cells accumulate and a rapid increase in body weight. Zebrafish express similar proteins involved in the transport of dietary fat to mammals, indicating that the zebrafish is a relevant model for the study of dietary fat uptake and processing. Understanding the dynamics of inflammatory cell migration to the intestine and vasculature and the signalling pathways involved in their activation as the acute intestinal inflammatory response induced by a HCD leads on to cardiovascular lesions may impact on the development of new therapeutic interventions. Developing this understanding forms the basis of this studentship. With tomographic imaging and the availability of transgenic strains of zebrafish bearing lineage associated markers for neutrophils and macrophages, we can visualise the recruitment of inflammatory cells and their interactions with mucosal epithelial cells and the vasculature in intact, live zebrafish, repeatedly imaging the same fish. This can be achieved using zebrafish larvae which are translucent and the transparent zebrafish mutant, Tra/Nac, which make juvenile fish accessible to live imaging. We have recently demonstrated that through feeding anti-sense morpholino oligomers (morpholinos) we can downregulate gene expression and cellular function locally within the intestine. Using this approach, local activation of the inflammasome was found to be critical for the accumulation of leukocytes in zebrafish intestine following an HCD and this is most interesting in light of similar inflammasome activation being observed in the myeloid cells of atherosclerotic plaques. Using the novel route of morpholino delivery and/or by microinjection in the one cell stage embryo, we propose to selectively knockdown genes associated with pro-inflammatory responses and/or the processing of dietary fat. Further, we will add specific pharmacological inhibitors to the water to target inflammatory or fat processing events in the intestine. Fish will be imaged over time to evaluate the dynamic composition of inflammatory cell recruitment and mucosal epithelial cell function as well as the longer term effects on vessels following an HCD.
To summarise, the primary objectives of this project are:
To further develop, characterise and validate zebrafish as model in which to assess and analyse the acute and chronic effects of an HCD
To begin to explore the cells and inflammatory pathways activated by an HCD and assess their impact on the effects of a longer-term HCD
This experimental approach, which is non-invasive unlike methods required for imaging mammalian intestinal and vascular inflammation, impacts on the 3Rs through both refinement of current experimental procedures which largely involve mice and in the reduction in the number of procedures that would be needed for comparable experiments in mice.