Characterisation of TH17-type cytokines and their receptors in rainbow trout, Oncorhynchus mykiss

The substantial increase in production by the aquaculture industry in the last few decades has been accompanied by a greater demand for prophylactic measures to control fish health, since fish diseases are a major impediment for the profitability of fish farms. To date vaccination has played a major role in the success of fish farming, offering resistance to a limited number of infectious diseases. In order to expand the availability of effective vaccines, a better knowledge of the piscine immune system is crucial. Although a tremendous effort has been put into studies of the fish immune system over recent years, our understanding is still far behind when compared to that of mammals, with many gaps in knowledge still requiring to be addressed. To gain a better understanding of fish immune responses, this thesis has focused on characterising a number of immune molecules considered crucial for disease resistance against extracellular pathogens, in a commercially important species, the rainbow trout (Oncorhynchus mykiss). Adaptive immunity is regulated by a group of specialised lymphocytes, the T Helper (T H) cells. They exert their helper functions through the release of a range of cytokines, which can be used to further categorise them into subsets. One of the latest subsets to be characterised was the T H 17 lineage, which secretes various cytokines, including interleukin (IL )-17 A and IL-22, pivotal in the eradication of extracellular pathogens. To gain a better insight into TH17-type cytokines and their responses in fish, Chapters II and III focused on the characterisation of such molecules, through expression studies, and by analysing their bioactivity as recombinant proteins for the rust time in piscine species. Homology studies confirmed that both molecules are likely to belong to the IL- 22 and IL-17 cytokine families, noting that the trout IL-17 AlP protein shared a close relationship with other piscine IL-17 AlP2 molecules. Interestingly, analysis of their biological activities in splenocyte primary cultures indicated that the two proteins had an effect on the expression of antimicrobial peptides, with IL-22 displaying a potentially more important role than IL-17 AlF. Cytokines are only able to conduct their effects through binding to specific cell surface receptors, acting as ligands. The presence of these particular receptors dictates which cells can be targeted by such cytokines and highlights the importance of the ligand-receptor interaction. To further understand the already identified cytokines, or their family members, a range of receptors was characterised in Chapter IV, with two of them (CRFB4 and IL-17RA) being potentially involved in the signalling of IL-22 and IL-17 AlF ligands. Although homology studies confirmed they belong to the IL-17 and class II cytokine receptor families, orthology of all nine receptors to mammalian homologues could not be inferred. Therefore, further work is required to achieve a better understanding of the homology of these molecules with the mammalian receptors, and their potential involvement with IL-22 and IL-17 AlF intracellular signalling. TH17 cells are characterised not only by the unique range of effector cytokines they secrete, but also through the presence of a master transcription factor, the retinoid-related orphan receptor (ROR)-yt. In Chapter V, two members of the ROR family were characterised, sharing 90% identity between each other. Trout ROR-y1 and -y2 were found to have a close relationship with known ROR-y family members, sharing a particularly high homology with the mammalian splice variant ROR-yt. This suggests that the piscine molecules are likely to be homologues of ROR-yt. However, it still remains to be elucidated whether they can drive piscine TH17 cell differentiation and indeed trigger a TH17 response in fish. T Helper (TH) cells are vital in promoting immune responses. Thus, Chapter VI aimed at developing a potentially effective antibody which could recognise the CD4 marker, typically expressed by this cell subset. The polyclonal antibody, produced using a recombinant protein approach, revealed encouraging results. It was found by Western blot analysis that this antibody could recognise proteins with a molecular weight approximating to that expected for the trout CD4 molecule and its potential splice variant. However, to confirm these observations, further work is still required t? better characterise this antibody and the cells it targets. The results of this thesis have revealed that despite the similarities between the mammalian and piscine immune systems, clear differences exist and also that fish have developed unique molecules and mechanisms not found in mammals. It is therefore of great interest to understand in more detail the role of these novel components so that future progress can be made towards gaining a better comprehension of fish immunity and help advance new vaccination strategies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo