Final Report Summary - SMARTFISH (Study of specific cell mediated immunity and vaccine optimization against bacterial and viral infections in trout (Oncorhynchus mykiss)) Fish are one of the most widely used low-cost protein sources in many parts of the world. However, it is clear that the availability of fish harvested from capture fisheries to support the growing demand for fish protein will be inadequate. Thus, the world will need to turn to producing fish, i.e. aquaculture. The control and prevention of infectious diseases is a major goal in fish farming due to the important economical losses they cause. Therefore, knowledge of the fish immune system is of practical importance.This project focused on the study of the rainbow trout (Oncorhynchus mykiss) immune system, since it is one of the most valuable specie farmed in the European Union. With the recent sequencing of several fish genomes, the number of known cytokine genes in fish has considerably expanded. Cytokines are single polypeptides or glycoproteins that act as signalling molecules within the immune system, where they have a key role in regulating immune responses. This project was focused on the study of interleukins 2 (IL-2) and 21 (IL-21) in rainbow trout, since these molecules are closely related and they are a key marker of specific cell-mediated immunity. Both cytokines are produced by T-lymphocytes (T helper), being crucial for lymphocyte-specific responses.The first objective of this project has been the study of interleukin 2 (IL-2), a central cytokine in the regulation of T-cell responses. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines were analyzed. IL-2 is an important immunomodulatory cytokine that primarily promotes proliferation, activation and differentiation of T cells. The present work describes the rainbow trout IL-2 cDNA and characterises the molecule in terms of expression analysis and production of the recombinant protein. IL-2 expression was induced by the T cell mitogen PHA (phytohaemagglutinin) and by the mixed leucocyte reaction (MLR, where genetic differences between individuals drive this T cell response. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. Preliminary analysis of the rIL-2 activity shows that it affects potentially important regulators of IL-2 action, such as STAT5 and Blimp-1, as well as affecting IFN and gIP expression, and having a positive feedback on its own expression.After the characterisation of IL-2 in rainbow trout, the project focused on IL-21, since IL-21 and IL-2 are closely linked. The main producers of IL-21 are activated CD4+ T cells and NKT cells. IL-21 is also highly expressed in Th17 cells and can drive the differentiation of Th17 cells in concert with TGF-beta. IL-21 has profound effects on regulation of the differentiation and/or maintenance of polarized Th cells. In this project, we have sequenced and functionally analysed the IL-21 molecule in rainbow trout. The trout IL-21 (IL-21) gene has a six exon/five intron structure, can be induced by bacterial and viral infection, and is synergistically increased by treatment with both calcium ionophore and PMA. Recombinant trout IL-21 (rIL-21) was produced and shown to induce the expression of IFN-?, IL-10, and IL-22 in head kidney cells and splenocytes, the signature cytokines for Th1-, Th2- and Th17-type responses, respectively. Trout rIL-21 increased the expression of T cell and B-cell marker genes in HK cultures where expression in unstimulated cells declined with time. We next investigated the signalling pathways involved in the trout rIL-21 induced expression of IFN-?, IL-10 and IL-22 and found that multiple pathways are involved in trout IL-21 signalling. Thus, inhibition experiments revealed that the activation of Jaks (mainly Jak1 and 3) and STAT3 was responsible for trout rIL-21 action although other pathways may also contribute to trout rIL-21 induction of IL-10 (Akt1/2 pathway) and IL-22 (Akt1/2 and PI3). Documenti correlati Final Report - SMARTFISH (Study of specific cell mediated immunity and vaccine optimization against bacterial and viral infections in trout (Oncorhynchus mykiss))
