Final Report Summary - FISHIL10 (Host and viral Interleukin-10 in fish: evolution of a regulatory cytokine) Interleukin-10 (IL-10) is a regulatory cytokine of the immune system that acts limiting and ultimately terminating the inflammatory response in order to minimize self-inflicted damage by the host. Being such an important cytokine, mammalian IL-10 was thoroughly studied and its bioactivity and cellular sources are well-known and understood. At the start of this project, the coding sequence for an IL-10 gene was described for several fish species, but little knowledge about its bioactivity had been acquired.Interestingly, an IL-10 homologue has been identified in the genome of koi herpes virus (KHV), a highly virulent virus of carp; representing the first viral IL-10 identified in a non-mammalian virus. This ‘hijacking’ phenomenon is not uncommon to herpes viruses: viral IL-10 sequences have been identified in the genome of Human Epstein-Barr-Virus and Cytomegalovirus, both herpes viruses. At the start of this project, no information was available on the bioactivity of the khvIL-10. Taking into account the known background information, the overall aim of this project was to study the biological activity of both host and viral IL-10 in fish to assess a) whether fish IL-10 is also a prototypical anti-inflammatory cytokine like it is in mammals and b) determine whether the viral IL-10 has retained all or part of the regulatory activities of host IL-10. The project had three main objectives:1. Identification of leukocyte sub-types that produce carp IL-10 (cIL-10).2. Biological activity of cIL-10 and of khvIL-10 on carp leukocytes in vitro. 3. In vivo analysis of the role of cIL-10 and khvIL-10 during infection using resistant and susceptible carp.1) During the course of the last two years, the cellular sources of fish IL-10 (objective 1) have been described in carp. The prototypical IL-10 producing leukocytes, macrophages, T cells and some subsets of B cells were found in fish. Interestingly, carp neutrophils also seem to be a very important source of IL-10 unlike mammalian neutrophils, where there is a huge controversy about these cells being able to even express this gene. This discovery provides a new point of view to be considered regarding the evolution of the immune system.2) Carp and KHV IL-10 were shown to possess the prototypical inhibitory activities of this cytokine (objective 2). They inhibit the main functions of phagocytes and the expression of pro-inflammatory genes. Carp IL-10 was also shown to promote the proliferation of defined subsets of B and T cells; an observation reported for the first time in fish. 3) The in vivo experiment (objective 3) could not be performed to the exact same set-up as proposed in the project. Following discussion with the Ethical Committee the expression and production of IL-10 protein by different cellular sources and in different organs was described during a parasite rather than viral infection. This completed the knowledge about the importance of this cytokine and the knowledge on the phase of infection vital to avoid an excessive immune response with detrimental effects on the host. Besides the objectives proposed in the project, extra work has been performed. In collaboration with Leiden University, The Netherlands, a first version of the common carp genome was published which helped us to identify a second isoform of carp IL-10, which was subsequently studied in parallel with the original one described in the project. Although the bioactivity of this second isoform appeared similar, the expression pattern is different and shows a complex regulation of this cytokine in carp. The signaling pathway of IL-10 was also studied to fill the knowledge gap and fully understand the activity of both, fish IL-10 and viral IL-10. The knowledge obtained during this project will help further understand the fish immune system and indirectly benefit fish heath in growing aquaculture setting. Common carp, the species of interest in this study, is worldwide the most cultured fish species for food consumption and infections with Koi Herpesvirus can be severely detrimental. Therefore, these results are instrumental, not only for a better understanding of the fish immune system, but also to understand KHV biology and may lead to improved future vaccination strategies and prophylactic measures.
