Differential expression, modulation and bioactivity of distinct fish IL-12 isoforms: implication towards the evolution of Th1-like immune responses.

IL-12 is a heterodimeric cytokine composed of an α-chain (p35) and a ß-chain (p40). Primarily produced by APCs, IL-12 induces IFN-γ production in T, B and NK cells. IL-12 drives Th1-cell differentiation and IFN-γ secretion to promote cell-mediated immunity, which is essential in the defence against intracellular pathogens. The importance of IL-12 in Th1 responses is echoed by its targeted suppression by intracellular pathogens evading cell-mediated immunity. IL-12 subunits have been identified recently in fish, although reported bioactivities are limited to higher vertebrates. Here, we report the cloning of a p35 gene and two divergent p40 genes (p40b and p40c), capable of producing two functional IL-12 isoforms (p35/p40b and p35/p40c) in rainbow trout. Trout IL-12 isoforms possess distinct bioactivities with respect to the induction of IFN-γ, IL-10 and p40c expression. Trout IL-12 isoforms are differentially expressed and modulated in vivo, exhibiting specific gene expression profiles in bacterial, viral and parasitic infection models, and in vitro in stimulated macrophage and leucocyte cultures. These data imply that alternative or additional pathogen-specific Th-like cell populations may exist in fish. This study will facilitate a broader understanding of the evolutionary processes driving host-pathogen interactions and Th1-like immune responses in lower vertebrates.

Identification of the salmonid IL-17A/F1a/b, IL-17A/F2b, IL-17A/F3 and IL-17N genes and analysis of their expression following in vitro stimulation and infection.

This study identifies four new IL-17A/F isoforms in salmonids, as well as IL-17N. IL-17A/F1 and IL-17A/F2 are each represented by two paralogues, with a predicted pseudogene of IL-17N also apparent in the salmonid genome. Analysis of the sequences and genes of the known IL-17A/F and IL-17N molecules suggests that IL-17N is a member within the IL-17A/F subfamily. Analysis of factors that modulated the expression of these genes showed that PHA and PMA were good inducers of salmon IL-17A/F1a and IL-17A/F2a, with rIL-21 a potent stimulator of IL-17A/F1a and IL-17A/F3. The potential involvement of these isoforms during responses post-vaccination and infection was also studied. In unvaccinated control fish, Yersinia ruckeri infection resulted in a marked up-regulation of IL-17A/F1a and IL-17N in the spleen and head kidney and IL-17A/F2a and IL-17A/F3 in the spleen. In the vaccinated fish, only one significant increase was seen relative to control fish, of IL-17A/F2a in the gills, whether the fish were challenged with Y. ruckeri or given the saline placebo. It was also apparent in the gills and head kidney that the level of IL-17A/F1b remained elevated in the Y. ruckeri-challenged fish at a time when it had decreased in saline-injected fish. The relative importance of these isoforms for disease resistance remains to be determined.

Identification and characterisation of the IL-27 p28 subunits in fish: Cloning and comparative expression analysis of two p28 paralogues in Atlantic salmon Salmo salar.

Interleukin (IL)-27 is an IL-6/IL-12 family member with pro-inflammatory and anti-inflammatory properties. It is a heterodimeric cytokine composed of an α-chain p28 and a ß-chain Ebi3 (Epstein-Barr virus induce gene 3). The p28 subunit can also be secreted as a monomer and function as IL-30 that acts as an inhibitor of IL-27 signalling. At present, the p28 gene has only been described in mammals. Thus, for the first time outwith mammals, we have identified seven p28 molecules in six divergent teleost fish species, Atlantic salmon, two cichlids, two cyprinids and a yellowtail. The fish p28 molecules have higher similarities to mammalian p28 than other IL-6/12 family members. Critical residues involved in the interaction with Ebi3 and the receptor gp130 are highly conserved. The prediction that these are true orthologues is supported by phylogenetic and synteny analysis. Two p28 paralogues (p28a and p28b) sharing 72% aa identity have been identified and characterised in Atlantic salmon. There are multiple upstream ATGs in the 5'-UTR and ATTTA motifs in the 3'-UTR of both cDNA sequences, suggesting regulation at the post-transcriptional and translational level. Both salmon p28 genes are highly expressed in immune relevant tissues, such as thymus, gills, spleen and head kidney. Conversely salmon Ebi3 is highly expressed in other organs, such as liver and caudal kidney. The expression of p28 but not Ebi3 was induced by PAMPs and recombinant cytokines in head kidney cells, and in spleen by Poly I:C challenge in vivo. The dissociation of the expression and modulation of p28 and Ebi3 suggest that both p28 and Ebi3 may be secreted alone or with other partners.

Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 ß-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1ß in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

The expanding repertoire of the IL-12 cytokine family in teleost fish: Identification of three paralogues each of the p35 and p40 genes in salmonids, and comparative analysis of their expression and modulation in Atlantic salmon Salmo salar.

Interleukin (IL)-12 family cytokines are heterodimers of an α-chain (p19, p28 and p35) and a ß-chain (p40 and Ebi3), present as IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3), and play key roles in immune responses in mammals. One p35 and up to three p40 genes have been cloned in some fish species. The identification of three active p35 genes, along with three p40 paralogues in salmonids in the current study further expands the repertoire of IL-12, IL-23 and IL-35 molecules in these species. The multiple p35 genes in teleost fish appear to have arisen via whole genome duplications. The different paralogues of the subunits are divergent, and differentially expressed and modulated by PAMPs and proinflammatory cytokines, hinting that distinct isoforms could be produced in response to infection. Therefore, the expanded IL-12 cytokine family may provide an unprecedented level of regulation to fine tune the immune response in fish.

Identification of IL-34 in teleost fish: differential expression of rainbow trout IL-34, MCSF1 and MCSF2, ligands of the MCSF receptor.

The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2-33.8%) and mammals (22.2-31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34. Comparative expression study of the three trout MCSFR ligands revealed that IL-34, MCSF1 and MCSF2 are differentially expressed in tissues and cell lines. The expression of MCSF1 and MCSF2 showed great variance in different tissues and cell lines, suggesting a role in the differentiation and maintenance of specific macrophage lineages in specific locations. The relatively high levels of IL-34 expression across different tissues suggests a homeostatic role of IL-34 for the macrophage lineage in fish. One striking observation in the present study was the lack of induction of MCSF1 and MCSF2 expression but the quick induction of IL-34 expression by PAMPs and inflammatory cytokines in cell lines and primary head kidney macrophages in rainbow trout. In a parasitic proliferative kidney disease (PKD) model, the expression of IL-34 but not the dominant MCSF2 was affected by PKD, suggesting an involvement of macrophage function in this disease model. Thus IL-34 expression is sensitive to inflammatory stimuli and may regulate macrophage biology once up-regulated.

Cloning of the IL-1ß3 gene and IL-1ß4 pseudogene in salmonids uncovers a second type of IL-1ß gene in teleost fish.

To date two closely related interleukin-1ß genes (IL-1ß1 and IL-ß2) have been found in salmonids. The cloning of trout and salmon IL-1ß3, and a salmon IL-1ß4 pseudogene reveals that two types of IL-1ß genes exist in teleost species. Type I teleost IL-1ß genes, including salmonid IL-1ß3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1ß genes, e.g. salmonid IL-1ß1-2, lack one or two coding exons at their 5'-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1ß genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1ß3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1ß3 also has a role in inflammation and host defence.