IL-4 and IL-13 are closely related canonical type-2 cytokines in mammals and have overlapping bioactivities via shared receptors. They are frequently activated together as part of the same immune response and are the signature cytokines produced by T-helper (Th)2 cells and type-2 innate lymphoid cells (ILC2), mediating immunity against extracellular pathogens. Little is known about the origin of type-2 responses, and whether they were an essential component of the early adaptive immune system that gave a fitness advantage by limiting collateral damage caused by metazoan parasites. Two evolutionary related type-2 cytokines, IL-4/13A and IL-4/13B, have been identified recently in several teleost fish that likely arose by duplication of an ancestral IL-4/13 gene as a consequence of a whole genome duplication event that occurred at the base of this lineage. However, studies of their comparative expression levels are largely missing and bioactivity analysis has been limited to IL-4/13A in zebrafish. Through interrogation of the recently released salmonid genomes, species in which an additional whole genome duplication event has occurred, four genomic IL-4/13 loci have been identified leading to the cloning of three active genes, IL-4/13A, IL-4/13B1 and IL-4/13B2, in both rainbow trout and Atlantic salmon. Comparative expression analysis by real-time PCR in rainbow trout revealed that the IL-4/13A expression is broad and high constitutively but less responsive to pathogen-associated molecular patterns (PAMPs) and pathogen challenge. In contrast, the expression of IL-4/13B1 and IL-4/13B2 is low constitutively but is highly induced by viral haemorrhagic septicaemia virus (VHSH) infection and during proliferative kidney disease (PKD) in vivo, and by formalin-killed bacteria, PAMPs, the T cell mitogen PHA, and the T-cell cytokines IL-2 and IL-21 in vitro. Moreover, bioactive recombinant cytokines of both IL-4/13A and B were produced and found to have shared but also distinct bioactivities. Both cytokines rapidly induce the gene expression of antimicrobial peptides and acute phase proteins, providing an effector mechanism of fish type-2 cytokines in immunity. They are anti-inflammatory via up-regulation of IL-10 and down-regulation of IL-1β and IFN-γ. They modulate the expression of cellular markers of T cells, macrophages and B cells, the receptors of IFN-γ, the IL-6 cytokine family and their own potential receptors, suggesting multiple target cells and important roles of fish type-2 cytokines in the piscine cytokine network. Furthermore both cytokines increased the number of IgM secreting B cells but had no effects on the proliferation of IgM+ B cells in vitro. Taken as a whole, fish IL-4/13A may provide a basal level of type-2 immunity whilst IL-4/13B, when activated, provides an enhanced type-2 immunity, which may have an important role in specific cell-mediated immunity. To our knowledge this is the first in-depth analysis of the expression, modulation and bioactivities of type-2 cytokines in the same fish species, and...

Mammalian interleukin (IL)-2 is a cytokine centrally involved in the differentiation and survival of CD4+ T helper subsets and CD4+ T regulatory cells and in activation of cytotoxic effector lymphocytes. In bony fish, IL2 orthologs have been identified with an additional divergent IL2-Like gene on the same locus present in several fish species. We report here two divergent IL2 paralogs, IL2A and IL2B, in salmonids that originated from the whole genome duplication event in this fish lineage. The salmonid IL2 paralogs differ not only in sequence but also in exon sizes. The IL-2 isoforms that are encoded have disparate pI values and may have evolved to preferentially bind specific IL-2 receptors. Rainbow trout IL2 paralogs are highly expressed in thymus, spleen, gills, kidney and intestine, important tissues/organs in fish T cell development and function. Their expression in peripheral blood leukocytes (PBL) is low constitutively but can be upregulated by the mixed leukocyte reaction, by the T cell mitogen phytohemagglutinin and by signal mimics of T cell activation (phorbol 12-myristate 13-acetate and calcium ionophore). Both trout IL-2 isoforms promoted PBL proliferation and sustained high-level expression of CD4 and CD8, suggesting that trout IL-2 isoforms are T cell growth/survival factors mainly expressed by activated T cells. The recombinant proteins for these two trout IL2 paralogs have been produced in E. coli and possess shared but also distinct bioactivities. IL-2A, but not IL-2B, induced IL12P35A1 and CXCR1 expression in PBL. IL-2B had a stronger effect on upregulation of the T helper 1 (Th1) cytokine interferon-γ (IFNγ) and could sustain CD8α and CD8β expression levels. Nevertheless, both cytokines upregulated key Th1 (IFNγ1, IFNγ2, TNFα2 and IL12) and T helper 2 (Th2) cytokines (IL4/13B1 and IL4/13B2), cytokine and chemokine receptors and the antimicrobial peptide cathelicidin-1 but had limited effects on T helper 17 cytokines and TGFβ1 in PBL. They could also enhance PBL phagocytosis. These results suggest, for the first time in fish, that IL-2 isoforms may have an important role in regulating Th1 and Th2 cell development, and innate and adaptive host defenses in fish, and shed light on lineage-specific expansion, evolution, and functional diversification of IL2 in vertebrates.

IL-15 is a member of the common γ-chain family of cytokines that possess a heterogeneous repertoire of activities on various cells of the immune system. We report here the first functional characterization of a fish IL-15 in rainbow trout. The trout IL-15 gene is 6-kb long and contains six exons and five introns that transcribe into a 1.2-kb mRNA containing seven out-of-frame AUG initiation codons and translate into a 193-aa peptide. Potential sites for transcriptional activators and repressors have been identified in the trout IL-15 gene. Like IL-15 from other species, trout IL-15 is closely linked to an INPP4B gene, but there is also a BCL10 gene located between the IL-15 and INPP4B genes. Three alternative splicing variants of the trout IL-15 gene have also been identified and their expression in vivo was studied. Trout IL-15 expression is present in all the tissues and cell lines studied. Recombinant trout IFN-γ selectively increased IL-15 expression but had little effect on other cytokines such as IL-1β and IL-11. Recombinant trout IL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-γ expression, with little, if any, effect on IL-1β expression. This effect was quite long-lived, and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-γ.

The intracellular suppressors of cytokine signaling (SOCS) family members, including CISH and SOCS1 to 7 in mammals, are important regulators of cytokine signaling pathways. So far, the orthologues of all the eight mammalian SOCS members have been identified in fish, with several of them having multiple copies. Whilst fish CISH, SOCS3, and SOCS5 paralogues are possibly the result of the fish-specific whole genome duplication event, gene duplication or lineage-specific genome duplication may also contribute to some paralogues, as with the three trout SOCS2s and three zebrafish SOCS5s. Fish SOCS genes are broadly expressed and also show species-specific expression patterns. They can be upregulated by cytokines, such as IFN-γ, TNF-α, IL-1β, IL-6, and IL-21, by immune stimulants such as LPS, poly I:C, and PMA, as well as by viral, bacterial, and parasitic infections in member- and species-dependent manners. Initial functional studies demonstrate conserved mechanisms of fish SOCS action via JAK/STAT pathways.

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.

Cartilaginous fish are considered the most primitive living jawed vertebrates with a complex immune system typical of all jawed vertebrates. Cytokine homologs are found within jawless and bony fish, although no cytokine or cytokine receptor genes have been sequenced in cartilaginous fish. In this study the complete coding sequence of the small spotted catshark (Scyliorhinus canicula) IL-1β gene is presented that contains a short 5′ untranslated region (54 bp), a 903-bp open reading frame, a 379-bp 3′ untranslated region, a polyadenylation signal, and eight mRNA instability motifs. The predicted translation (301 amino acids) has highest identity to trout IL-1β (31.7%), with greatest homology within the putative 12 β-sheets. The IL-1 family signature is also present, but there is no apparent signal peptide. As with other nonmammalian IL-1β sequences, the IL-1-converting enzyme cut site is absent. Expression of the IL-1β transcript is detectable by RT-PCR in the spleen and testes, induced in vivo with LPS. Furthermore, a 7-fold increase of transcript levels in splenocytes incubated for 5 h with LPS was seen. The genomic organization comprises six exons and five introns with highest homology seen in exons encoding the largest amount of secondary structure per amino acid. Southern blot analysis suggests at least two copies of the IL-1β gene or genes related to the 3′ end of the IL-1β sequence are present in the catshark. The cloning of IL-1β in S. canicula, the first cytokine sequenced within cartilaginous fish, verifies previous bioactivity evidence for the presence of inflammatory cytokines.

Myxovirus resistance (Mx) proteins are interferon (IFN)-inducible Dynamin-like GTPases, which play an important role in antiviral immunity. Three Mx genes (Mx1-3) have been cloned previously in rainbow trout. In this study, an additional six Mx genes were cloned that reside in four chromosomal loci. Further bioinformatics analysis suggests the presence of three teleost Mx groups (TMG) each with a characteristic gene organisation. Salmonid Mx belong to TMG1 and TMG2. The increased salmonid Mx gene copies are due mainly to local gene duplications that happened before and after salmonid speciation, in a lineage/species specific manner. Trout Mx molecules have been diversified in the loop 1 and 4 regions, and in the nuclear localisation signal in loop 4. The trout Mx genes were shown to be differentially expressed in tissues, with high levels of expression of TMG1 (Mx1-4) in blood and TMG2 (Mx5-9) in intestine. The expression of the majority of the trout Mx genes was induced by poly IC in vitro and in vivo, and increased during development. In addition, induction by antiviral (IFN) and proinflammatory cytokines was studied, and showed that type I IFN, IFNγ and IL-1β can induce Mx gene expression in an Mx gene-, cytokine-and cell line-dependent manner. These results show that salmonids possess a large number Mx genes as well as complex regulatory pathways, which may contribute to their success in an anadromous life style.

Enteric redmouth disease (ERM or yersiniosis) is one of the most important diseases of salmonids and leads to significant economic losses. It is caused by the Gram-negative bacterium Yersinia ruckeri but can be controlled by bacterin vaccination. The first commercial ERM vaccine was licenced in 1976 and is one of the most significant and successful health practices within the aquaculture industry. Although ERM vaccination provides complete protection, knowledge of the host immune response to the vaccine and the molecular mechanisms that underpin the protection elicited is limited. In this report, we analysed the expression in spleen and gills of a large set of genes encoding for cytokines, acute phase proteins (APPs) and antimicrobial peptides (AMPs) in response to ERM vaccination in rainbow trout, Oncorhynchus mykiss. Many immune genes in teleost fish are known to have multiple paralogues that can show differential responses to ERM vaccination, highlighting the necessity to determine whether all of the genes present react in a similar manner. ERM vaccination immediately activated a balanced inflammatory response with correlated expression of both pro- and anti-inflammatory cytokines (eg IL-1β1-2, TNF-α1-3, IL-6, IL-8 and IL-10A etc.) in the spleen. The increase of pro-inflammatory cytokines may explain the systemic upregulation of APPs (eg serum amyloid A protein and serum amyloid protein P) and AMPs (eg cathelicidins and hepcidin) seen in both spleen and gills. We also observed an upregulation of all the α-chains but only one β-chain (p40B2) of the IL-12 family cytokines, that suggests specific IL-12 and IL-23 isoforms with distinct functions might be produced in the spleen of vaccinated fish. Notably the expression of Th1 cytokines (IFN-γ1-2) and a Th17 cytokine (IL-17A/F1a) was also up-regulated and correlated with enhanced expression of the IL-12 family α-chains, and the majority of pro- and anti-inflammatory cytokines, APPs and AMPs. These expression profiles may suggest that ERM vaccination activates host innate immunity and expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune response. A late induction of Th2 cytokines (IL-4/13B1-2) was also observed, that may have a homeostatic role and/or involvement in antibody production. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may provide essential information and function as biomarkers in future vaccine development in aquaculture.