Search

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.

Cytokines, produced at the site of entry of a pathogen, drive inflammatory signals that regulate the capacity of resident and newly arrived phagocytes to destroy the invading pathogen. They also regulate antigen presenting cells (APCs), and their migration to lymph nodes to initiate the adaptive immune response. When naive CD4+ T cells recognize a foreign antigen-derived peptide presented in the context of major histocompatibility complex class II on APCs, they undergo massive proliferation and differentiation into at least four different T-helper (Th) cell subsets (Th1, Th2, Th17, and induced T-regulatory (iTreg) cells in mammals. Each cell subset expresses a unique set of signature cytokines. The profile and magnitude of cytokines produced in response to invasion of a foreign organism or to other danger signals by activated CD4+ T cells themselves, and/or other cell types during the course of differentiation, define to a large extent whether subsequent immune responses will have beneficial or detrimental effects to the host. The major players of the cytokine network of adaptive immunity in fish are described in this review with a focus on the salmonid cytokine network. We highlight the molecular, and increasing cellular, evidence for the existence of T-helper cells in fish. Whether these cells will match exactly to the mammalian paradigm remains to be seen, but the early evidence suggests that there will be many similarities to known subsets. Alternative or additional Th populations may also exist in fish, perhaps influenced by the types of pathogen encountered by a particular species and/or fish group. These Th cells are crucial for eliciting disease resistance post-vaccination, and hopefully will help resolve some of the difficulties in producing efficacious vaccines to certain fish diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, we have identified 421 molecules across the vertebrate spectrum and propose a unified nomenclature for CXC chemokines in fish, amphibians and reptiles based on phylogenetic analysis. Expanding on earlier studies in teleost fish, lineage specific CXC chemokines that have no apparent homologues in mammals were confirmed. Furthermore, in addition to the two subgroups of the CXCL8 homologues known in teleost fish, a third group was identified (termed CXCL8_L3), as was a further subgroup of the fish CXC genes related to CXCL11. Expression of the CXC chemokines found in rainbow trout, Oncorhynchus mykiss, was studied in response to stimulation with inflammatory and antiviral cytokines, and bacterial. Tissue distribution analysis revealed distinct expression profiles for these trout CXC chemokines. Lastly three of the trout chemokines, including two novel fish specific CXC chemokines containing three pairs of cysteines, were produced as recombinant proteins and their effect on trout leucocyte migration studied. These molecules increased the relative expression of CD4 and MCSFR in migrated cells in an in vitro chemotaxis assay., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mammalian gamma-chain (γC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common γC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rβ chain. Deficiency of γC signalling in mammals prevents CD4+ T cells from developing effector functions and CD8+ T cells from developing immunological memory. Thus γC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the γC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second γC and two IL-2Rβ chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian γC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbow trout, including γC itself, IL-2Rβ, IL-4Rα, IL-13Rα1, IL-13Rα2 and IL-2/15Rα, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish γC cytokines is beginning but made more difficult by the co-existence of so many paralogues of the ligands and their receptors. Initial functional studies have shown that fish γC cytokines can modulate the expression of key cytokines (e.g. interferon-γ, IL-10 and IL-22) of the adaptive immune response, and may thus have promise as adjuvants to improve vaccination efficiency in fish., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A relatively large repertoire of type I interferon (IFN) genes is apparent in rainbow trout/Atlantic salmon, that includes six different IFN subgroups (IFNa-IFNf) belonging to the three known type I IFN groups (1–3) in bony fish. Whether this is true for other salmonids, and how the various type I subgroups evolved in teleost fish was studied using the extensive genomic resources available for fish. This confirmed that salmonids, at least the Salmoninae, indeed have a complex (in terms of IFN subgroups present) and large (number of genes) IFN repertoire relative to other teleost fish. This is in part a consequence of the salmonid 4 R WGD that duplicated the growth hormone (GH) locus in which type I IFNs are generally located. Divergence of the IFN genes at the two GH loci was apparent but was not seen in common carp, a species that also underwent an independent 4 R WGD. However, expansion of IFN gene number can be found at the CD79b locus of some perciform fish (both freshwater and marine), with expansion of the IFNd gene repertoire. Curiously the primordial gene order of GH-IFNc-IFNb-IFNa-IFNe is largely retained in many teleost lineages and likely reflects the tandem duplications that are taking place to increase IFN gene number. With respect to the evolution of the IFN subgroups, a complex acquisition and/or loss has occurred in different teleost lineages, with complete loss of IFN genes at the GH or CD79b locus in some species, and reduction to a single IFN subgroup in others. It becomes clear that there are many variations to be discovered regarding the mechanisms by which fish elicit protective (antiviral) immune responses. • A model of type I IFN gene evolution in teleost fish is proposed. • The impact of WGD events on IFN loci was examined in detail. • The salmonid WGD generated two GH loci, that show evidence of significant divergence. • The appearance and loss of IFN subgroups during teleost evolution is apparent. • The IFN genes at the CD79b locus have expanded considerably in some perciformes. [ABSTRACT FROM AUTHOR]

Arsenic toxicity in an aquatic environment is a major concern, and its elimination has become a global challenge. In the current study, histopathology, serum biomarkers and cytokine gene expression were comparatively examined in fish fed with a control diet or diets containing Chlorella vulgaris (Ch) after exposure to sodium arsenite (NaAsO2) in Nile tilapia (Oreochromis niloticus) with the aim of evaluating the protective role of Ch against arsenite-induced toxicity. Severe histopathological alterations were evident in fish exposed to 7 ppm (parts per million) arsenite for 21 days, compared to unexposed fish. Levels of serum biomarkers ALT, AST, ALP, urea and creatinine were increased, but the levels of Na+, total proteins, albumins and globulins were decreased. Moreover, the expression of all the cytokine genes examined, including IL-1β (7-fold), TNF-α (14-fold) and TGF-β1 (13-fold), were significantly upregulated after arsenite exposure. However, in fish fed with diets containing 5% or 10% Ch, the histopathological alterations in the gills, liver and head kidney were reduced, the biomarkers were stabilized, and the upregulation of cytokine gene expression was lowered, with the high Ch diet (10%) showing more prominent effects. These results suggest the protective and therapeutic roles of Ch as a feed supplement in Nile tilapia against arsenic induced toxicity. [ABSTRACT FROM AUTHOR]

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non‐lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS‐222, metomidate and AQUI‐S upon the gill and general health of post‐smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS‐222 or AQUI‐S exhibited upregulation of osmoregulatory genes in the gill and AQUI‐S‐treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI‐S or metomidate, however MS‐222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS‐222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected. [ABSTRACT FROM AUTHOR]

Background: Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. Molecular Characterization of IRF10 in Three Fish Species: In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5’-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. Expression Analysis of IRF10 In Vivo and In Vitro : Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense. [ABSTRACT FROM AUTHOR]

The chemokine and chemokine receptor networks regulate leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. Comparative immunological studies have revealed that both chemokines and their receptors have expanded greatly in a species/lineage specific way. Of the 10 human CC chemokine receptors (CCR1-10) that bind CC chemokines, orthologues only to CCR6, 7, 9 and 10 are present in teleost fish. In this study, four fish-specific CCRs, termed as CCR4La, CCR4Lc1, CCR4Lc2 and CCR11, with a close link to human CCR1-5 and 8, in terms of amino acid homology and syntenic conservation, have been identified and characterized in rainbow trout ( Oncorhynchus mykiss ). These CCRs were found to possess the conserved features of the G protein-linked receptor family, including an extracellular N-terminal, seven TM domains, three extracellular loops and three intracellular loops, and a cytoplasmic carboxyl tail with multiple potential serine/threonine phosphorylation sites. Four cysteine residues known to be involved in forming two disulfide bonds are present in the extracellular domains and a DRY motif is present in the second intracellular loop. Signaling mediated by these receptors might be regulated by N-glycosylation, tyrosine sulfation, S-palmitoylation, a PDZ ligand motif and di-leucine motifs. Studies of intron/exon structure revealed distinct fish-specific CCR gene organization in different fish species/lineages that might contribute to the diversification of the chemokine ligand-receptor networks in different fish lineages. Fish-specific trout CCRs are highly expressed in immune tissues/organs, such as thymus, spleen, head kidney and gills. Their expression can be induced by the pro-inflammatory cytokines, IL-1β, IL-6 and IFNγ, by the pathogen associated molecular patterns, PolyIC and peptidoglycan, and by bacterial infection. These data suggest that fish-specific CCRs are likely to have an important role in immune regulation in fish. [ABSTRACT FROM AUTHOR]

Mammalian CCL20, or macrophage inflammatory protein-3α, can function as a homeostatic and inflammatory chemokine. In relation to the latter, it is responsible for the chemoattraction of lymphocytes and dendritic cells to mucosal immune sites under inflammatory and pathological conditions. CK1, CK8A and CK8B are rainbow trout (Oncorhynchus mykiss) CC chemokines that were reported previously to be phylogenetically related to mammalian CCL20. In the current study, an additional seven CCL20_L paralogues in rainbow trout are reported, that are divided into three subgroups and have been designated here as: CCL20_L1a (also referred to as CK1), CCL20_L1b1-2, CCL20_L2a (CK8A), CCL20_L2b (CK8B), CCL20_L3a, and CCL20_L3b1-4. Multiple CCL20_L genes were also identified in other salmonids that arose from both whole genome duplication and local gene duplication. Phylogenetic tree, homology and synteny analysis support that CCL20_L1-3 found in salmonids are also present in most teleosts arose from the 3 R whole genome duplication and in some species, local gene duplication. Like mammalian CCL20, rainbow trout CCL20_L molecules possess a high positive net charge with a pI of 9.34–10.16, that is reported to be important for antimicrobial activity. Rainbow trout CCL20_L paralogues are differentially expressed and in general highly expressed in mucosal tissues, such as gills, thymus and intestine. The expression levels of rainbow trout CCL20_L paralogues are increased during development and following PAMP/cytokine stimulation. For example, in RTS-11 cells CCL20_L3b1 and CCL20_L3b2 are highly up-regulated by LPS, Poly I:C, recombinant(r) IFNa and rIL-1β. Trout CCL20_L paralogues are also increased after Yersinia ruckeri infection or Poly I:C stimulation in vivo , with CCL20_L3b1 and CCL20_L3b2 again highly up-regulated. Overall, this is the first report of the complete CCL20 chemokine subfamily in rainbow trout, and the analysis of their expression and modulation in vitro and in vivo. These results suggest that teleosts possess divergent CCL20_L molecules that may have important roles in anti-viral/anti-bacterial defence and in mucosal immunity. • Ten CCL20_L genes are present in rainbow trout that can be classified into three groups CCL20_L1-3. • CCL20_L1-3 genes are present in most teleosts, with lineage/species-specific expansion. • The expansion of CCL20_L genes was via whole genome duplication and local gene duplication. • Trout CCL20_L genes are differentially expressed and in general highly expressed in mucosal tissues. • Trout CCL20_L genes are increased during development and following immune stimulation in vitro and in vivo. [ABSTRACT FROM AUTHOR]