Plasma Proteome Responses in Salmonid Fish Following Immunization.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.

Identification and expression modulation of a C-type lectin domain family 4 homologue that is highly expressed in monocytes/macrophages in rainbow trout (Oncorhynchus mykiss).

The C-type lectin domain containing (CLEC) receptors including CD209 are expressed in vivo by monocytes, monocyte-derived macrophages and dendritic cells and by in vitro generated monocyte-derived cells. This paper reports the cloning and sequencing of a lectin molecule, CLEC4T1, in rainbow trout that is a homologue of the CLEC4 family. The expression pattern of the CLEC4T1 was investigated in vivo after infection with a bacterial pathogen and in cultured macrophages after modulation with microbial mimics. Trout CLEC4T1 was highly expressed in spleen and head kidney following infection with Yersinia ruckeri. Expression could also be induced in macrophage cultures by LPS but not by Poly I:C, and suggests that the regulation of CLEC4T1 expression in trout varies according to the nature of the stimulant. A polyclonal CLEC4T1 antibody was generated and validated by Western blotting for use in evaluation of CLEC4T1(+) cells by flow cytometry analysis. Freshly isolated adherent trout head kidney cultures, potentially containing macrophages and dendritic cell precursors, showed an increase of CLEC4T1(+) cells (assessed by flow cytometry) upon stimulation with recombinant interleukin-4/13A. The results suggest that CLEC4T1 is a useful marker for further characterisation of monocyte derived antigen presenting cells in fish.

Molecular characterisation of four class 2 cytokine receptor family members in rainbow trout, Oncorhynchus mykiss.

The interleukin (IL)-10 cytokine family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 and the lambda/type III interferons. They are highly pleiotropic and mediate a variety of activities, including immune suppression and antibacterial immunity. To exert their functions they signal through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptors; IL-10R1, IL-20R1 or IL-22R1) and a subunit with a short intracellular domain (R2 type receptors; IL-10R2 or IL-20R2). In this study we report the identification of three R1 type receptors (named IL-10R1/CRFB7, IL-20R1a/CRFB8a and IL-20R1b/CRFB8b) and one R2 type receptor (named IL-10R2/CRFB4) in rainbow trout. The nomenclature of the receptors was supported by homology analysis, conserved motifs and phylogenetic tree analysis, confirming they belong to the piscine class 2 cytokine receptor family. For instance, they all displayed the presence of characteristic features, such as conserved fibronectin type-III domains. Expression analysis in tissues collected from healthy fish revealed different patterns of expression for each receptor, suggesting their potential involvement in different types of immune responses. When studying the modulation of the genes in cell lines and primary cultures, a greater effect was observed in the cell lines, where the expression of most receptors was affected by incubation with microbial mimics (LPS and PolyI:C) or the pro-inflammatory cytokine rIFN-γ. In addition, expression of the four receptors was modulated by viral infection, suggesting a potential involvement of such receptors and their ligands in antiviral defence.

Sequence and expression analysis of rainbow trout CXCR2, CXCR3a and CXCR3b aids interpretation of lineage-specific conversion, loss and expansion of these receptors during vertebrate evolution.

The chemokine receptors CXCR1-3 bind to 11 chemokines (CXCL1-11) that are clustered on the same chromosome in mammals but are largely missing in ray-finned fish. A second CXCR1/2, and a CXCR3a and CXCR3b gene have been cloned in rainbow trout. Analysis of CXCR1-R3 genes in lobe-finned fish, ray-finned fish and tetrapod genomes revealed that the teleostomian ancestor likely possessed loci containing both CXCR1 and CXCR2, and CXCR3a and CXCR3b. Based on this synteny analysis the first trout CXCR1/2 gene was renamed CXCR1, and the new gene CXCR2. The CXCR1/R2 locus was shown to have further expanded in ray-finned fish. In relation to CXCR3, mammals appear to have lost CXCR3b and birds both CXCR3a and CXCR3b during evolution. Trout CXCR1-R3 have distinct tissue expression patterns and are differentially modulated by PAMPs, proinflammatory cytokines and infections. They are highly expressed in macrophages and neutrophils, with CXCR1 and CXCR2 also expressed in B-cells.

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 and characterization of rainbow trout interleukin-17A/F2 (IL-17A/F2) and IL-17 receptor A: expression during infection and bioactivity of recombinant IL-17A/F2.

Lower vertebrates have been found to possess genes that have similar homology to both interleukin (IL)-17A and IL-17F, which have been termed IL-17A/F. In fish species, several of these genes can be present, but, to date, very little is known about their functional activity. This article describes the discovery and sequence analysis of a rainbow trout (Oncorhynchus mykiss) IL-17A/F2 molecule and an IL-17RA receptor. In addition, the bioactivity of the trout IL-17A/F2 is investigated for the first time in any species. The predicted IL-17A/F2 and IL-17RA proteins consist of 146 and 966 amino acids (aa), respectively, with both molecules containing conserved family motifs. Expression analysis revealed high constitutive expression of trout IL-17A/F2 in mucosal tissues from healthy fish, suggesting a potential role in mucosal immunity. When the modulation of IL-17A/F2 and IL-17RA in vitro was analyzed, it was observed that the two molecules were similarly affected. The expression of IL-17A/F2 was also induced in head kidney during bacterial, parasitic, and viral infections, revealing a possible function in defense against such pathogens. However, downregulation of IL-17RA was seen in some tissues and infections. The recombinant IL-17A/F2 protein was produced in Escherichia coli and was found to affect the expression of an antimicrobial peptide and the proinflammatory cytokines IL-6 and IL-8 in splenocytes. Consistent with mammalian IL-17 homologues, our expression and bioactivity results imply that trout IL-17A/F2 plays an important role in promoting inflammatory and host innate immune responses directed against different pathogen groups.

Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss.

This paper describes the cloning and characterisation of two retinoid-related orphan receptor (ROR)-γ homologues (ROR-γa1 and -γa2) in rainbow trout (Oncorhynchus mykiss). The coding region predicted for both homologues consists of 1410 base pairs (bp), which translate into two 469 amino acid (aa) proteins. The trout ROR-γs revealed a high conservation of both DNA- and ligand-binding domains (functional regions of the nuclear receptor family), and shared a high homology to mammalian ROR-γt. A phylogenetic tree containing ROR family members confirmed that both trout homologues clustered within the ROR-γ group. Both results suggested that these molecules are likely to be ROR-γ homologues, more similar to the mammalian splice variant ROR-γt than the full length ROR-γ. Expression analysis of tissues obtained from healthy fish revealed highest constitutive expression of trout ROR-γ in muscle, followed by the brain, heart and skin. This suggests that these genes may play an important role in such tissues. In vitro studies, using trout cell lines, demonstrated that ROR-γ is induced significantly by LPS and down-regulated by the presence of PolyI:C and recombinant interferon (IFN)-γ. Moreover, analysis of this gene in head kidney macrophages and mixed primary leucocyte cultures indicated that differences were apparent between the different cell types/sources used, indicating that its expression may be cell-type dependent. Additional studies to investigate the regulation of this gene in vivo demonstrated that its expression was significantly higher in vaccinated vs unvaccinated fish following bacterial (Yersinia ruckeri) challenge but it was down-regulated after a viral (VHSV) infection. This suggests a potential role of trout ROR-γ, a putative T(H)17 transcription factor, in protection against extracellular bacteria.

Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): salmonid IL-6Rα possesses a polymorphic N-terminal Ig domain with variable numbers of two repeats.

Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we have cloned both the IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-D6) and presence of intracellular motifs important for signal transduction to tetrapod gp130s. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine-binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, a phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra large D1 domain of the salmonid IL-6Rα molecules results partially from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in the cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness.

Cloning, expression analysis and bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-22.

This report describes the cloning and characterisation of rainbow trout (Oncorhynchus mykiss) interleukin (IL)-22, and presents studies of the functional activity of its recombinant protein for the first time in a non-mammalian species. The predicted IL-22 coding region consists of 522 nucleotides which translates into a 173 amino acid protein, that contains an IL-10 family signature which is reasonably well conserved with other vertebrate IL-22 molecules. Expression analysis in tissues from healthy fish revealed a higher constitutive expression of IL-22 in mucosal tissues, suggesting a potentially important role in mucosal immunity. In vitro studies demonstrated that IL-22 expression was induced significantly by PHA and PMA in splenocyte primary cultures 4h post-stimulation. Expression was also induced in the spleen upon infection of fish with the Gram-negative bacterium Yersinia ruckeri, suggesting a potential role of IL-22 in vivo in defence against bacterial diseases. The Escherichia coli produced recombinant IL-22 enhanced the expression of a number of antimicrobial peptides, promoting host innate immunity against microbes and revealing a biological similarity with its mammalian counterpart.

Identification of two FoxP3 genes in rainbow trout (Oncorhynchus mykiss) with differential induction patterns.

FoxP3 is a master transcription factor for the development and function of regulatory T cells in mammals, but little is known about this molecule in fish. Two paralogues of mammalian FoxP3 that share 83.9% identity at the amino acid level have been identified in rainbow trout (Oncorhynchus mykiss). The C-terminal region containing a Zn_C2H2 domain, a leucine zipper-like domain and a forkhead (FH) domain important for dimerization, nuclear translocation, and DNA binding, is well conserved between fish and other vertebrate FoxP3. However, the N-terminal of FoxP3 that is required for FoxP3-mediated repression of transcription is greatly diverged between fish, amphibians and monotreme mammals compared to eutherian mammals, suggesting that FoxP3 in fish, frog and platypus may have a different role to the human and mouse counterpart that defines the Treg cellular lineage and mediates the immune regulatory function. The expression of both trout (t) FoxP3a and tFoxP3b are detectable in all the 14 tissues examined without any significant difference except in muscle in which the expression of tFoxP3a was higher. Both tFoxP3a and tFoxP3b are highly expressed in thymus and in immune related organs including the spleen, kidney, gills and intestine, and are up-regulated by phytohaemagglutinin (PHA) in splenocytes and thymocytes. Whilst the up-regulated tFoxP3b expression induced by PHA was dose-dependent it required a higher PHA concentration to achieve maximal expression relative to tFoxP3a where the highest expression level was seen using 1 µg/ml PHA with higher concentrations having no further effects. In addition, the tFoxP3b expression increased during development from eyed eggs to fry, when it reached a comparable level to that of tFoxP3a. In contrast, tFoxP3a expression was at a high and almost constant level over all of the developmental stages examined. The high level of tFoxP3a expression in early development may be related to the relatively high constitutive level of tFoxP3a expression seen in muscle, perhaps suggesting novel roles of tFoxP3 in fish muscle. The structural and expression analysis suggests that the tFoxP3a and tFoxP3b are subject to differential modulation of expression and may have evolved novel functions. The identification of the two trout FoxP3 paralogues will help to clarify the existence of Treg cells and to dissect the T cell differentiation pathways in fish.

Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.