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Fish Cytokines and Immune Response

Written By

Sebastián Reyes-Cerpa, Kevin Maisey, Felipe Reyes-López, Daniela Toro-Ascuy, Ana María Sandino and Mónica Imarai

Submitted: March 22nd, 2012 Published: November 21st, 2012

DOI: 10.5772/53504

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1. Introduction

The immune system can be defined as a complex system that protects the organism against organisms or substances that might cause infection or disease. One of the most fascinating characteristics of the immune system is its capability to recognize and respond to pathogens with significant specificity. Innate and adaptive immune responses are able to recognize foreign structures and trigger different molecular and cellular mechanisms for antigen elimination. The immune response is critical to all individuals; therefore numerous changes have taken place during evolution to generate variability and specialization, although the immune system has conserved some important features over millions of years of evolution that are common for all species. The emergence of new taxonomic categories coincided with the diversification of the immune response. Most notably, the emergence of vertebrates coincided with the development of a novel type of immune response. Apparently, vertebrates inherited innate immunity from their invertebrate ancestors [1].

In higher vertebrates, the immune system consists of primary and secondary lymphoid organs with distinct compartments and morphology located in anatomically distinct sites. The thymus and bone marrow constitute the primary lymphoid organs, while the spleen, lymph nodes, and mucosal associated lymphoid tissue (MALT) comprise the secondary lymphoid organs [2].

Fish are a heterogeneous group divided into three classes: Agnatha (jawless fish such as the hagfish and lampreys), Chrondrichthyes (cartilaginous fish such as sharks, rays and skates) and Osteichthyes (bony fish) [3]. As in all vertebrates, fish have cellular and humoral immune responses and organs, the main function of which is immune defence. Most generative and secondary lymphoid organs in mammals are also found in fish, except for lymphatic nodules and bone marrow [3].

The head kidney or pronephros has hematopoietic functions [3, 4], and unlike in higher vertebrates, it is the immune organ involved in phagocytosis [5], antigen processing, production of IgM [6, 7] and immune memory through melanomacrophagic centres [8, 9]. The thymus, another lymphoid organ situated near the opercular cavity in teleosts, produces T lymphocytes involved in allograft rejection, stimulation of phagocytosis and antibody production by B cells [10, 11]. The spleen is a large, blood-filtering organ that undergoes increasing structural complexity in order to augment its efficiency in trapping and processing antigens [12-15]. Melanomacrophage centres are present for clearance of ingested material and can be surrounded by immunoglobulin-positive cells, especially after immunization [8]. Proliferation of granular cells has also been observed in association with ellipsoids and melanomacrophage centres after immunization [16].

1.1. Innate and adaptive immune response

The development of an immune system is essential for the survival of living organisms. In vertebrates, immunity can be divided into two components, the innate immune response and the adaptive immune response. The innate immune response is the initial line of defence against infection, which includes physical barriers and cellular response. The adaptive immune response is capable of specific antigen recognition and is responsible for the secondary immune response.

The innate immune system recognizes conserved molecular structures common to pathogenic microorganisms such as polysaccharides, lipopolysaccharides (LPS), peptidoglycans, bacterial DNA, and double-strand viral RNA, among others, through their interaction with specific receptors like toll receptors (TLRs). These mechanisms of recognition may lead directly to successful removal of pathogens, for instance by phagocytosis, or may trigger additional protective responses through induction of adaptive immune responses [17]. Cells of the innate immune system have a diverse array of functions. Some cells are phagocytic, allowing them to engulf and degrade pathogenic particles. Other cells produce and secrete cytokines and chemokines that can stimulate and help guide the migration of cells and further direct the immune response [18].

The adaptive system recognizes foreign structures by means of two cellular receptors, the B cell receptor (BCR) and the T cell receptor (TCR). Adaptive immunity is highly regulated by several mechanisms. It increases with antigen exposure and produces immunological memory, which is the basis of vaccine development and the preventive function of vaccines [19, 20]. The adaptive response generally starts days after infection and is capable of recognizing specific protein motifs of peptides, which leads to a response that increases in both speed and magnitude with each successive exposure [21]. The main effector cells of the adaptive immune response are the lymphocytes, specifically B cells and T cells. When B cells are activated, they are capable of differentiating into plasma cells that can secrete antibodies. Upon activation T cells differentiate into either helper T cells or cytotoxic T cells. Helper T cells are capable of activating other cells of the adaptive immune response such as B cells and macrophages, while cytotoxic T cells upon activiation are able to kill cells that have been infected [22].

1.2. Fish immune response

Immune responses in fish have not been as well characterized as they have in higher vertebrates. Consequently, there is not enough information about the components of the fish immune system and its function and regulation. Key immune mammalian homologous genes have been identified in several fish species, suggesting that the fish immune system shares many features with the mammalian system. For example, the identification of α and β T cell receptor genes (TCR) [23], key T cell markers such as CD3, CD4, CD8, CD28, CD40L, and a great number of cytokines and chemokines [24-26] suggest that T helper (Th)1, Th2 and Th17 and the regulatory subset Treg are present in fish. Some cell subsets have been better studied mainly because their activity can be easily differentiated and measured, as in the case of cytotoxic cells [27] and macrophages [28, 29]. Finally, B cells have been much more studied due to the availability of monoclonal antibodies that have been isolated and identified by a number of techniques [30, 31]. Phenotypic characterization of leukocytes has been hampered mainly by the lack of membrane cell markers [32, 33]. Researchers anticipate developing antibodies for cell lineage markers of fish immunocompetent cells that can be used to isolate and characterize immune cells to obtain insights into their regulation and role in immune response [34-36].

Antibodies in teleosts play a key role in the immune response. In general, IgM is the main immunoglobulin in teleosts that can elicit effective specific humoral responses against various antigens. For IgM, one gene alone can generate as many as six structural isoforms. Therefore, diversity is the result of structural organization rather than genetic variability [37]. Recently, several reports have provided evidence for the existence of IgD/IgZ/IgT in fish [38-41]. Interestingly, B cells from rainbow trout and salmon have high phagocytic capacity, suggesting a transition in B lymphocyte during evolution in which a key cell type of the innate immunity and phagocytosis evolved into a highly specialized component of the adaptive immune response in higher vertebrates [42, 43].

1.3. Fish cytokines

Cytokines are secreted proteins with growth, differentiation, and activation functions that regulate the nature of immune responses. Cytokines are involved in several steps of the immune response, from induction of the innate response to the generation of cytotoxic T cells and the production of antibodies. In higher vertebrates, the combination of cytokines that are secreted in response to an immune stimulation induces the expression of immune-related genes through multiple signalling pathways, which contributes to the initiation of the immune response. Cytokines can modulate immune responses through an autocrine or paracrine manner upon binding to their corresponding receptors [44].

Cytokines have overlapping and sometimes contradictory pleiotropic functions that make their classification difficult. Cytokines are produced by macrophages, lymphocytes, granulocytes, DCs, mast cells, and epithelial cells, and can be divided into interferons (IFNs), interleukins (ILs), tumor necrosis factors (TNFs), colony stimulating factors, and chemokines [45]. They are secreted by activated immune-related cells upon induction by various pathogens, such as parasitic, bacterial, or viral components [46]. Macrophages can secret IL-1, IL-6, IL-12, TNFα, and chemokines such as IL-8 and MCP-1, all of which are indispensable for macrophage, neutrophil, and lymphocyte recruitment to the infected tissues and their activation as pathogen eliminators [47]. Meanwhile, cytokines released by phagocytes in tissues can also induce acute phase proteins, including mannose-binding lectin (MBL) and C-reactive protein (CRP), and promote migration of DCs [48].

Fish appear to possess a repertoire of cytokines similar to those of mammals. To date several cytokine homologues and suppressors have been cloned in fish species [24, 25, 49]. Some cytokines described in fish are TNFα, IL-1β, IL-6 or IFN.

Current knowledge of fish cytokines is based on mammal models of the cytokines network and their complex interactions. In this review we included the pro-inflammatory cytokines associated with innate and adaptive immunity, regulatory cytokines and anti-inflammatory cytokines.

1.4. Pro-inflammatory fish cytokines

1.4.1. Tumour necrosis factor α (TNFα)

TNFα (tumour necrosis factor alpha) is a pro-inflammatory cytokine that plays an important role in diverse host responses, including cell proliferation, differentiation, necrosis, apoptosis, and the induction of other cytokines. TNFα can induce either NF-kB mediated survival or apoptosis, depending on the cellular context [50]. TNFα mediates powerful anti-microbial responses, including inducing apoptosis, killing infected cells, inhibiting intracellular pathogen replication, and up-regulating diverse host response genes. Many viruses have evolved strategies to neutralize TNFα by direct binding and inhibition of the ligand or its receptor or modulation of various downstream signalling events [51].

TNFα has been identified, cloned, and characterized in several bony fish, including Japanese flounder [52], rainbow trout [53, 54], gilthead seabream [55], carp [56] catfish [57], tilapia [58], turbot [59] and goldfish [60]. These studies have revealed the existence of some obvious differences from their mammalian counterpart, such as the presence of multiple isoforms of TNFα in some teleost species [54, 56] the high constitutive expression of this gene in different tissues of healthy fish and its relatively poor up-regulation by immune challenge in vitro and in vivo [53, 55, 57]. However, the most unexpected and interesting difference between fish and mammal TNFα concerns the weak in vitro effects of TNFα on phagocyte activation in goldfish [60], rainbow trout [57], turbot [59] and gilthead seabram [61]. This weak in vitro activity of fish TNFα sharply contrasts with the powerful actions exerted by the i.p. injection of recombinant TNFα in gilthead seabream, which includes the recruitment of phagocytes to the injection site, with a concomitant strong increase in their respiratory burst [61]. Apparently endothelial cells are the main target cells of fish TNFα, suggesting that TNFα is mainly involved in the recruitment of leukocytes to the inflammatory foci rather than in their activation [62]. Despite the above, differential expression has been observed in studies with rainbow trout leucocytes, which have shown increased response to different pro-inflammatory stimuli, as human recombinant TNFα [63], LPS [53, 64], zimosan and muramyl dipeptide as a peptidoglycan constituent of both gram-positive and gram-negative bacteria [64]. Moreover, it is known that Infectious Pancreatic Necrosis Virus (IPNV)-mediated up-regulation of TNFα regulates both the Bad/Bid-mediated apoptotic pathway and the RIP1 (receptor-interacting protein-1)/ROS-mediated secondary necrosis pathway [65].

1.4.2. Interleukin 1 family

In mammals, the 11 members of the Interleukin-1 family include IL-1α (IL-1F1), IL-1β (IL-1F2), IL-1 receptor antagonist (IL-1ra/IL-1F3), IL-18 (IL-1F4), IL-1F5-10 and IL-33 (IL-1F11). These molecules tend to be either pro-inflammatory or act as antagonists that inhibit the activities of particular family members [66]. Despite these semantic issues, to date only two clear homologues of these molecules have been discovered in fish, IL-1β and IL-18 [24]. Interleukin 1β

IL-1β is one of the earliest expressed pro-inflammatory cytokines and enables organisms to respond promptly to infection by inducing a cascade of reactions leading to inflammation. Many of the effector roles of IL-1β are mediated through the up- or down-regulation of expression of other cytokines and chemokines [67]. Mammalian IL-1β is produced by a wide variety of cells, but mainly by blood monocytes and tissue macrophages. IL-1β was the first interleukin to be characterized in fish and has since been identified in a number of fish species, such as rainbow trout [68], carp [69], sea bass [70], gilt head seabram [71], haddock [72], tilapia [73]. A second IL-1β gene (IL-1beta2] has been identified in trout [74]

In mammals pro-IL-1β remains cytosolic and requires cellular proteases to release the mature peptide. It is known that the peptide is cleaved by the IL-1β converting enzyme (ICE)[75]. However, the aspartic acid residue for which this enzyme has specificity is not present in all fish genes sequenced to date. Nevertheless, using a combination of multiple alignments and analysis of the N-terminal sequences of known mature peptides, it is possible to predict fish gene cutting sites. In trout, this gives a mature peptide of 166 and 165 aminoacids for IL-1β1 and IL-1β2 [76].

Like its mammalian counterpart, teleost IL-1β has been found to be regulated in response to various stimuli, such as LPS or poly I:C [68, 70-74, 77-81]. The biological activity of recombinant IL-1β (rIL-1β) has been studied in several fish species, indicating that fish IL-1β is involved in the regulation of immune relevant genes, lymphocyte activation, migration of leucocytes, phagocytosis and bactericidal activities [77, 81-84]. Interleukin 18

In mammals, IL-18 is mainly produced by activated macrophages. It is an important cytokine with multiple functions in innate and acquired immunity [85-87]. One of its primary biological properties is to induce interferon gamma (IFNγ) synthesis in Th1 and NK cells in synergy with IL-12 [88, 89]. It promotes T and NK cell maturation, activates neutrophils and enhances Fas ligand-mediated cytotoxicity [90-92]. Like IL-1β, it is synthesized as an inactive precursor of approximately 24 kDa and is stored intracellularly. Activation and secretion of IL-18 is mainly effected through specific cleavage of the precursor after D35 by caspase 1, also termed the IL-1β-converting enzyme (ICE), which is believed to be one of the key processes regulating IL-18 bioactivity [93, 94]. Some other enzymes, including caspase 3 and neutrophil proteinase 3, also cleave the IL-18 precursor to generate active or inactive mature molecules [95, 96].

IL-18 was discovered in fish by analysis of sequenced fish genomes (fugu) and EST databases (medaka) [97, 98]. An alternative splicing form of the IL-18 mRNA was discovered in trout that may have an important role in regulating IL-18 expression and processing in this species. This form shows a lower constitutive expression relative to the full length transcript, but unlike the full length transcript, it increases in response to LPS and polyI:C stimulation in the RTG-2 fibroblast cell line [98]. The expression level of the full length transcript can increase in response to LPS plus IL- 1b in head kidney leucocyte cultures, and by IFNγ in RTS-11 cells [99].

1.4.3. Other pro-inflammatory cytokines Interleukin 6

A number of other interleukins are considered pro-inflammatory, some of which are released during the cytokine cascade that follows bacterial infection. Of these IL-6 is one of the best known, and is itself a member of the IL-6 family of cytokines that includes IL-11 and IL-31, as well as cytokines such as mammalian CNTF, LIF, OSM, CT-1 and CT-2 [24]. Whilst the homology of known fish molecules with many of these IL-6 family members is not conclusive [100], true homologues appear to be present in at least in the cases of IL-6 and IL-11 [24]. IL-6 is produced by a diverse group of cells including T lymphocytes, macrophages, fibroblasts, neurons, endothelial and glial cells. The pleiotropic effects of IL-6 are mediated by a 2-subunit receptor [101] and include the regulation of diverse immune and neuro-endocrine processes. IL-6 has been implicated in the control of immunoglobulin production, lymphocyte and monocyte differentiation, chemokine secretion and migration of leukocytes to inflammation sites [102-104].

IL-6 was first discovered in fugu by analysis of the genome sequence [105] and subsequently in other species as part of EST analysis of immune gene-enriched cDNA libraries [106-108]. However, little is known about the function and signalling pathways of IL-6 in fish. Interestingly, trout IL-6 expression in macrophages is reported to be induced by LPS, poly I:C and IL-1β in the macrophage cell line RTS-11, as well as in head kidney macrophages [109]. Moreover, IL-6 induces the expression of itself, so it can act in an autocrine and paracrine fashion to increase its expression, with the potential to both amplify and exacerbate the inflammatory response. However, IL-6 can significantly down-regulate the expression of trout TNFα1, TNFα2, and IL-1β, suggesting a potential role of trout IL-6 in limiting host damage during inflammation [109]. Interleukin 11

In mammals, IL-11 is produced by many cell types throughout the body. Basal and inducible IL-11 mRNA expression can be detected in fibroblasts, epithelial cells, chondrocytes, synoviocytes, keratinocytes, endothelial cells, osteoblasts and certain tumour cells and cell lines [110]. Viral [111] and bacterial infection [112] and cytokine stimulation (IL-1, TNFα and TGF-β1] induce IL-11 expression. IL-11 acts on multiple cell types, including hemotopoietic cells, hepatocytes, adipocytes, intestinal epithelial cells, tumour cells, macrophages, and both osteoblasts and osteoclasts. In the hematopoietic compartment IL-11 supports multilineage and committed progenitors, contributing to myeloid, erythroid, megakaryocyte and lymphoid lineages [113]. IL-11 is also an anti-inflammatory cytokine that inhibits the production of pro-inflammatory cytokines from lipopolysaccharde (LPS)-stimulated macrophages [114]. In combination with its trophic effects on the gastrointestinal epithelium, IL-11 plays an important role in the protection and restoration of gastrointestinal mucosa [115, 116].

The teleostean IL-11 orthologue has been found to consist of duplicate IL-11 genes, named IL-11a and IL-11b [117], with expression patterns indicating that both divergent forms of teleostean IL-11 play roles in antibacterial and antiviral defence mechanisms of fish [117-119]. In trout, IL-11 molecule is grouped with IL-11a and is constitutively expressed in intestine and gills and is highly up-regulated at other immune sites (spleen, head kidney, liver) following bacterial infection. In vitro, the macrophage-like RTS-11 cell line has shown enhanced IL-11 expression in response to LPS, bacteria, poly I:C and rIL-1β [118]. In carp, IL-11a is modulated by LPS, ConA and peptidoglycan in head kidney macrophages [117, 120] and cortisol has been found to inhibit IL-11 expression on its own and in combination with LPS [117]. In contrast to carp IL-11a, which shows low levels of constitutive expression in blood leucocytes, IL-11b in Japanese flounder shows higher expression at this site, and strong up-regulation was found in response to rhabdovirus infection in kidney cells [119]. This suggests that these paralogues have some complementarity of function related to their differential expression, although study of both forms in a single experiment is still required [24].

1.5. Chemokines

Chemokines are a superfamily of approximately 40 different small secreted cytokines that direct the migration of immune cells to infection sites. Their activity is coordinated by binding to G-protein-linked receptors with seven transmembrane domains. Four distinct subgroups make up the chemokine superfamily. These are designated as CXC (or a), CC (or b), C (or g) and CX3C (or d), which are defined by the arrangement of the first two cysteine residues within their peptide structure. The CC subfamily can be further subdivided according to the total number of cysteine residues, as some members of this group contain four cysteines whilst the remainder possesses six (and are known as the C6-b group). Similarly, the CXC subfamily contains two subgroups based on whether or not the first two cysteines are preceded by a Glu-Leu-Arg (ELR) motif associated with specificity to neutrophils [76, 121].

1.5.1. Interleukin 8

An important chemokine related to the pro-inflammatory process is CXCL-8, also called interleukin 8, this chemokine is a member of the CXC chemokine subfamily and attracts neutrophils, T lymphocytes and basophils in vitro, but not macrophages or monocytes [122]. Many cell-types, including macrophages, produce IL-8 in response to a variety of stimuli (LPS, cytokines and viruses). The neutrophil-attracting ability of IL-8 can be attributed to the presence of the ELR motif adjacent to the CXC motifs at its N-terminus, presumably by affecting its binding to specific receptors [123, 124]. In contrast, CXC chemokines lack an ELR motif and specifically attract lymphocytes but not neutrophils. The biological effects of IL-8 on neutrophils include increased cytosolic calcium levels, respiratory burst, a change in neutrophil shape and chemotaxis[125].

The fish IL-8 has been found in flounder [126], trout [125, 127], catfish [128], and lamprey [129]. In vitro stimulation of a trout macrophage cell line (RTS-11) [125] or in vivo intraperitoneal challenge [78] with either LPS or poly I:C did result in clear up-regulation of IL-8 expression. Moreover, induction of IL-8 expression in primary cultures of rainbow trout leukocytes stimulated for 24 hours with LPS and TNFα confirms that this fish chemokine is associated with inflammatory response, as has been suggested in mammals [127]. Interestingly, the ELR motif associated with the neutrophil-attracting ability is absent from the lamprey molecule and it is similar in flounder, where CXCL8 also lacks the ELR motif and appears to be regulated by a bacterial mechanism, since its transcript has only been detected in the major immune organs (spleen and head kidney) of an LPS stimulated flounder. The case of the trout is different, although there is also no ELR preceding the CXC motif, it has a very similar motif (DLR) in this position [130]. The human CXCL8 molecule, where the ELR motif has been mutated to DLR, retains neutrophil-attracting ability, albeit at lower potency [123]. Consequently, it is possible that the trout molecule has similar chemotactic activity to that of mammalian CXCL8 [130].

1.6. The interleukin 2 family

The IL-2 subfamily of cytokines signals via the common gamma chain (gC or CD132), a member of the type I cytokine receptor family expressed in most leucocytes.These cytokines in mammals include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-2, IL-4, IL-9 and IL-21 are all cytokines released from Th cells, which affect their responses [24], whilst IL-7 and IL-15 are particularly important for the maintenance of T cell memory [131]. To date molecules with homology to all of these have been found in fish, except IL-9 [24].

1.6.1. Interleukin 2

Interleukin-2 (IL-2 is an important immunomodulatory cytokine that primarily promotes proliferation, activation and differentiation of T cells [132]. IL-2, initially known as T-cell growth factor (TCGF), is synthesized and secreted mainly by Th1 cells that have been activated by stimulation by certain mitogens or by interaction of the T-cell receptor with the antigen/MHC complex on the surface of antigen-presenting cells [133-135]. Although CD4 T cells are the major source of IL-2 production in response to TCR stimulation, transient induction of IL-2 mRNA and production of the protein has been detected in murine dendritic cells activated by gram-negative bacteria [136]. IL-2 can also be produced by B cells in certain situations [137, 138]. The produced IL-2 promotes the expansion and survival of activated T cells and is also required for the activation of natural killer (NK) cells [139] and for immunoglobulin (Ig) synthesis by B cells [140].

The IL-2 gene has been detected only recently in fish by analysis of the fugu genome sequence, which also identified IL-21 as a neighbouring gene, as in mammals, providing the first direct evidence for the existence of a true IL-2 homologue in bony fish [141]. The gene has a 4 exon/3 intron organisation, as in mammals, and showed no constitutive expression in a range of tissues examined. However, injection of Fugu with poly I:C induced expression of IL-2 in the gut and gills [141]. Moreover, IL-2 could be induced in head kidney cell cultures stimulated with PHA, and in T-cell enriched cultures isolated from PBL when stimulated with B7-H3 or B7- H4 Ig fusions proteins in the presence of PHA [24, 142]. IL-2 has since been cloned in rainbow trout [143, 144]. The trout IL-2 was significantly up-regulated in head kidney leucocytes by the T cell mitogen PHA and in classical mixed leucocyte reactions and in vivo following infection with bacteria (Y. ruckeri) or the parasite Tetracapsuloides bryosalmonae. More importantly, the recombinant trout IL-2 produced in Escherichia coli was shown to induce expression of two transcription factors (STAT5 and Blimp-1) known to be involved in IL-2 signalling in mammals [143], as well as interferon-g (IFNγ) and IL-2 itself, and a CXC chemokine known to be induced by IFNγ, termed a IFNγ-inducible protein (γIP) [145].

1.6.2. Interleukin 4

Interleukin-4 IL-4 is a pleiotropic cytokine produced by T cells, mast cells, and basophils and is known to regulate an array of functions in B cells, T cells, macrophages, hematopoietic and non-hematopoietic cells [146, 147]. IL-4 serves as a key cytokine in driving Th2 differentiation and mediating humoral immunity, allergic responses and certain autoimmune diseases [148]. The IL-4 gene is conserved evolutionally in the animal kingdom and has been isolated from various animals including humans [149], mice [150, 151] and bovines [152], in which the IL-4 locus has been mapped in a region adjacent to those of IL-5 and IL-13 on the same chromosome [153, 154].

Teleost fish have two genes of the IL-4/13 family, IL-4/13A and IL-4/13B, which are situated on separate chromosomes in regions that duplicated during the fish-specific whole genome duplication (FS-WGD) around 350 million years ago [155, 156]. A few IL-4-like genes have been found in fish to date. The first was discovered by searching the Tetraodon nigroviridis genome [157]. In this work, IL-4 was constitutively expressed in head kidney, spleen, liver, brain, gill, muscle and heart. The ubiquitous expression of IL-4 is consistent with a postulated role in immune cytokines regulation. Stimulating the fish with a mixed stimulant containing ConA, PHA and PMA significantly up-regulated the expression of IL-4, which suggests that IL-4 is involved in the immune inflammatory responses triggered by mitogens [157], as in mammals, where it has been observed that this mitogen increases IL-4 expression [158]. However, the homology (amino acid identity) of this molecule was very low [12–15%), making it difficult to be sure it is an IL-4 homologue, although clearly related to Th2-type cytokines [24]. In fugu, T cell enriched PBL was found to express more IL-4/13A and IL-4/13B after stimulation with recombinant B7 molecules [142]. In zebrafish a recombinant IL4/13B was shown to increase the number of IgT-positive and CD209-positive cells in blood [159, 160], and in zebrafish spleen the expression of IL-4/13B and transcription factor related to Th2 immune response as GATA-3, and STAT6 was simultaneously enhanced after PHA stimulation [161]. The IL-4/13A gene was identified in trout and salmon [162], where the tissue distribution of salmonid IL-4/13A and GATA-3 expression were compared to the expression of IL-4, IL-13, and GATA-3 in mice. High levels of these transcripts were found in both salmonid and murine thymus, while constitutive IL-4/13A richness of skin and respiratory tissue was found in salmonids but not in mice. Experiments with isolated cells from gill and pronephros (head kidney) indicated that trout IL-4/13A is mainly expressed by surface IgM-negative cells, readily inducible by PHA but not by poly I:C, and regulated differently from the Th1 cytokine IFNγ gene. In mammals, IL-5 is also considered a Th-2 type cytokine and along with IL-3 and GM-CSF it signals through receptors with a common γ-chain (γC). None of these cytokines have been discovered in fish to date [24].

1.6.3. Interleukin 7

The cytokine IL-7 plays several important roles during lymphocyte development, survival, and homeostatic proliferation [163]. It is produced by many different stromal cell types, including epithelial cells of the thymus and the intestine [164-166]. There is only one report on IL-7 in fish, for the fugu molecule that was discovered using a gene synteny approach by searching with the mammalian IL-7 gene neighbours C8orf70 and PKIA. Fugu IL-7 shows constitutive expression in head kidney, spleen, liver, intestine, gill and muscle, with expression shown to increase in head kidney cultures stimulated with LPS, poly I:C or PHA [24, 167].

1.6.4. Interleukin 15

The central action of IL-15 cytokine is on T-cells, dendritic cells and NK cells. IL- 15 is an important regulator of the innate immune response to infection and autoimmune disease conditions. This gene shares activities with IL-2 and utilizes IL-2R β and γ units [45].

Two genes with homology to IL-15 have been discovered in fish. One shows similar gene organisation and synteny to mammalian and chicken IL-15, and has been termed IL-15. The second gene, which has a 4-exon structure and is in a different genome location, has been termed IL-15-like [168-170]. They show differential expression patterns in terms of the tissues where constitutive expression is apparent, and in terms of inducibility in PBL, with IL-15L being refractory to induction [168]. Two alternative splice variants of IL-15L (IL-15La and IL-15Lb) have also been described [170]. Trout IL-15, which has subsequently been cloned and sequenced, was strongly induced by rIFNγ in two trout cell lines (RTS-11 and RTG-2). rIL-15 could up-regulate IFNγ expression in splenic leucocytes, suggesting a positive feedback loop exists in fish between these two cytokines. Interestingly, unstimulated head kidney leucocytes were not responsive to rIL-15, at least in terms of the IFNγ expression level [171].

1.6.5. Interleukin 21

Interleukin 21 (IL-21) is a newly recognized member of IL-2 cytokine family that utilizes the common γ-chain receptor subunit for signal transduction [172-174]. In humans and other mammals, IL-21 is produced by both Th1 and Th2 cells [172, 175, 176]. IL-21 has pleiotropic effects on both innate and adaptive immune responses and can act on CD4+ and CD8+ T cells, B cells, NK cells, dendritic cells (DC), myeloid cells, and other tissue cells. IL-21 enhances the proliferation of anti–CD3-stimulated T cells and acts in concert with other γc cytokines to enhance the growth of CD4+ T cells [177]. IL-21–producing CD4+ T cells exhibit a stable phenotype of IL-21 production in the presence of IL-6 but retain the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions [178]. IL-21 stimulates CD8+ T cell proliferation and synergizes with IL-15 in promoting CD8+ T cell expansion in vitro and their antitumor effects in vivo [177, 179]. B cells that encounter IL-21 in the context of Ag-specific (BCR) stimulation and T cell co/stimulation undergo class-switch recombination and differentiate into Ab-producing plasma cells. In contrast, B cells encountering IL-21 during nonspecific TLR stimulation or without proper T cell help undergo apoptosis [180].

Since its discovery in fugu as a gene neighbour of IL-2 [141], IL-21 has been reported in tetraodon [181, 182] and rainbow trout [182]. Fugu IL-21 shows low constitutive expression. However, stimulation of isolated kidney leucocytes with PHA induced IL-21 expression. IL-21 was also up-regulated at mucosal sites as gill and gut when fish were injected with LPS or poly I:C [141]. Similarly, in tetraodon IL/21 expression is low but detectable in the gut, gonad and gills of healthy fish, and is induced in the kidney, spleen and skin following LPS injection [181]. In trout IL-21 expression is highest in gills and intestine, and is induced in vivo by bacterial (Y. ruckeri) and viral (VHSV) infection [182]. Relative to IL-2, induction of IL-21 expression in head kidney cells appears more rapidly but has shorter duration after stimulation. The trout rIL-21 has also been produced and shown to increase the expression of IL-10, IL-22 and IFNγ, and to a lesser extent IL-21, and to maintain the expression levels of key lymphocyte markers in primary cultures [182]. Thus, IL-21 may act as a survival factor for fish T and B cells [24].

1.7. The interleukin 10 family

Interleukin-IL-10 is an anti-inflammatory cytokine and a member of the class II cytokine family that also includes IL-19, IL-20, IL-22, IL-24, IL-26 and the interferons [183]. Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads to diverse biological effects through the activation of signal transducer and activator of transcription (STAT) factors [184].

1.7.1. Interleukin 10

Interleukin-10 (IL-10) was discovered initially as an inhibitory factor for the production of Th1 cytokines. Subsequently, pleiotropic inhibitory and stimulatory effects on various types of blood cells were described for IL-10, including its role as a survival and differentiation factor for B cells. IL-10, which is produced by activated monocytes, T cells and other cell types like keratinocytes, appears to be a crucial factor for at least some forms of peripheral tolerance and a major suppressor of the immune response and inflammation. The inhibitory function of IL-10 is mediated by the induction of regulatory T cells [185].

IL-10 was discovered in fish by searching the fugu genome. The translation showed 42–45% similarity to mammalian molecules with very low constitutive expression in tissues [186]. IL-10 has since been cloned in several other fish species including carp [187] zebrafish [188], rainbow trout [189], sea bass [190, 191] and cod [79]. Such studies have shown that IL-10 expression can be increased by LPS stimulation, by bacterial infection, by bath administration of immunostimulants [192] and by IPNV infection which may be associated with mechanisms of immune evasion [78].

1.7.2. Interleukin 20 (IL-20Like)

In mammals, IL-20 was discovered as a new member of the IL-10 family of cytokines. IL-20 shares the highest amino-acid sequence identity with IL-10, IL-24 and IL-19. It is secreted by immune cells and activated epithelial cells like keratinocytes. A high expression of the corresponding IL-20 receptor chains has been detected on epithelial cells. In terms of function, IL-20 might therefore mediate crosstalk between epithelial cells and tissue-infiltrating immune cells under inflammatory conditions [193].

In fish, the gene of IL-20 has been described in putterfish [183], zebrafish [194] and trout [195]. In the latter work, the IL-20 gene, called IL-20-like (IL-20L) has been described as having a high level of expression in immune related tissues and in the brain, suggesting an important role of the fish IL-20L molecule in both the immune and nervous systems. Although the exact cell types expressing IL-20L have yet to be defined, macrophages express IL-20L. Moreover, IL-20L expression in the macrophage cell line RTS-11 is modulated by pro-inflammatory cytokines, signalling pathway activators, microbial mimics and the immuno-suppressor dexamethasone. These data suggest that trout IL-20L plays an important role in the cytokine network. The increased expression of IL-20L was only detected at late stages (4–24 h) of LPS stimulation in RTS-11 cells and in spleen 24–72 h after infection with Yersinia ruckeri, which suggests that the increased expression of IL-20L by LPS and infection is via the rapid increase of pro-inflammatory cytokines (e.g., IL-1β) and other factors known to occur [195].

1.7.3. Interleukin 22/26

In mammals, interleukin-22 is secreted by Th17 cells [196], as well as by a subset of NK cells, designated as NK22 [197]; and even by some Th1 cells [198]. Studies have suggested there is a distinct Th22 cell lineage [199, 200]. Many of the same cytokines that induce differentiation and proliferation of IL-17-producing cells also lead to the secretion of IL-22 by Th17 cells, NK22 cells, and putative Th22 cells, including IL-6, IL-23, IL- 1β, TGF-β, and TNFα [201]. IL-17 and IL-22 are therefore frequently produced together in response to infections [202]. Interleukin-22 interacts with a heterodimeric receptor, IL-10R2/IL-22R1 [203], which is expressed on a variety of non-lymphoid cells, especially epithelial cells. Ligation of this receptor leads to both protective and detrimental effects. In synergy with IL-17, IL-22 induces pro-inflammatory cytokines in human bronchial epithelial cells against Klebsiella pneumoniae infection [204] and in colonic myofibroblasts [205]. Independently or in synergy with IL-17, IL-22 acts in defence against intestinal infection of mice with Citrobacter rodentium [206]. Moreover, IL-22 has been implicated in intestinal homeostasis keeping commensal bacteria contained in anatomical niches, which is key to our symbiotic relationship and normal intestinal physiology. However, the mechanisms that restrict colonization to specific niches are unclear. David Artis and colleagues have described a crucial role for IL-22-producing innate lymphoid cells (ILCs) in preventing lymphoid-resident commensal bacteria from escaping their niche and causing inflammation [207].

IL-26 can be produced by primary T cells, NK cells and T cell clones following stimulation with specific antigen or mitogenic lectins. IL-26 was initially shown by several groups to be co-expressed with IL-22 [208]. IL-26 is co-expressed with IFNγ and IL-22 by human Th1 clones, but not by Th2 clones. It was subsequently found that IL-26 is co-expressed with IL-17 and IL-22 by Th17 cells, an important subset of CD4+ T-helper cells that are distinct from Th1 and Th2 cells [209-211]. More recently, a novel subset of CD56+ NKp44+ NK cells was identified that co-expresses IL-22 and IL-26, especially following treatment with IL-23 [212]. Furthermore, a different subset of immature NK cells was described that do not express CD56 or NKp44 but do express CD117 and CD161 and constitutively express IL-22 and IL-26 [213].

The mechanisms that regulate transcription of the human IL-26 gene are so far largely undefined. It is possible and perhaps likely that expression of the IL-26 gene is induced in an IL-23-dependent manner because IL-23 is known to induce differentiation of Th17 cells, and IL-23 amplifies expression of IL-17 and IL-22 by Th17 cells [214].

In fish, the IFNγ locus was discovered using a gene synteny approach, and was first reported for fugu [215]. It contained a homologue of IL-22/26, that later studies of the zebrafish genome revealed to be two genes, one with clear homology to IL-22 and one with somewhat less clear homology to IL-26 [216]. The IL-22 gene was expressed constitutively in intestine and gills in all the treated and non-treated tissues. The gene was also expressed in kidney and spleen in LPS and PolyI:C-treated tissues, respectively, while IL-26 was expressed only in intestine treated with PolyI:C without expression [216]. IL-22 expression has been correlated with disease resistance in haddock vaccinated against V. anguillarum, with a strong constitutive expression in gills in vaccinated fish but not in control fish 24 hours post bath challenge, resulting in complete protection in fish vaccinated [217]. Moreover, IL-22, a cytokine released by Th-17 cells in mammals, is also interesting, and such responses are thought to be crucial for protection against extracellular microbes and at mucosal sites [218]. This coupled with the recent discovery of novel gill-associated immune tissue in fish [219] may provide a clue to a potential mechanism of resistance elicited by the V. anguillarum vaccination [24].

1.8. The interleukin 17 family

Interleukin-17 and a related family of genes are known to have pro-inflammatory actions and are associated with diseases [220]. After the discovery of the human IL- 17 gene [221], five cellular paralogs of IL-17 were identified, namely IL-17B, C, D, E and F [222-227]. These paralogs, identified by ESTs, genomics and proteomic databases, share identities of 20–50% with IL-17A gene. Human IL-17 A and F are present in tandem in opposite transcriptional orientation on the same chromosome 6p12, while IL-17B (Chr 5q24), IL-17C (Chr 16q24), IL-17D (Chr 13q11) and IL-17E (Chr 14q11) are dispersed. The structural similarities lead to the classification of IL-17 A, B, C, D, E, and F genes to a larger IL-17 sub-family [45]. Several IL-17 family members have been discovered in teleost fish, but homology to mammalian genes has not always been easy to assign. Two IL-17A or F homologue genes (IL-17A/F) have been found on the same chromosome. However, it has been difficult to determine which gene codes IL-17A and F. This gene in zebrafish was named IL-17A/F1 and 2. Furthermore, another IL-17A or F homologue gene (IL-17A/F3) has been found in zebrafish localized on a chromosome different from that of IL-17A/F1 and 2 [228]. In addition to those in zebrafish, IL-17A or F homologue genes have been found in rainbow trout [229], Atlantic salmon [230], pufferfish (IL-17A/F1, 2 and 3) [231], and medaka (IL-17A/F1, 2 and 3) [232].

The tissue distribution of the fugu IL-17 gene family also differs. In particular, IL-17 family genes are highly expressed in the head kidney and gills. Moreover, expression of IL-17 family genes is significantly up-regulated in the lipopolysaccharide-stimulated head kidney, suggesting that Fugu IL-17 family members are involved in inflammatory responses [231]. In Atlantic salmon IL-17D expression is widely distributed in tissues, with the highest levels of expression in testis, ovary and skin. Infection with A. salmonicida by injection increases IL-17D expression levels in the head kidney (but not the spleen) in a time-dependent manner. Skin and kidney showed an increased IL-17D expression level in fish given a cohabitation challenge with A. salmonicida [230]. The two trout IL-17C genes show some degree of differential expression within tissues, with IL-17C1 being more dominant in the gills and skin, whilst IL- 17C2 is more dominant in the spleen, head kidney and brain. Expression of both genes increases significantly with bacterial infection, although the increased expression of IL-17C2 is greater in terms of fold change. Similarly, both genes could be up-regulated in the trout RTS-11 cell line by LPS, poly I:C, calcium ionophore and rIL-1β, with IL-17C2 showing higher fold increases in all cases [229].

1.9. Interleukin 12

IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits. It can mediate a number of different activities, including stimulation of IFNγ secretion from resting lymphocytes, NK cell stimulation and cytolytic T cell maturation. Perhaps most crucially, IL-12 also affects the progression of uncommitted T cells to either the Th1 lineage, which in general is characterized by secretion of lymphokines associated with cell-mediated rather than humoral immunity [233].

The p35 and p40 subunits were discovered in fish by analysis of the fugu genome [234]. The p35 locus is quite well conserved, with Schip1 being the immediate neighbour in all cases. This association has allowed p35 to be cloned by gene walking from Schip1 from fish species for which no genome sequence is available [24, 235]. The p40 subunit in fugu is constitutively expressed in all the tissues examined, except muscle, and no increases in expression were seen 3 h after injection with poly I:C or LPS. This constitutive and broad expression distribution of the p40 subunit suggests that it may be expressed in most cell types. The expression of the p35 subunit is more limited in its tissue expression and is induced after injection with poly I:C in the head kidney and the spleen, but not after injection with LPS. These results show that there are differences from the mammalian data in fugu IL-12 subunit expression. Further investigation will be required to show whether this is unique to fugu, if IL-12 is involved more in antiviral defence in fish and if the two subunits are regulated differently from their regulation in the mammalian system [234].

1.10. Transforming growth factor β (TGF-β)

TGF-β is a pleiotropic cytokine that regulates cell development, proliferation, differentiation, migration, and survival in various leukocyte lineages including lymphocytes, dendritic cells, NK cells, macrophages and granulocytes [236, 237]. In the mammalian immune system, TGF-β1 is a well-known suppressive cytokine and its dominant role is to maintain immune tolerance and suppress autoimmunity [238, 239]. The potent immunosuppressive effects of TGF-β1 are mediated predominantly through its multiple effects on T cells: TGF-β1 suppress Th1 and Th2 cell proliferation, while it promotes T regulatory cell generation by inducing Foxp3 expression. On the other hand, TGF-β also promotes immune responses by inducing the generation of Th17 cells [236, 240, 241]. Therefore, the regulatory roles of TGF-β as a positive or negative control device in immunity are widely acknowledged in mammals [238, 240, 241].

In teleost, despite the lack of extensive investigation on the functional role of TGF-β, some recent studies have revealed that TGF-β1 also exterts powerful immune depressing effects on activated leukocytes, as it does in mammals. For instance, TGF-β1 significantly blocks TNFα-induced activation of macrophage in goldfish and common carp, but induces the proliferation of the goldfish fibroblast cell line CCL71 [242, 243]. In grass carp, TGF-β1 down-regulates LPS/PHA-stimulated the proliferation of peripheral blood lymphocyte by contrast with the stimulatory effect of TGF-b1 alone in the same cells [244]. In red sea bream, similar phenomenon was observed during leukocyte migration under TGF-β1 treatment, with or without LPS challenges [245]. These findings not only define TGF-β1 as an immune regulator in teleost, but also indicate that TGF-β1 may have retained similar functions in immunity during the evolution of vertebrates [246].

1.11. Interferons

Interferons genes are involved in mediating cellular resistance against viral pathogens and modulating innate and adaptive immune systems. Broadly, IFNs are classified into two main groups called type I and type II [45]. Type I IFN includes the classical IFNα/β, which is induced by viruses in most cells, whereas type II IFN is only composed of a single gene called IFNγ and is produced by NK cells (NK cells) and T lymphocytes in response to interleukin-12 (IL-12), IL-18, mitogens or antigens [247]. Structurally both IFN types belong to the class II a-helical cytokine family, but have different 3-dimensional structures and bind to different receptors [248].

Two IFNs (IFNα1 and IFNα2) have been cloned from Atlantic salmon and characterized with respect to sequence, gene structure, promoter, antiviral activity and induction of ISGs [249-252]. Salmon IFNα1 induces both Mx and ISG15 proteins in TO cells and thus has properties similar to mammalian IFNα/β and IFNλ [251, 252]. Furthermore, salmon IFNα1 induces potent antiviral activity against the IPNV in vitro [251], but this protection has not been observed in vivo, despite a high level of expression of IFNα detected in spleen and head kidney of Atlantic salmon challenged intraperitoneally with IPNV [78].

At least three type I IFNs have been discovered in rainbow trout. The IFN1 (rtIFN1) and rtIFN2 show high sequence similar to Atlantic salmon IFNα1 and IFNα2, which contains two cysteines. On the other hand, rtIFN3 contains four cysteines, which further confirms the relationship between mammalian IFNα and fish IFNs. Recombinant rtIFN1 and rtIFN2 have both been shown to up-regulate expression of Mx and inhibit VHSV replication in RTG-2 cells. In contrast, recombinant rtIFN3 has been found to be a poor inducer of Mx and antiviral activity. Interestingly, the three rtIFNs show differential expression in cells and tissues [253]. This suggests that the three trout IFNs have different functions in the immune system of fish, which is an interesting subject for further research [254].

IFNγ has been identified in several fish species, including rainbow trout and Atlantic salmon [215, 216, 248, 255-257]. In contrast to the type I IFNs, fish and mammalian IFNγ are similar in exon/intron structure and display gene synteny. However, some fish species also possess a second IFNγ subtype named IFN gamma rel, which is quite different from the classical IFNγ [258]. Rainbow trout and carp IFNγ have several functional properties in common with mammalian IFNγ, including the ability to enhance respiratory burst activity, nitric oxide production, and phagocytosis of bacteria in macrophages [257-259]. Far less is known about the antiviral properties of fish IFNγ. However, it has been reported that it induces antiviral activity against both IPNV and the Salmon Alpha Virus (SAV) in salmon cell lines [260]

1.12. Tools for fish cytokine analysis

The major strategy of functional genomics is to identify the types of responses to specific pathogens based on cytokines expression as a predictor of profile immune response, which began by using suppressive subtractive hybridization as major tools at the beginning of the immunogenomics and upgrade to platforms of wide screening that allow identify thousands of EST’s that are differentially regulated in their expression and that allow identifying potential candidates as biomarkers in the progression of the immune response at differential environmental conditions, not only against pathogens, but also in captivity stress conditions that affect the fisheries production.

1.13. Suppressive subtractive hybridization (SSH)

One of the most important biological processes in higher eukaryotes against external stimuli is the response mediated by differential gene expression. To understand the molecular regulation of these processes, the relevant subsets of differentially expressed genes of interest must be identified, cloned, and studied in detail using specific molecular techniques. In this matter, subtractive cDNA hybridization has been a powerful approach to identify and isolate cDNAs of differentially expressed genes [261-263]. Numerous cDNA subtraction methods have been reported. In general, they involve hybridization of cDNA from one population (tester) to excess of mRNA (cDNA) from another population (driver) and then separation of the unhybridized fraction (target) from the common hybridized sequences. One of these tools is a PCR-based technique called representational difference analysis, which does not require physical separation of single-strand (ss) and double-strand (ds) cDNAs. Representational difference analysis has been applied to enrich genomic fragments that differ in size or representation [264] and to clone differentially expressed cDNAs [265]. However, representational difference analysis has the problem of the wide differences in abundance of individual mRNA species so that multiple rounds of subtraction are needed [265]. Other strategies, such as mRNA differential display [266] and RNA fingerprinting by arbitrary primed PCR [267], are potentially faster methods for identifying differentially expressed genes, but both of these methods have high levels of false positives [268] that bias high-copy-number mRNA [269], which can inappropriate in experiments where only a few genes are expected to vary [268]. One of the techniques most often used to establish differential expression pattern between two conditions is suppression subtractive hybridization (SSH), which selectively amplifies target cDNA fragments (differentially expressed) and simultaneously suppresses non-target DNA amplification. The method is based on the fact that long inverted terminal repeats attached to DNA fragments can selectively suppress amplification of undesirable sequences in PCR procedures [270]. This method overcomes the problem of differences in mRNA abundance by incorporating a hybridization step that normalizes (equalizes) sequence abundance during the course of subtraction by standard hybridization kinetics [271]. Two types of SSH are possible: forward SSH, when the reaction involves the hybridization of cDNA from one population indicated as the evaluated phenotype (tester) to excess of mRNA (cDNA) from a control phenotype (driver); and reverse SSH, when the conditions described above are inverted. Together, the two processes are called reciprocal SSH.

Different works have been done with SSH to evaluate fish immune response at the gene expression level against challenges with bacteria-derived pathogen-associated molecular patterns (PAMP) like LPS [272, 273] and whole bacteria like Aeromonas salmonicida [274, 275], Listonella anguillarum [276], Edwardsiella tarda [277], and Vibrio parahaemolyticus [278] (Table 1).

A critical step in any immune response is the recognition of invading organisms. This is mediated by many proteins, including pattern recognition receptors (PRR), which recognize and bind to molecules present on the surface of microorganisms. LPS is an essential cell wall component of gram-negative bacteria and is recognized by PRR, triggering a series of responses that lead to the activation of the host defence system. These PRRs include a number of toll-like receptors, as well as other cell-surface and cytosolic receptors that, upon stimulation, modulate immunity [279, 280]. In LPS-stimulated yellow grouper spleen a subtracted cDNA library was constructed using SSH. The contigs and singlets obtained were analyzed and a low number of immune-related genes were found [272]. In Asian seabass the up-regulation of differentially expressed genes like pro-inflammatory cytokines and related receptors, such as TNF receptor super family member 14 (TNFRSF14), IL-31 receptor A (IL31RA), chemokine receptor-like 1 (CMKLR1), chemokine (C-X-C motif) receptor 3 (CXCR3), chemokine (C-C motif) receptor 7 (CCR7) and chemokine (C-C motif) ligand 25 (CCL25), was identified at 24h post-challenge by bacterial LPS in spleen Complement components were also identified [273]. These genes are a solid basis for a better understanding of immunity in Asian seabass and for developing effective strategies for immune protection against infections in that species.

Infection of Atlantic salmon by A. salmonicida was observed to stimulate an acute-phase response (APR) as part of the innate immune defence system to infection, whose gene expression pattern was remarkably observed in liver at 7 days post-infection [275] indicating that the liver appears to be the main source of APPs in fish, as in mammals. Not surprisingly, the liver gene expression pattern observed in other fish species against L. anguillarum [276], E. tarda [277], and V. parahaemolyticus [278]. The APR is characterized by alterations in the levels of plasma proteins referred to as acute-phase proteins (APPs), as well as the secretion of some other innate defence molecules important for innate immunity, such as complementary systems [281-283]. In Atlantic cod stimulated with atypical A. salmonicida (formalin-killed) interleukin-1β (IL-1- β), interleukin-8 (IL-8), CC chemokine type 3, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin were identified in the forward spleen SSH library. Atlantic cod IRF1 was constitutively expressed at low levels, and expression was significantly elevated in spleen and head kidney at 24 h following A. salmonicida stimulation, with the highest levels of induction observed in the spleen [274]. The target IRFI genes, as well as their importance in innate immune responses in fish, have not yet been determined, although the expression of IRF1 in teleost macrophages can be induced by both IFNγ and IL-1β, with IFNγ being a much more potent inducer of IRF1 than IL-1β [99].

Microorganism Fish Pathogen Tissue/Cell type Infection route Reference
Bacteria Atlantic salmon Aeromonas salmonicida A449 Liver, head kidney and spleen Intraperitoneal Tsoi et al., 2004
Yellow grouper LPS (E. coli) Spleen Intraperitoneal Wang et al., 2007
Atlantic cod Aeromonas salmonicida (formalin-killed) Head kidney, Spleen Intraperitoneal Feng et al., 2009
Asian seabass LPS (E. coli) Spleen Intraperitoneal Xia and Yue 2010
Ayu Listonella anguillarum Liver Intraperitoneal Li et al., 2011
Japanese flounder Edwardsiella tarda (NE8003) PBL Intraperitoneal Matsuyama et al., 2011
Marine medaka Vibrio parahaemolyticus Liver Intraperitoneal Bo et al., 2012
Virus Mandarian fish ISKNV Spleen Intramuscular He et al., 2006
Sea bream Nodavirus (strain 475-9/99) Brain Intramuscular Dios et al., 2007
Atlantic cod poly I:C Spleen Intraperitoneal Rise et al., 2008
Sea bass SBNNV Head kidney Intramuscular Poisa-Beiro et al., 2009
Atlantic cod ACNNV Brain Intraperitoneal Rise et al., 2010
Orange-spotted grouper SGIV Spleen Intraperitoneal Xu et al., 2010

Table 1.

Transcriptomics studies on fish after treatments with bacteria or virus in vivo analyzed with SSH. LPS: Lipopolysaccharide; ISKNV: Infectious spleen and kidney necrosis virus; poly I:C: polyriboinosinic polyribocytidylic acid; SBNNV: Sea bass nervous necrosis virus; ACNNV: Atlantic cod nervous necrosis virus; SGIV: Singapure grouper iridovirus; PBL: Peripheral blood leukocytes.

SSH has been in several investigations to evaluate fish gene expression patterns against challenges with PAMPs, such as polyriboinosinic polyribocytidylic acid (poly I:C) [284], Infectious Spleen and Kidney Necrosis Virus (ISKNV) [285], Nodavirus [286-288], and Singapore grouper iridovirus (SGIV) [289].

Spleen gene expression in mandarin fish at 4 days post-infection with ISKNV of Mx protein, interferon-inducible protein Gig-2, and viperin (interferon-inducible and antiviral protein) was up-regulated, suggesting IFN pathway stimulation after ISKNV infection [285]. Also, two inflammatory cytokine genes, CC chemokine and IL-8, were found in the forward SSH library, whereas the CD59/Neurotoxin/Ly-6-like protein gene was down-regulated. In mammals, CD59 is a complement regulatory protein, which can inhibit complement activation and membrane attack complex (MAC) formation on autologous cells [290], suggesting that down-regulation in the ISKNV-infected host cells may make these cells more sensitive to complement attack, mounting an anti-virus mechanism of the host [285].

In orange-spotted grouper after 5 days of infection with Singapore grouper iridovirus (SGIV) novel genes were annotated as immune-related, such as C-type lectin, epinecidin, and complement components C3 and C9. Interestingly, the most abundant clone was C-type lectin, and the microarray results at 1, 5 and 9 days post-infection indicated that its expression was up-regulated in liver, spleen and kidney [289]. Lectins are multivalent carbohydrate-binding proteins that function as important pattern-recognition receptors (PRR) and have been isolated and characterized in fish [291-294]. C-type lectin represents a very large family, most members of which are able to bind PAMP and microorganisms themselves through sugar moieties and play important roles in non-self recognition and clearance of invading microorganisms. The up-regulation of C-type lectin in different organs with immunological functions confirmed as SSH as microarrays suggest an important role in the development of control strategies against SGIV infection.

The SSH method was used to generate a subtracted cDNA library enriched in gene transcripts differentially expressed after 1 day post-infection in the brains of sea bream infected with nodavirus. Most of the expressed sequence tags (ESTs) differentially expressed in infected tissues fell into gene categories related to cell structure, transcription, cell signalling or different metabolic routes. Other interesting putative homologies corresponded to genes expressed in stress responses, such as heat shock proteins (Hsp-70) and to immune-related genes such as the Fms-interacting protein, TNFα-induced protein, interferon-induced with helicase C domain protein (mda-5), which in mammals play an important role in the synthesis and secretion of IFN type I [295]. Another nodavirus, sea bass nervous necrosis virus (SBNNV) was studied to identify genes potentially involved in antiviral immune defence in sea bass head kidney using the SSH technique [287]. The results of up-regulated EST from sea bass head kidney SSH showed significant similarities with immune genes, such as β-2 microglobulin, heat shock protein 90 (Hsp-90), IgM, MHC class I and class II, and β-galactoside-binding lectin, identified as a member of the galectin family and closely related to the galectin-1 group (Sbgalectin-1). When the recombinant protein (rSbgalectin-1) was produced and functional assays were conducted, a decrease in IL-1β, TNFα, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared to those infected with the nodavirus alone, suggesting a potential anti-inflammatory protective role of Sbgalectin-1 during viral infection. A similar nodavirus, the Atlantic cod nervous necrosis virus (ACNNV), was studied to evaluate the transcript expression responses in the Atlantic cod (Gadus morhua) brain to asymptomatic high nodavirus carrier state [288]. In the forward brain SSH library was identified with significant similarity to genes with immune-relevant functional annotations the interferon stimulated gene 15 (ISG15), IL-8 variant 5, DEXH (Asp-Glu-X-His) box polypeptide 58 (DHX58; LGP2), radical Sadenosyl methionine domain-containing 2 (RSAD2; viperin), β-2-microglobulin (B2M), chemokine CXC-like protein, signal transducer and activator of transcription 1 (STAT1), and CC chemokine type 2. Interestingly, ISG15, DHX58, RSAD2, and sacsin (SACS) transcripts are all strongly upregulated by both high nodavirus carriage and intraperitoneal poly I:C stimulation, suggesting a similar host response is significantly induced in the brain by both nodavirus and poly I:C. This expression pattern is corroborated when the response of Atlantic cod spleen is evaluated against poly I:C stimulation, showing the up-regulation of ISG15, RSAD2, LGP2 and other transcripts such as MHC class I, and IRF1, 7, and 10, indicating that Atlantic cod recognize dsRNA and mount a interferon pathway response [284].

1.14. Microarrays

Microarray analysis measures the expression of large numbers of genes in parallel. This methodology, which combines hypotheses-driven and hypotheses-free research strategies, is used to infer molecular mechanisms, classify samples, and diagnose and search for novel biomarkers. With the use of standard platforms, laboratory protocols and procedures for processing of primary data, the results of microarrays analyses are well suited for database management and meta-analysis across multiple experiments, whilst data mining is based on powerful statistical procedures with support from functional and structural annotations of genes [296].

The Atlantic salmon is of particular importance to the global aquaculture industry. Salmonid cDNA microarrays were constructed shortly after large-scale sequencing of salmon and trout cDNA libraries by several research institutes. One of the projects related to salmon sequencing is GRASP (Genomics Research on Atlantic Salmon Project), an initiative funded by Genome Canada that is intended to improve understanding of physiological and evolutionary processes influencing the survival and phenotype of salmonids and other fish in natural and aquaculture environments. The first salmonid GRASP microarray platform (GRASP-1), containing 7356 salmonid elements representing 3557 different cDNAs (3.7K), was obtained from 80,388 ESTs, principally from cDNA libraries [298] of different salmon species such as Atlantic salmon, rainbow trout, Chinook salmon, sockeye salmon, and lake whitefish cDNA libraries. The second version of the GRASP microarray platform (GRASP-2) was developed and contained cDNAs representing 16,006 genes (16K). The genes identified in the array have been stringently selected from Atlantic salmon and rainbow trout EST databases representing a wide variety of different classes of genes [297]. Finally, a new expanded salmonid cDNA microarray (GRASP-3) of 32,000 features (32K) was created where 69% of the total EST collection used was from Atlantic salmon [298]. The Aleksei Krasnov’s group designed the rainbow trout microarray (SFA1.0) by identifying a relatively small number of genes (1300 genes; 1.3K) using clones from normalized and subtracted cDNA libraries, as well as genes selected by the functional categories of Gene Ontology for inclusion in a microarray aimed at characterizing transcriptome responses to environmental stressors [299] to maximize the presence of transcripts related directly to immune response in rainbow trout, because of which this platform is also called Immunochip (SFA1.0 immunochip). The updated SFA platform (1.8K; SFA2.0 immunochip) was specially designed for studies of responses to pathogens and stressors and has substantially improved coverage of immune genes [300]. Another cDNA platform in commercial fish species has been designed in Japanese flounder [301] and European flounder [302], turbot [303], and sole [304].However despite impressive achievements, cDNA platforms suffer from limitations and disadvantages. At present most research groups working with salmonids and other aquaculture species do not have full access to clones required for fabrication of cDNA microarrays. Maintenance and PCR amplification of large clone sets is expensive and time consuming, while the risk of errors is high [296]. Probably the most important drawback of cDNA microarrays is their limited ability to discriminate paralogs since long probes cross-hybridize with highly similar transcripts from members of multi-gene families [305]. In salmonids this problem is aggravated by the large number of expressed gene duplicates. These complications can be resolved with oligonucleotide microarrays (ONM) that also provide greater accuracy and reproducibility of analyses. Until recently, the use of ONM platforms was hampered by the cost, but they are now rapidly replacing cDNA platforms. Construction of ONM platforms begins with establishment of mRNA sequence sets for comprehensive coverage of transcriptomes with low redundancy. The next stage is identifying genes by searching protein databases and annotating them according to functions, pathways and structural features. For successful development and use of ONM, it is necessary to define the gene composition and optimum number of spot replicates and to choose criteria for quality assessment [296].

Because of the commercial importance of salmonid species, there is special interest gene expression pattern against different pathogens. Initially salmonid (rainbow trout) ONM contained 1672 elements, representing more than 1400 genes [306]. Currently, one of the most often used ONM platforms to evaluate the response against different conditions and pathogens is the custom salmon ONM (SIQ-3), based on the Agilent Technology system (21K in 4x44K format). Because limited availability of peripheral blood leukocyte (PBL) markers is a well-recognised problem of fish immunology, this platform compares the transcriptomes of PBL and other tissues to search for genes with preferential expression in leukocytes [296], making it a very significant tool to evaluate the response to pathogens in Atlantic salmon. Another ONM platform based on 500K ESTs Atlantic salmon and 250K ESTs rainbow trout [298] is the cGRASP 44K salmonid oligo array (Agilent eArray), although no studies employing this platform have been published yet. Another ONM has been designed in fish model organisms like zebrafish and in commercial fish species such as channel catfish and turbot [307].

Functional genomic studies based on evaluating immune responses, also called immunogenomics, have been conducted in vivo to evaluate the response to different pathogens at the systemic level in different organs, especially the liver and head kidney. The functional genomic approach has been used with Salmo salar and Oncorhynchus mykiss, where PAMPs, whole bacteria and viruses are the most studied pathogens. Here we present different works in fish challenged by bacteria or viruses where differential gene expression profiles were evaluated using microarray platforms with special emphasis on in vivo fish immune response (Table 2).

1.15. Studies with bacterial pathogens, PAMPs and cytokine network interactions

One of the most commonly studied bacterial pathogens is Aeromonas salmonicida, a gram-negative bacteria and the causative agent of furunculosis. In fact prior to the development of species-specific cDNA microarrays a preliminary study used a human microarray (GENEFILTERS GF211) to explore the liver response in Atlantic salmon infected using a cohabitation model [275]. Only 4 mRNAs were consistently up-regulated (p < 0.01) from the 241 positively identified spots with a clearly detectable hybridization signal, none of them related to cytokine expression. This was probably due to the lack of sequence homology, a problem commonly associated with cross-species cDNA hybridization. Thus the creation of species-specific platforms was a key step in fish immunology. Using a custom Atlantic salmon cDNA microarray (NRC-IMB) consisting of over 4,000 different cDNA amplicons, the first results for challenge with Aeromonas salmonicida were reported in 2005 [275]. The study described a cohabitation challenge and identified 16 up-regulated mRNAs in all three tissues studied (spleen, liver and head kidney), whereas 2 and 19 mRNAs were identified as down-regulated in the head kidney and liver, respectively. The authors found that genes related to the acute phase response were up-regulated in spleen and head kidney of infected salmon, indicating that the infected fish underwent a typical acute phase response to infection.

The effects of an Aeromonas salmonicida infection were recently reported in turbot, Scolphtalmus maximus, [307]. Using a custom designed oligonucleotide-microarray (8x15K), the authors identified a set of 48 differentially regulated mRNAs in the spleen of challenged fish at 3 dpi, mostly related to the acute-phase and the stress/defence immune response. A study using channel and blue catfish explored the effects of a gram-negative bacterial infection on the acute phase response (APR) [308]. The authors showed up-regulation of mRNA transcripts involved in iron homeostasis, transport proteins, complement components and inflammatory and humoral immune response, indicating that conserved APR occurs as part of the innate immune response in both catfish species. Interestingly, a more acute response was observed composed of several immune pathways in the blue but not the channel catfish. More studies are required to elucidate expression patterns resulting from gram-negative bacterial infection of phylogenetically similar and different fish are required to describe common and divergent responses. This could lead to the development of marker systems, consensus on the APR in fish and treatments tailored to certain species, all of which have significant applied interest.

The activity of LPS from gram-negative bacteria, a common membrane-associated PAMP used in immunological studies, has been explored in several fish species. These studies include effects on the spleen in channel catfish [285], rainbow trout head kidney [309], and liver in the Senegalese sole [310]. Using a 19K oligonucleotide microarray (ONM) it was observed that some pro-inflammatory mRNAs in the catfish spleen were up-regulated very quickly, principally between 2 and 4 hours post-injection with LPS, whereas immunoglobulin- (2h post-injection) and antigenic presentation-related mRNA transcripts were repressed 24h post-injection [311]. A similar inhibition was reported in head kidney of rainbow trout, where the suppression of major cellular processes, including immune function and an initial stress reaction, was followed by a proliferative hematopoietic-type/biogenesis response 3 dpi [309]. However, in the Senegalese sole a clear up-regulation of transcripts related to the immune response was reported 24 hpi in the liver [310]. These results collectively highlight the diversity of responses observed at the tissue level and reflect the nature of the immune system that is diffusely located throughout many organ compartments.

Microorganism Fish Pathogen Tissue/Cell type Resource Platform Reference
Bacteria Atlantic salmon Aeromonas salmonicida (A449) Liver cDNA GENE- FILTERS GF211 Tsoi et al., 2003
Aeromonas salmonicida (A449) Head kidney/liver/spleen cDNA NRC-IMB Ewart et al., 2005
Aeromonas salmonicida Liver/spleen cDNA SFA-2 Skugor et al., 2009
Aeromonas salmonicida (Brivax II) Liver cDNA TRAITS/SPG Martin et al., 2010
Piscirickettsia salmonis Head kidney cDNA GRASP-1 Rise et al., 2004
Chinook salmon Vibrio anguillarum Head kidney cDNA GRASP-1 Ching et al., 2010
Rainbow Trout Vibrio anguillarum (FDKC) Liver ONM OSUrbt Gerwick et al., 2007
LPS (E.coli 026:B6) Head kidney cDNA SFA-1 MacKenzie et al., 2008
LPS (E. coli 026:B6) HK macrophages cDNA SFA-1 Mackenzie et al., 2008
LPS, PGN (E. coli 0111:B4) HK macrophages cDNA SFA-2 Boltaña et al., 2011
PGN (E. coli 0111:B4, K12) HK macrophages cDNA SFA-2 Boltaña et al., 2011
LPS, PGN (E. coli 0111:B4)/poly I:C Erythrocytes cDNA SFA-2 Morera et al., 2011
Brook trout LPS (E. coli 026:B6) HK macrophages cDNA SFA-1 Mackenzie et al., 2006
Channel catfish LPS (E.coli 0127:B8 ) Spleen ONM UMSMED-1 Li et al., 2006
Edwardsiella ictaluri (MS-S97-773) Gills/head kidney/liver/skin/spleen ONM UMSMED-2 Peatman et al., 2007
Blue catfish Edwardsiella ictaluri (MS-S97-773) Liver ONM UMSMED-2 Peatman et al., 2008
Japanese flounder Edwardsiella tarda (NE9505) Head kidney cDNA Japanese flounder custom-3 Yasuike et al., 2010
Streptococcus iniae (02; FKC) Head kidney cells cDNA Japanese flounder custom-3 Dumrongphol et al., 2008
Mycobacterium bovis (TUMSAT-Msp001) FKC Kidney cells cDNA Japanese flounder custom-3 Kato el al., 2010
Zebrafish Mycobacterium marinum (M, E11) Whole fish ONM MWG - Sigma Genosys - Affimetrix Meijer et al., 2005
Mycobacterium marinum (E11; Mma20) Whole fish ONM ZF Agilent Van der Sar et al., 2009
Streptococcus suis (HA9801) Whole fish ONM Affimetrix Zebrafish GeneChip Wu et al., 2010
Turbot Aeromonas salmonicida Spleen ONM Turbot custom Agilent Millán et al., 2010
Solea LPS (E. coli 011:B4) Liver cDNA GENIPOL-1 Osuna-Jimenez et al., 2009
Virus Atlantic salmon ISAV (Glesvaer 2/90) Gills/heart/liver/spleen cDNA SFA-2 Jorgensen et al., 2008
ISAV (Glesvaer2/90) Heart/PBL ONM SIQ-3 Krasnov et al., 2011
Chinook salmon ISAV NA-HRP4 (970) Head kidney cDNA GRASP-3 Leblanc et al., 2010
Rainbow Trout IHNV (32/87)/attenuated IHNV Head kidney cDNA SFA-1 MacKenzie et al., 2008
IHNV (strain 220-90) Head kidney cDNA GRASP-2 Purcell et al., 2011
Japanese flounder VHSV (KRRV9822) Head kidney cells cDNA Japanese flounder custom-1 Byon et al., 2005
VHSV (KRRV9822) Head kidney cells cDNA Japanese flounder custom-2 Byon et al., 2006
HIRRV (8601H) Kidney cells cDNA Japanese flounder custom-1 Yasuike et al., 2007
HIRRV (8601H) Kidney cells cDNA Japanese flounder custom-3 Yasuike et al., 2010
Zebrafish VHSV (07.71) Fin/head kidney/liver/spleen ONM ZF Agilent Encinas et a., 2010
Turbot Nodavirus (AH95)/poly (I:C) Kidney cDNA Turbot custom Park et al., 2009

Table 2.

Transcriptomics studies on fish after treatments with bacteria or virus in vivo analyzed with microarrays. FDKC: Formaldehyde –killed cells; LPS: Lipopolysaccharide; FKC: Formalin-killed cells; ISAV: Infectious salmon anemia virus; PD: Pancreas disease; CMS: Cardiomyopathy syndrome; HSMI: Heart and skeletal muscle inflammation; IHNV: Infectious hematopoietic necrosis virus; VHSV: Viral hemorrhagic septicemia virus; HIRRV: Hirame rhabdovirus; poly I:C: polyriboinosinic polyribocytidylic acid; PBL: Peripheral blood leukocytes; ONM: oligonucleotide microarray.

For gram-positive infections in fish at the level of transcriptome analyses, infection of zebrafish with Streptococcus suis is the only model reported [272]. Streptococcus suis is a pathogen associated with zoonosis reported in several countries [312, 313]. The Affymetrix Zebrafish GeneChip was used to identify 125 up-regulated transcripts where the most significant pathways were antigen processing and presentation, leukocyte trans-endothelial migration and the proteosome. The authors suggested that the target list obtained could serve as infection markers for gram-positive infection in fish.

Undoubtedly, the identification of prognostic biomarkers for disease resistance is a major aim for aquaculture. Functional genomics has the potential to identify such potential tools. Disease resistance is normally measured by challenge with the pathogen of interest and assessing the cumulative mortalities. Surviving fish or non-challenged siblings from the same family are then considered ‘resistant’. Because this process is costly there is a need for non-lethal methodologies of measuring resistance, ideally based on molecular determinants of resistance. An initial example of this approach used the GRASP 3.7K cDNA array to identify in vitro macrophage and in vivo head kidney biomarkers in response to Piscirickettsia salmonis infection, yielding a number of 11 regulated genes common to both challenges. The researchers proposed 19 highly regulated transcripts as potential biomarkers to evaluate the efficacy of vaccines against Piscirickettsia salmonis [314]. C-type lectin 2-1, a gene whose product is involved in endocytosis and the C/EBP-driven inflammatory response [315] was identified and has been identified in almost all reports in which bacterial preparations have been used to challenge live fish [275, 309, 314, 316, 317]. Another study aimed at identifying biomarkers at the transcriptional level described differences between triploid and diploid Chinook salmon under live Vibrio anguillarum challenge using the GRASP 3.7K cDNA microarray [318]. Twelve annotated mRNAs were identified as showing significant differences between diploid and triploid fish. The authors however were unable to provide a description of the underlying mechanisms to explain the observed reduced immune function of triploid salmon.

Individual variation is a major hurdle for the development of prognostic markers as both genetic and epigenetic factors must be taken into account. The utility of, for example, C-type lectin in salmon, and other potential biomarkers in other species for bacterial disease resistance, requires further development. The future publication of several fish genomes coupled to array platforms with a much increased transcript representation could provide an exciting route to further develop this strategy by combining both functional and structural genomics for species of commercial interest with a sequenced genome. Several studies have attempted to correlate gene expression profiles with the activity of bacterins (killed bacteria preparations) used to vaccinate fish in culture. Most studies have concentrated on the rainbow trout and Japanese flounder [262, 277, 319, 320]. In trout, intraperitoneal administration of killed V. anguillarum resulted in identifying 36 differentially expressed transcripts [320]. Most of the identified targets are involved in inflammatory response and respond to a broad range of stimuli. This suggests that these targets have little use as markers for vaccination, contrary to previous descriptions in other studies. Both the second and fourth versions of the Japanese flounder cDNA microarray have been used to address vaccination [262, 277, 319]. The results of experimental infection with Gram-negative E. tarda indicated that a formalin-killed preparation reduced mortality in vaccinated fish from 90% to 20% [277]. However, a correlation between the transcriptome and the efficacy of vaccination could not be identified.

The effects of a commercial vaccine for Atlantic salmon (a six-component oil-adjuvant vaccine from PHARMAQ) were evaluated to correlate vaccine protection to high and low resistance to furunculosis. The authors did not find any association between either group and suggested that “outcomes of vaccination depend largely on the ability of host to prevent the negative impacts of immune response and to repair damages” [305]. Although this study did not identify correlations between vaccination and gene expression profiles, the potential of a functional genomics approach to evaluate the efficacy and underlying mechanisms of vaccination is highlighted. In terms of the immune response and the resulting complexity in expression patterns resulting from multiple cell types and different tissue responses, the investigator has the potential to obtain a clearer ‘image’ of the biological response from global expression data. A key objective is therefore to increase the available genomic resources much facilitated by next generation sequencing technologies to form a more robust representation of the immune system among different fish species. Furthermore, the increasing use of ONM platforms will also improve comparison across species as data sets become more easily comparable.

It remains difficult to compare microarray experiments across distinct platforms. In this respect, Meijer et al. 2005, evaluated host transcriptome profiling to Mycobacterium marinum infection of adult zebrafish employing three oligonucleotides platforms (MWG, Sigma Genosys, and Affimetrix). At a significance level of P < 1.00E-5, there were differences among the platforms in the total number of more than 2-fold up-regulated genes, whereas the 2-fold down-regulated genes were in a similar range. Evaluation of the distribution of infection-induced genes over different categories reveled some divergence in the set from MWG, probably due to the abundance of genes of the same UniGene cluster. As well, from the total overlap of 4,138 UniGene clusters among the three microarrays, only 66 and 93 genes were up- and down-regulated, respectively [321]. With this antecedent, the same group generated a new platform (Agilent 44K) that includes their 22K probes, a 16K set probes similar to the Sigma-Compugen oligonucleotide library, and 6K set of probes for selected genes of interest indentified by previous data mining of zebrafish transcript and genome databases [322], and they evaluated the transcriptome response to acute and chronic infection by Mycobacterium marinum. This important effort in combing different platforms makes it clear that not all relevant genes, including immune-related ones, are represented in all platforms. Consequntly, new efforts are necessary to broaden our understanding of the immune response in fish challenged with a pathogen of interest.

1.16. Wide screening in fish challenged with viral pathogens

Two studies have reported host responses to IHNV with the SFA and GRASP platforms [309, 323]. The potential mechanisms responsible for host-specific virulence were assessed in rainbow trout infected with high (M) and low virulence (U) strains of IHNV. A marked down-regulation in biological processes, including the immune response, lymphocyte activation, response to stress, transcription and translation, together with a greater viral load (M), suggest that the higher virulence is due to the ability to suppress the immune response via the transcriptional and translational machinery of cell [323]. Furthermore, in rainbow trout was compared the expression profiles of IHNV and attenuated IHNV were compared in rainbow trout over a short time frame of one and three days post-challenge. At 3 dpi, a significant change in the transcriptional program of head kidney revealed an immunological shift orientated toward the activation of adaptive immunity. This shift was IHNV-dependent as determined by differences between the attenuated and virulent IHNV specific expression profiles. The rapid systemic spreading of IHNV inhibited TNFα, MHC class I, and several macrophage and cell cycle/differentiation markers and favored a MHC class II, immunoglobulin and MMP/TBX4-enhanced immune response [309].

Parallel studies were conducted with a cDNA microarray enriched with 213 immune-related genes to study the immune response and the efficacy of DNA vaccines containing the viral G proteins of VHSV and HIRRV administered intramuscularly in the Japanese flounder [301, 324]. As expected, all DNA vaccines containing the viral G glycoprotein conferred specific protection to the challenged fish one month after vaccination. It is suggested that the protection occurs via the IFN type I system due to the number of IFN-related genes up-regulated in both studies, ISG-15, interferon-stimulated gene 56kDa (ISG56) and the Mx protein. In both studies, VHSV and HIRRV in the Japanese flounder, the majority of differentially up-regulated genes were identified between 3 and 7 days post-vaccination (dpv), including the less effective DNA vaccine containing N protein of HIRRV. Interestingly, Mx, an antiviral protein commonly used as a marker for antiviral activity in animal species, was consistently up-regulated across vaccinations [301]. In a similar observation, IRF-3, Mx, Vig-1 and Vig-8 were up-regulated in trout at the site of DNA vaccination against IHNV at 7 dpv [323]. In turbot challenged with nodavirus, both Mx and IFN-inducible proteins were identified 24 hpi [303]. These and the previously described results suggest that both the host-expressed viral glycoprotein and the virulent rhadovirus induce a systemic anti-viral state indicative of non-specific IFN type1 innate immune response and that this canonical response is conserved among all fish. However, the mechanisms to develop a specific cytotoxic T or B lymphocyte-mediated humoral response in fish vaccinated with plasmid DNA-IHNV G that confers protective immunity have not been identified [323].

In direct relation to the above, a significant increase in transcript markers for adaptive immunity was reported in Atlantic salmon during ISA virus (ISAV) infection [325]. Importantly, a progressive increase was observed in IgZ mRNA parallel to a decrease in IgM expression that peaked > 30 days post-infection. This coordinated increase in a group of genes related to B lymphocyte differentiation and maturation and activation of T lymphocyte-mediated immunity, including CD4, TGF-β, CD8α and IFNγ, provides strong evidence for the coordinated regulation of the two arms of the immune system in response to viral infection. An important technological contribution derived from the above study was the development of an ONM for Atlantic salmon (SIQ-3). The first assessment of the performance of these arrays was carried out in Atlantic salmon for the study of virus-responsive genes from samples infected with ISA, salmonid alpha virus/PD-virus, cardiomyopathy syndrome (CMS) agent, heart and skeletal muscle inflammation (HSMI) and PBL from fish infected with ISAV. Some 95 up-regulated transcripts were identified. Most of the regulated transcripts are related directly to the immune response or associated with antiviral response [296]. As previously mentioned, the creation of species-specific platforms has been a key challenge for the study of the immune response against pathogens. Despite impressive achievements, cDNA microarrays suffer from limitations and disadvantages, the most important drawback being the limited ability to discriminate between paralogs as long cDNA probes cross-hybridize with highly similar transcripts from members of multi-gene families [305]. Furthermore this information needs to be supplemented to establish if the increased level of detected transcripts is consistent with specific protein synthesis. Recently, a study employed a combined proteomic and transcriptomic approach to evaluate the immune response against VHS [326]. In the fins of infected fish a series of mRNA transcripts principally related to complement components, immunoglobulin-related proteins, and macrophages were up-regulated (> 2-fold), whereas in parallel using two dimensional differential gel electrophoresis (2D-DIGE), enzymes of the glycolytic pathway and some proteins related to cytoskeletal remodelling and apoptosis (such as annexin A1a) increased with infection. However, very few proteins related to anti-viral response were identified.


2. Concluding remarks

A complex network exists to regulate the innate and adaptive immune responses of fish from the various cytokines that have been reported. The study of the functional activity of these cytokines is in progress and it will be interesting to know whether mammalian Th1, Th2, Th17 and Treg responses are present in fish, regulating specific cell-mediated immunity. The recombinant production of these cytokines and antibodies against them will be the next challenge in understanding the balance of such immune responses and aid in the effective design of therapeutic strategies to manipulate the fish immune system. towards humoral or cellular immunity in response to specific antigen stimulation, vaccine strategies, functional diets to increase the quality of fishery production and predict the health of cultured fish.

The study of functional genomics in fish has provided substantial data on species of commercial interest. The major aim has been to functionally identify the intensity of responses to specific pathogens and their associated molecular components and to identify transcripts in a whole organism or specific tissue that contribute to such responses. However, the complex biology of the immune response, in which different spatial-temporal expression occurs in multiple cell types at distinct body locations, makes complete mapping of a response difficult and expensive. Moreover, considering that arrays are only as good as the transcripts represented upon them. Thus, the representation of transcripts relevant to the immune response is intimately linked to gene discovery efforts through large-scale sequencing projects, where strategies like SSH contribute not only to understanding transcriptomic response against specific pathogens but also to gene discovery. In this area, access to high-throughput NGS technology has increased in recent years and promises to make an important contribution to understanding immune response in fish. The major task now is the meta-analysis of transcriptomic data to delineate responses common among fish species to specific pathogen groups and highly specific responses. This approach will reveal host specific expression profiles and facilitate the identification of prognostic markers for diseases.



The authors are grateful for the support of VRID-USACH, INNOVA-CORFO 07CN13PBT-90, INNOVA-CORFO 09MCSS6691, INNOVA-CORFO 09MCSS6698 and CONICYT Fellowships to K. Maisey and D. Toro-Ascuy.


  1. 1. Flajnik MF, Du Pasquier L. Evolution of the Immune System. 6 th ed. Paul WE, editor: Lippincott Williams & Wilkins; 2008.
  2. 2. Akirav EM, Liao S, Ruddle NH. Lymphoid Tissues and Organs. 6 th ed. Paul WE, editor: Lippincott Williams & Wilkins; 2008.
  3. 3. Zapata AG, Chibá A, Varas A. Cells and tissues of the immune system of fish. In: Iwama G, Nakanishi T, editors. The fish immune system Organism, pathogen and environment: Academic Press; 1996. p. 1-62.
  4. 4. Meseguer J, Lopez-Ruiz A, Garcia-Ayala A. Reticulo-endothelial stroma of the head-kidney from the seawater teleost gilthead seabream (Sparus aurata L.): an ultrastructural and cytochemical study. Anat Rec. 1995 Mar;241(3):303-9.
  5. 5. Danneving BH, Lauve A, Press MC, Landsverk T. Receptor-mediated endocytosis and phagocytosis by rainbow trout head kidney sinusoidal cells. Fish Shellfish Immunol. 1994;4:3-18.
  6. 6. Brattgjerd S, Evensen O. A sequential light microscopic and ultrastructural study on the uptake and handling of Vibrio salmonicida in phagocytes of the head kidney in experimentally infected Atlantic salmon (Salmo salar L.). Vet Pathol. 1996 Jan;33(1):55-65.
  7. 7. Kaattari SL, Irwin MJ. Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires. Dev Comp Immunol. 1985 Summer;9(3):433-44.
  8. 8. Herraez MP, Zapata AG. Structure and function of the melano-macrophage centres of the goldfish Carassius auratus. Vet Immunol Immunopathol. 1986 Jun;12(1-4):117-26.
  9. 9. Tsujii T, Seno S. Melano-macrophage centers in the aglomerular kidney of the sea horse (teleosts): morphologic studies on its formation and possible function. Anat Rec. 1990 Apr;226(4):460-70.
  10. 10. Bowden TJ, Cook P, Rombout JH. Development and function of the thymus in teleosts. Fish Shellfish Immunol. 2005 Nov;19(5):413-27.
  11. 11. Zapata AG, Amemiya CT. Phylogeny of lower vertebrates and their immunological structures. Curr Top Microbiol Immunol. 1999;248:67-107.
  12. 12. Espenes A, Press CM, Dannevig BH, Landsverk T. Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss). Cell Tissue Res. 1995 Oct;282(1):41-8.
  13. 13. Vallejo AN, Miller NW, Harvey NE, Cuchens MA, Warr GW, Clem LW. Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation. Dev Immunol. 1992;3(1):51-65.
  14. 14. Vallejo AN, Miller NW, Clem LW. Cellular pathway(s) of antigen processing in fish APC: effect of varying in vitro temperatures on antigen catabolism. Dev Comp Immunol. 1992 Sep-Oct;16(5):367-81.
  15. 15. Ellis AE. Antigen trapping in the spleen and kidney of the plaice, Pleuronectes platessa. J Fish Dis. 1980;3:413-26.
  16. 16. Agius C, Roberts RJ. Melano-macrophage centres and their role in fish pathology. J Fish Dis. 2003 Sep;26(9):499-509.
  17. 17. Medzhitov R. The Innate Immune System. 6 th ed. Paul WE, editor: Lippincott Williams & Wilkins; 2008.
  18. 18. Janeway CA, Jr. How the immune system protects the host from infection. Microbes Infect. 2001 Nov;3(13):1167-71.
  19. 19. McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigen-specific memory B cell development. Annu Rev Immunol. 2005;23:487-513.
  20. 20. Sallusto F, Lanzavecchia A. Heterogeneity of CD4+ memory T cells: functional modules for tailored immunity. Eur J Immunol. 2009 Aug;39(8):2076-82.
  21. 21. Dixon B, Stet RJ. The relationship between major histocompatibility receptors and innate immunity in teleost fish. Dev Comp Immunol. 2001 Oct-Dec;25(8-9):683-99.
  22. 22. Abbas AK, Lichtman AH, Pillai S. Cells and Tissues of the Adaptive Immune System. 6 th ed: Saunders; 2007.
  23. 23. Castro R, Bernard D, Lefranc MP, Six A, Benmansour A, Boudinot P. T cell diversity and TcR repertoires in teleost fish. Fish Shellfish Immunol. 2011 Nov;31(5):644-54.
  24. 24. Secombes CJ, Wang T, Bird S. The interleukins of fish. Dev Comp Immunol. 2011 Dec;35(12):1336-45.
  25. 25. Wang T, Gorgoglione B, Maehr T, Holland JW, Vecino JL, Wadsworth S, et al. Fish Suppressors of Cytokine Signaling (SOCS): Gene Discovery, Modulation of Expression and Function. J Signal Transduct. 2011;2011:905813.
  26. 26. Alejo A, Tafalla C. Chemokines in teleost fish species. Dev Comp Immunol. 2011 Dec;35(12):1215-22.
  27. 27. Nakanishi T, Toda H, Shibasaki Y, Somamoto T. Cytotoxic T cells in teleost fish. Dev Comp Immunol. 2011 Dec;35(12):1317-23.
  28. 28. Boltana S, Donate C, Goetz FW, MacKenzie S, Balasch JC. Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages. Fish Shellfish Immunol. 2009 Apr;26(4):651-61.
  29. 29. Hanington PC, Hitchen SJ, Beamish LA, Belosevic M. Macrophage colony stimulating factor (CSF-1) is a central growth factor of goldfish macrophages. Fish Shellfish Immunol. 2009 Jan;26(1):1-9.
  30. 30. Salinas I, Zhang YA, Sunyer JO. Mucosal immunoglobulins and B cells of teleost fish. Dev Comp Immunol. 2011 Dec;35(12):1346-65.
  31. 31. Zhang YA, Salinas I, Oriol Sunyer J. Recent findings on the structure and function of teleost IgT. Fish Shellfish Immunol. 2011 Nov;31(5):627-34.
  32. 32. Randelli E, Buonocore F, Scapigliati G. Cell markers and determinants in fish immunology. Fish Shellfish Immunol. 2008 Oct;25(4):326-40.
  33. 33. Rombout JH, Huttenhuis HB, Picchietti S, Scapigliati G. Phylogeny and ontogeny of fish leucocytes. Fish Shellfish Immunol. 2005 Nov;19(5):441-55.
  34. 34. Araki K, Akatsu K, Suetake H, Kikuchi K, Suzuki Y. Characterization of CD8+ leukocytes in fugu (Takifugu rubripes) with antiserum against fugu CD8alpha. Dev Comp Immunol. 2008;32(7):850-8.
  35. 35. Toda H, Saito Y, Koike T, Takizawa F, Araki K, Yabu T, et al. Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish. Dev Comp Immunol. 2011 Jun;35(6):650-60.
  36. 36. Takizawa F, Dijkstra JM, Kotterba P, Korytar T, Kock H, Kollner B, et al. The expression of CD8alpha discriminates distinct T cell subsets in teleost fish. Dev Comp Immunol. 2011 Jul;35(7):752-63.
  37. 37. Kaattari S, Evans D, Klemer J. Varied redox forms of teleost IgM: an alternative to isotypic diversity? Immunol Rev. 1998 Dec;166:133-42.
  38. 38. Wilson M, Bengten E, Miller NW, Clem LW, Du Pasquier L, Warr GW. A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4593-7.
  39. 39. Stenvik J, Jorgensen TO. Immunoglobulin D (IgD) of Atlantic cod has a unique structure. Immunogenetics. 2000 May;51(6):452-61.
  40. 40. Danilova N, Bussmann J, Jekosch K, Steiner LA. The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat Immunol. 2005 Mar;6(3):295-302.
  41. 41. Hansen JD, Landis ED, Phillips RB. Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proc Natl Acad Sci U S A. 2005 May 10;102(19):6919-24.
  42. 42. Li J, Barreda DR, Zhang YA, Boshra H, Gelman AE, Lapatra S, et al. B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nat Immunol. 2006 Oct;7(10):1116-24.
  43. 43. Overland HS, Pettersen EF, Ronneseth A, Wergeland HI. Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol. 2010 Jan;28(1):193-204.
  44. 44. Wang T, Huang W, Costa MM, Secombes CJ. The gamma-chain cytokine/receptor system in fish: more ligands and receptors. Fish Shellfish Immunol. 2011 Nov;31(5):673-87.
  45. 45. Savan R, Sakai M. Genomics of fish cytokines. Comp Biochem Physiol Part D Genomics Proteomics. 2006 Mar;1(1):89-101.
  46. 46. Salazar-Mather TP, Hokeness KL. Cytokine and chemokine networks: pathways to antiviral defense. Curr Top Microbiol Immunol. 2006;303:29-46.
  47. 47. Svanborg C, Godaly G, Hedlund M. Cytokine responses during mucosal infections: role in disease pathogenesis and host defence. Curr Opin Microbiol. 1999 Feb;2(1):99-105.
  48. 48. DeVries ME, Ran L, Kelvin DJ. On the edge: the physiological and pathophysiological role of chemokines during inflammatory and immunological responses. Semin Immunol. 1999 Apr;11(2):95-104.
  49. 49. Whyte SK. The innate immune response of finfish--a review of current knowledge. Fish Shellfish Immunol. 2007 Dec;23(6):1127-51.
  50. 50. Rahman MM, McFadden G. Modulation of tumor necrosis factor by microbial pathogens. PLoS Pathog. 2006 Feb;2(2):e4.
  51. 51. Benedict CA, Banks TA, Ware CF. Death and survival: viral regulation of TNF signaling pathways. Curr Opin Immunol. 2003 Feb;15(1):59-65.
  52. 52. Hirono I, Nam BH, Kurobe T, Aoki T. Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus. J Immunol. 2000 Oct 15;165(8):4423-7.
  53. 53. Laing KJ, Wang T, Zou J, Holland J, Hong S, Bols N, et al. Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha. Eur J Biochem. 2001 Mar;268(5):1315-22.
  54. 54. Zou J, Wang T, Hirono I, Aoki T, Inagawa H, Honda T, et al. Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol. 2002 Mar;26(2):161-72.
  55. 55. Garcia-Castillo J, Pelegrin P, Mulero V, Meseguer J. Molecular cloning and expression analysis of tumor necrosis factor alpha from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics. 2002 Jun;54(3):200-7.
  56. 56. Saeij JP, Stet RJ, de Vries BJ, van Muiswinkel WB, Wiegertjes GF. Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev Comp Immunol. 2003 Jan;27(1):29-41.
  57. 57. Zou J, Secombes CJ, Long S, Miller N, Clem LW, Chinchar VG. Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Dev Comp Immunol. 2003 Dec;27(10):845-58.
  58. 58. Praveen K, Evans DL, Jaso-Friedmann L. Constitutive expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in regulation of cell-mediated cytotoxicity. Mol Immunol. 2006 Feb;43(3):279-91.
  59. 59. Ordas MC, Costa MM, Roca FJ, Lopez-Castejon G, Mulero V, Meseguer J, et al. Turbot TNFalpha gene: molecular characterization and biological activity of the recombinant protein. Mol Immunol. 2007 Jan;44(4):389-400.
  60. 60. Grayfer L, Walsh JG, Belosevic M. Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha. Dev Comp Immunol. 2008;32(5):532-43.
  61. 61. Garcia-Castillo J, Chaves-Pozo E, Olivares P, Pelegrin P, Meseguer J, Mulero V. The tumor necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner. Cell Mol Life Sci. 2004 Jun;61(11):1331-40.
  62. 62. Roca FJ, Mulero I, Lopez-Munoz A, Sepulcre MP, Renshaw SA, Meseguer J, et al. Evolution of the inflammatory response in vertebrates: fish TNF-alpha is a powerful activator of endothelial cells but hardly activates phagocytes. J Immunol. 2008 Oct 1;181(7):5071-81.
  63. 63. Hardie LJ, Chappell LH, Secombes CJ. Human tumor necrosis factor alpha influences rainbow trout Oncorhynchus mykiss leucocyte responses. Vet Immunol Immunopathol. 1994 Jan;40(1):73-84.
  64. 64. Iliev DB, Liarte CQ, MacKenzie S, Goetz FW. Activation of rainbow trout (Oncorhynchus mykiss) mononuclear phagocytes by different pathogen associated molecular pattern (PAMP) bearing agents. Mol Immunol. 2005 Jun;42(10):1215-23.
  65. 65. Wang WL, Hong JR, Lin GH, Liu W, Gong HY, Lu MW, et al. Stage-specific expression of TNFalpha regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells. PLoS One. 2011;6(2):e16740.
  66. 66. Dinarello C, Arend W, Sims J, Smith D, Blumberg H, O'Neill L, et al. IL-1 family nomenclature. Nat Immunol. 2010 Nov;11(11):973.
  67. 67. Dinarello CA. Interleukin-1. Cytokine Growth Factor Rev. 1997 Dec;8(4):253-65.
  68. 68. Zou J, Grabowski PS, Cunningham C, Secombes CJ. Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site. Cytokine. 1999 Aug;11(8):552-60.
  69. 69. Fujiki K, Shin DH, Nakao M, Yano T. Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1 beta, high affinity immunoglobulin E Fc receptor gamma subunit and serum amyloid A. Fish Shellfish Immunol. 2000 Apr;10(3):229-42.
  70. 70. Scapigliati G, Buonocore F, Bird S, Zou J, Pelegrin P, Falasca C, et al. Phylogeny of cytokines: molecular cloning and expression analysis of sea bass Dicentrarchus labrax interleukin-1beta. Fish Shellfish Immunol. 2001 Nov;11(8):711-26.
  71. 71. Pelegrin P, Garcia-Castillo J, Mulero V, Meseguer J. Interleukin-1beta isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines. Cytokine. 2001 Oct 21;16(2):67-72.
  72. 72. Corripio-Miyar Y, Bird S, Tsamopoulos K, Secombes CJ. Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus). Mol Immunol. 2007 Feb;44(6):1361-73.
  73. 73. Lee DS, Hong SH, Lee HJ, Jun LJ, Chung JK, Kim KH, et al. Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus. Comp Biochem Physiol A Mol Integr Physiol. 2006 Mar;143(3):307-14.
  74. 74. Pleguezuelos O, Zou J, Cunningham C, Secombes CJ. Cloning, sequencing, and analysis of expression of a second IL-1beta gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics. 2000 Oct;51(12):1002-11.
  75. 75. Cerretti DP, Kozlosky CJ, Mosley B, Nelson N, Van Ness K, Greenstreet TA, et al. Molecular cloning of the interleukin-1 beta converting enzyme. Science. 1992 Apr 3;256(5053):97-100.
  76. 76. Secombes CJ, Wang T, Hong S, Peddie S, Crampe M, Laing KJ, et al. Cytokines and innate immunity of fish. Dev Comp Immunol. 2001 Oct-Dec;25(8-9):713-23.
  77. 77. Lu DQ, Bei JX, Feng LN, Zhang Y, Liu XC, Wang L, et al. Interleukin-1beta gene in orange-spotted grouper, Epinephelus coioides: molecular cloning, expression, biological activities and signal transduction. Mol Immunol. 2008 Feb;45(4):857-67.
  78. 78. Reyes-Cerpa S, Reyes-Lopez FE, Toro-Ascuy D, Ibanez J, Maisey K, Sandino AM, et al. IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence. Fish Shellfish Immunol. 2012 Feb;32(2):291-300.
  79. 79. Seppola M, Larsen AN, Steiro K, Robertsen B, Jensen I. Characterisation and expression analysis of the interleukin genes, IL-1beta, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.). Mol Immunol. 2008 Feb;45(4):887-97.
  80. 80. Zou J, Holland J, Pleguezuelos O, Cunningham C, Secombes CJ. Factors influencing the expression of interleukin-1 beta in cultured rainbow trout (Oncorhynchus mykiss) leucocytes. Dev Comp Immunol. 2000 Sep-Oct;24(6-7):575-82.
  81. 81. Buonocore F, Forlenza M, Randelli E, Benedetti S, Bossu P, Meloni S, et al. Biological activity of sea bass (Dicentrarchus labrax L.) recombinant interleukin-1beta. Mar Biotechnol (NY). 2005 Nov-Dec;7(6):609-17.
  82. 82. Hong S, Zou J, Crampe M, Peddie S, Scapigliati G, Bols N, et al. The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1 beta. Vet Immunol Immunopathol. 2001 Aug 30;81(1-2):1-14.
  83. 83. Peddie S, Zou J, Cunningham C, Secombes CJ. Rainbow trout (Oncorhynchus mykiss) recombinant IL-1beta and derived peptides induce migration of head-kidney leucocytes in vitro. Fish Shellfish Immunol. 2001 Nov;11(8):697-709.
  84. 84. Peddie S, Zou J, Secombes CJ. Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of Ergosan. Vet Immunol Immunopathol. 2002 May;86(1-2):101-13.
  85. 85. Dinarello CA, Fantuzzi G. Interleukin-18 and host defense against infection. J Infect Dis. 2003 Jun 15;187 Suppl 2:S370-84.
  86. 86. Gracie JA, Robertson SE, McInnes IB. Interleukin-18. J Leukoc Biol. 2003 Feb;73(2):213-24.
  87. 87. Sugawara I. Interleukin-18 (IL-18) and infectious diseases, with special emphasis on diseases induced by intracellular pathogens. Microbes Infect. 2000 Aug;2(10):1257-63.
  88. 88. Micallef MJ, Ohtsuki T, Kohno K, Tanabe F, Ushio S, Namba M, et al. Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production. Eur J Immunol. 1996 Jul;26(7):1647-51.
  89. 89. Okamura H, Nagata K, Komatsu T, Tanimoto T, Nukata Y, Tanabe F, et al. A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. Infect Immun. 1995 Oct;63(10):3966-72.
  90. 90. Dao T, Mehal WZ, Crispe IN. IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J Immunol. 1998 Sep 1;161(5):2217-22.
  91. 91. Leung BP, Culshaw S, Gracie JA, Hunter D, Canetti CA, Campbell C, et al. A role for IL-18 in neutrophil activation. J Immunol. 2001 Sep 1;167(5):2879-86.
  92. 92. Xu D, Trajkovic V, Hunter D, Leung BP, Schulz K, Gracie JA, et al. IL-18 induces the differentiation of Th1 or Th2 cells depending upon cytokine milieu and genetic background. Eur J Immunol. 2000 Nov;30(11):3147-56.
  93. 93. Ghayur T, Banerjee S, Hugunin M, Butler D, Herzog L, Carter A, et al. Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature. 1997 Apr 10;386(6625):619-23.
  94. 94. Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming MA, et al. Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science. 1997 Jan 10;275(5297):206-9.
  95. 95. Akita K, Ohtsuki T, Nukada Y, Tanimoto T, Namba M, Okura T, et al. Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J Biol Chem. 1997 Oct 17;272(42):26595-603.
  96. 96. Sugawara S, Uehara A, Nochi T, Yamaguchi T, Ueda H, Sugiyama A, et al. Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells. J Immunol. 2001 Dec 1;167(11):6568-75.
  97. 97. Huising MO, Stet RJ, Savelkoul HF, Verburg-van Kemenade BM. The molecular evolution of the interleukin-1 family of cytokines; IL-18 in teleost fish. Dev Comp Immunol. 2004 May 3;28(5):395-413.
  98. 98. Zou J, Bird S, Truckle J, Bols N, Horne M, Secombes C. Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss). Eur J Biochem. 2004 May;271(10):1913-23.
  99. 99. Martin SA, Zou J, Houlihan DF, Secombes CJ. Directional responses following recombinant cytokine stimulation of rainbow trout (Oncorhynchus mykiss) RTS-11 macrophage cells as revealed by transcriptome profiling. BMC Genomics. 2007;8:150.
  100. 100. Wang T, Secombes CJ. Identification and expression analysis of two fish-specific IL-6 cytokine family members, the ciliary neurotrophic factor (CNTF)-like and M17 genes, in rainbow trout Oncorhynchus mykiss. Mol Immunol. 2009 Jul;46(11-12):2290-8.
  101. 101. Taga T, Kishimoto T. Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol. 1997;15:797-819.
  102. 102. Hirano T. Interleukin 6 and its receptor: ten years later. Int Rev Immunol. 1998;16(3-4):249-84.
  103. 103. Inoue K, Takano H, Shimada A, Morita T, Yanagisawa R, Sakurai M, et al. Cytoprotection by interleukin-6 against liver injury induced by lipopolysaccharide. Int J Mol Med. 2005 Feb;15(2):221-4.
  104. 104. Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol. 2003 Jan;24(1):25-9.
  105. 105. Bird S, Zou J, Savan R, Kono T, Sakai M, Woo J, et al. Characterisation and expression analysis of an interleukin 6 homologue in the Japanese pufferfish, Fugu rubripes. Dev Comp Immunol. 2005;29(9):775-89.
  106. 106. Iliev DB, Castellana B, Mackenzie S, Planas JV, Goetz FW. Cloning and expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus mykiss). Mol Immunol. 2007 Mar;44(7):1803-7.
  107. 107. Nam BH, Byon JY, Kim YO, Park EM, Cho YC, Cheong J. Molecular cloning and characterisation of the flounder (Paralichthys olivaceus) interleukin-6 gene. Fish Shellfish Immunol. 2007 Jul;23(1):231-6.
  108. 108. Castellana B, Iliev DB, Sepulcre MP, MacKenzie S, Goetz FW, Mulero V, et al. Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata). Mol Immunol. 2008 Jul;45(12):3363-70.
  109. 109. Costa MM, Maehr T, Diaz-Rosales P, Secombes CJ, Wang T. Bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-6: effects on macrophage growth and antimicrobial peptide gene expression. Mol Immunol. 2011 Sep;48(15-16):1903-16.
  110. 110. Czupryn MJ, McCoy JM, Scoble HA. Structure-function relationships in human interleukin-11. Identification of regions involved in activity by chemical modification and site-directed mutagenesis. J Biol Chem. 1995 Jan 13;270(2):978-85.
  111. 111. Bartz H, Buning-Pfaue F, Turkel O, Schauer U. Respiratory syncytial virus induces prostaglandin E2, IL-10 and IL-11 generation in antigen presenting cells. Clin Exp Immunol. 2002 Sep;129(3):438-45.
  112. 112. Kernacki KA, Goebel DJ, Poosch MS, Hazlett LD. Early cytokine and chemokine gene expression during Pseudomonas aeruginosa corneal infection in mice. Infect Immun. 1998 Jan;66(1):376-9.
  113. 113. Nandurkar HH, Robb L, Begley CG. The role of IL-II in hematopoiesis as revealed by a targeted mutation of its receptor. Stem Cells. 1998;16 Suppl 2:53-65.
  114. 114. Trepicchio WL, Bozza M, Pedneault G, Dorner AJ. Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production. J Immunol. 1996 Oct 15;157(8):3627-34.
  115. 115. Orazi A, Du X, Yang Z, Kashai M, Williams DA. Interleukin-11 prevents apoptosis and accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation. Lab Invest. 1996 Jul;75(1):33-42.
  116. 116. Liu Q, Du XX, Schindel DT, Yang ZX, Rescorla FJ, Williams DA, et al. Trophic effects of interleukin-11 in rats with experimental short bowel syndrome. J Pediatr Surg. 1996 Aug;31(8):1047-50; discussion 50-1.
  117. 117. Huising MO, Kruiswijk CP, van Schijndel JE, Savelkoul HF, Flik G, Verburg-van Kemenade BM. Multiple and highly divergent IL-11 genes in teleost fish. Immunogenetics. 2005 Jul;57(6):432-43.
  118. 118. Wang T, Holland JW, Bols N, Secombes CJ. Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss. FEBS J. 2005 Mar;272(5):1136-47.
  119. 119. Santos MD, Yasuike M, Kondo H, Hirono I, Aoki T. Teleostean IL11b exhibits complementing function to IL11a and expansive involvement in antibacterial and antiviral responses. Mol Immunol. 2008 Jul;45(12):3494-501.
  120. 120. Ribeiro CM, Hermsen T, Taverne-Thiele AJ, Savelkoul HF, Wiegertjes GF. Evolution of recognition of ligands from Gram-positive bacteria: similarities and differences in the TLR2-mediated response between mammalian vertebrates and teleost fish. J Immunol. 2010 Mar 1;184(5):2355-68.
  121. 121. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, et al. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev. 2000 Mar;52(1):145-76.
  122. 122. Mukaida N, Harada A, Matsushima K. Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine Growth Factor Rev. 1998 Mar;9(1):9-23.
  123. 123. Hebert CA, Vitangcol RV, Baker JB. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem. 1991 Oct 5;266(28):18989-94.
  124. 124. Clark-Lewis I, Schumacher C, Baggiolini M, Moser B. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J Biol Chem. 1991 Dec 5;266(34):23128-34.
  125. 125. Laing KJ, Zou JJ, Wang T, Bols N, Hirono I, Aoki T, et al. Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss. Dev Comp Immunol. 2002 Jun;26(5):433-44.
  126. 126. Lee EY, Park HH, Kim YT, Choi TJ. Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceous). Gene. 2001 Aug 22;274(1-2):237-43.
  127. 127. Sangrador-Vegas A, Lennington JB, Smith TJ. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization. Cytokine. 2002 Jan 21;17(2):66-70.
  128. 128. Chen L, He C, Baoprasertkul P, Xu P, Li P, Serapion J, et al. Analysis of a catfish gene resembling interleukin-8: cDNA cloning, gene structure, and expression after infection with Edwardsiella ictaluri. Dev Comp Immunol. 2005;29(2):135-42.
  129. 129. Najakshin AM, Mechetina LV, Alabyev BY, Taranin AV. Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines. Eur J Immunol. 1999 Feb;29(2):375-82.
  130. 130. Laing KJ, Secombes CJ. Chemokines. Dev Comp Immunol. 2004 May 3;28(5):443-60.
  131. 131. Osborne LC, Abraham N. Regulation of memory T cells by gammac cytokines. Cytokine. 2010 May;50(2):105-13.
  132. 132. Sogo T, Kawahara M, Ueda H, Otsu M, Onodera M, Nakauchi H, et al. T cell growth control using hapten-specific antibody/interleukin-2 receptor chimera. Cytokine. 2009 Apr;46(1):127-36.
  133. 133. Morgan DA, Ruscetti FW, Gallo R. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science. 1976 Sep 10;193(4257):1007-8.
  134. 134. Smith KA. T-cell growth factor. Immunol Rev. 1980;51:337-57.
  135. 135. Smith KA. Interleukin-2: inception, impact, and implications. Science. 1988 May 27;240(4856):1169-76.
  136. 136. Granucci F, Vizzardelli C, Pavelka N, Feau S, Persico M, Virzi E, et al. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat Immunol. 2001 Sep;2(9):882-8.
  137. 137. Walker E, Leemhuis T, Roeder W. Murine B lymphoma cell lines release functionally active interleukin 2 after stimulation with Staphylococcus aureus. J Immunol. 1988 Feb 1;140(3):859-65.
  138. 138. Gaffen SL, Wang S, Koshland ME. Expression of the immunoglobulin J chain in a murine B lymphoma is driven by autocrine production of interleukin 2. Cytokine. 1996 Jul;8(7):513-24.
  139. 139. Yu TK, Caudell EG, Smid C, Grimm EA. IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway. J Immunol. 2000 Jun 15;164(12):6244-51.
  140. 140. Gold MR, DeFranco AL. Biochemistry of B lymphocyte activation. Adv Immunol. 1994;55:221-95.
  141. 141. Bird S, Zou J, Kono T, Sakai M, Dijkstra JM, Secombes C. Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny. Immunogenetics. 2005 Mar;56(12):909-23.
  142. 142. Sugamata R, Suetake H, Kikuchi K, Suzuki Y. Teleost B7 expressed on monocytes regulates T cell responses. J Immunol. 2009 Jun 1;182(11):6799-806.
  143. 143. Diaz-Rosales P, Bird S, Wang TH, Fujiki K, Davidson WS, Zou J, et al. Rainbow trout interleukin-2: cloning, expression and bioactivity analysis. Fish Shellfish Immunol. 2009 Sep;27(3):414-22.
  144. 144. Zhang YA, Hikima J, Li J, LaPatra SE, Luo YP, Sunyer JO. Conservation of structural and functional features in a primordial CD80/86 molecule from rainbow trout (Oncorhynchus mykiss), a primitive teleost fish. J Immunol. 2009 Jul 1;183(1):83-96.
  145. 145. Laing KJ, Bols N, Secombes CJ. A CXC chemokine sequence isolated from the rainbow trout Oncorhynchus mykiss resembles the closely related interferon-gamma-inducible chemokines CXCL9, CXCL10 and CXCL11. Eur Cytokine Netw. 2002 Oct-Dec;13(4):462-73.
  146. 146. Banchereau J, Bidaud C, Fluckiger AC, Galibert L, Garrone P, Malisan F, et al. Effects of interleukin 4 on human B-cell growth and differentiation. Res Immunol. 1993 Oct;144(8):601-5.
  147. 147. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986 Apr 1;136(7):2348-57.
  148. 148. Finkelman FD, Urban JF, Jr. The other side of the coin: the protective role of the TH2 cytokines. J Allergy Clin Immunol. 2001 May;107(5):772-80.
  149. 149. Yokota T, Otsuka T, Mosmann T, Banchereau J, DeFrance T, Blanchard D, et al. Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5894-8.
  150. 150. Noma Y, Sideras P, Naito T, Bergstedt-Lindquist S, Azuma C, Severinson E, et al. Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter. Nature. 1986 Feb 20-26;319(6055):640-6.
  151. 151. Lee F, Yokota T, Otsuka T, Meyerson P, Villaret D, Coffman R, et al. Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2061-5.
  152. 152. Heussler VT, Eichhorn M, Dobbelaere DA. Cloning of a full-length cDNA encoding bovine interleukin 4 by the polymerase chain reaction. Gene. 1992 May 15;114(2):273-8.
  153. 153. Li-Weber M, Krammer PH. Regulation of IL4 gene expression by T cells and therapeutic perspectives. Nat Rev Immunol. 2003 Jul;3(7):534-43.
  154. 154. Ansel KM, Djuretic I, Tanasa B, Rao A. Regulation of Th2 differentiation and Il4 locus accessibility. Annu Rev Immunol. 2006;24:607-56.
  155. 155. Ohtani M, Hayashi N, Hashimoto K, Nakanishi T, Dijkstra JM. Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish. Immunogenetics. 2008 Jul;60(7):383-97.
  156. 156. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, et al. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature. 2004 Oct 21;431(7011):946-57.
  157. 157. Li JH, Shao JZ, Xiang LX, Wen Y. Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: the first evidence of Th2-type cytokine in fish. Mol Immunol. 2007 Mar;44(8):2078-86.
  158. 158. Secrist H, Egan M, Peters MG. Tissue-specific regulation of IL-4 mRNA expression in human tonsil. J Immunol. 1994 Feb 1;152(3):1120-6.
  159. 159. Lin AF, Xiang LX, Wang QL, Dong WR, Gong YF, Shao JZ. The DC-SIGN of zebrafish: insights into the existence of a CD209 homologue in a lower vertebrate and its involvement in adaptive immunity. J Immunol. 2009 Dec 1;183(11):7398-410.
  160. 160. Hu YL, Xiang LX, Shao JZ. Identification and characterization of a novel immunoglobulin Z isotype in zebrafish: implications for a distinct B cell receptor in lower vertebrates. Mol Immunol. 2010 Jan;47(4):738-46.
  161. 161. Mitra S, Alnabulsi A, Secombes CJ, Bird S. Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio. FEBS J. 2010 Jan;277(1):128-47.
  162. 162. Takizawa F, Koppang EO, Ohtani M, Nakanishi T, Hashimoto K, Fischer U, et al. Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes suggests that these tissues form Th2-skewed immune environments. Mol Immunol. 2011 Jul;48(12-13):1360-8.
  163. 163. Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J Immunol. 2005 Jun 1;174(11):6571-6.
  164. 164. Alves NL, Richard-Le Goff O, Huntington ND, Sousa AP, Ribeiro VS, Bordack A, et al. Characterization of the thymic IL-7 niche in vivo. Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1512-7.
  165. 165. Mazzucchelli RI, Warming S, Lawrence SM, Ishii M, Abshari M, Washington AV, et al. Visualization and identification of IL-7 producing cells in reporter mice. PLoS One. 2009;4(11):e7637.
  166. 166. Repass JF, Laurent MN, Carter C, Reizis B, Bedford MT, Cardenas K, et al. IL7-hCD25 and IL7-Cre BAC transgenic mouse lines: new tools for analysis of IL-7 expressing cells. Genesis. 2009 Apr;47(4):281-7.
  167. 167. Kono T, Bird S, Sonoda K, Savan R, Secombes CJ, Sakai M. Characterization and expression analysis of an interleukin-7 homologue in the Japanese pufferfish, Takifugu rubripes. FEBS J. 2008 Mar;275(6):1213-26.
  168. 168. Bei JX, Suetake H, Araki K, Kikuchi K, Yoshiura Y, Lin HR, et al. Two interleukin (IL)-15 homologues in fish from two distinct origins. Mol Immunol. 2006 Mar;43(7):860-9.
  169. 169. Fang W, Xiang LX, Shao JZ, Wen Y, Chen SY. Identification and characterization of an interleukin-15 homologue from Tetraodon nigroviridis. Comp Biochem Physiol B Biochem Mol Biol. 2006 Mar;143(3):335-43.
  170. 170. Gunimaladevi I, Savan R, Sato K, Yamaguchi R, Sakai M. Characterization of an interleukin-15 like (IL-15L) gene from zebrafish (Danio rerio). Fish Shellfish Immunol. 2007 Apr;22(4):351-62.
  171. 171. Wang T, Holland JW, Carrington A, Zou J, Secombes CJ. Molecular and functional characterization of IL-15 in rainbow trout Oncorhynchus mykiss: a potent inducer of IFN-gamma expression in spleen leukocytes. J Immunol. 2007 Aug 1;179(3):1475-88.
  172. 172. Parrish-Novak J, Dillon SR, Nelson A, Hammond A, Sprecher C, Gross JA, et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature. 2000 Nov 2;408(6808):57-63.
  173. 173. Asao H, Okuyama C, Kumaki S, Ishii N, Tsuchiya S, Foster D, et al. Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex. J Immunol. 2001 Jul 1;167(1):1-5.
  174. 174. Vosshenrich CA, Di Santo JP. Cytokines: IL-21 joins the gamma(c)-dependent network? Curr Biol. 2001 Mar 6;11(5):R175-7.
  175. 175. Chtanova T, Tangye SG, Newton R, Frank N, Hodge MR, Rolph MS, et al. T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol. 2004 Jul 1;173(1):68-78.
  176. 176. Wurster AL, Rodgers VL, Satoskar AR, Whitters MJ, Young DA, Collins M, et al. Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon gamma-producing Th1 cells. J Exp Med. 2002 Oct 7;196(7):969-77.
  177. 177. Leonard WJ, Zeng R, Spolski R. Interleukin 21: a cytokine/cytokine receptor system that has come of age. J Leukoc Biol. 2008 Aug;84(2):348-56.
  178. 178. Suto A, Kashiwakuma D, Kagami S, Hirose K, Watanabe N, Yokote K, et al. Development and characterization of IL-21-producing CD4+ T cells. J Exp Med. 2008 Jun 9;205(6):1369-79.
  179. 179. Frederiksen KS, Lundsgaard D, Freeman JA, Hughes SD, Holm TL, Skrumsager BK, et al. IL-21 induces in vivo immune activation of NK cells and CD8(+) T cells in patients with metastatic melanoma and renal cell carcinoma. Cancer Immunol Immunother. 2008 Oct;57(10):1439-49.
  180. 180. Konforte D, Simard N, Paige CJ. IL-21: an executor of B cell fate. J Immunol. 2009 Feb 15;182(4):1781-7.
  181. 181. Wang HJ, Xiang LX, Shao JZ, Jia S. Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis. Cytokine. 2006 Aug;35(3-4):126-34.
  182. 182. Wang T, Diaz-Rosales P, Costa MM, Campbell S, Snow M, Collet B, et al. Functional characterization of a nonmammalian IL-21: rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-gamma, IL-10, and IL-22. J Immunol. 2011 Jan 15;186(2):708-21.
  183. 183. Lutfalla G, Roest Crollius H, Stange-Thomann N, Jaillon O, Mogensen K, Monneron D. Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: the class II cytokine receptors and their ligands in mammals and fish. BMC Genomics. 2003 Jul 17;4(1):29.
  184. 184. Fickenscher H, Hor S, Kupers H, Knappe A, Wittmann S, Sticht H. The interleukin-10 family of cytokines. Trends Immunol. 2002 Feb;23(2):89-96.
  185. 185. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683-765.
  186. 186. Zou J, Clark MS, Secombes CJ. Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes. Immunogenetics. 2003 Aug;55(5):325-35.
  187. 187. Savan R, Igawa D, Sakai M. Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L. Eur J Biochem. 2003 Dec;270(23):4647-54.
  188. 188. Zhang DC, Shao YQ, Huang YQ, Jiang SG. Cloning, characterization and expression analysis of interleukin-10 from the zebrafish (Danio rerion). J Biochem Mol Biol. 2005 Sep 30;38(5):571-6.
  189. 189. Inoue Y, Kamota S, Ito K, Yoshiura Y, Ototake M, Moritomo T, et al. Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) interleukin-10 cDNAs. Fish Shellfish Immunol. 2005 Apr;18(4):335-44.
  190. 190. Buonocore F, Randelli E, Bird S, Secombes C, Facchiano A, Constantini S, et al. Interleukin-10 expression by real-time PCR and homology modelling analysis in the European sea bass (Dicentrarchus Labrax L.). Aquaculture. 2007;270(1-4):512 - 22.
  191. 191. Pinto RD, Nascimento DS, Reis MI, do Vale A, Dos Santos NM. Molecular characterization, 3D modelling and expression analysis of sea bass (Dicentrarchus labrax L.) interleukin-10. Mol Immunol. 2007 Mar;44(8):2056-65.
  192. 192. Zhang Z, Swain T, Bogwald J, Dalmo RA, Kumari J. Bath immunostimulation of rainbow trout (Oncorhynchus mykiss) fry induces enhancement of inflammatory cytokine transcripts, while repeated bath induce no changes. Fish Shellfish Immunol. 2009 May;26(5):677-84.
  193. 193. Wegenka UM. IL-20: biological functions mediated through two types of receptor complexes. Cytokine Growth Factor Rev. 2010 Oct;21(5):353-63.
  194. 194. Stein C, Caccamo M, Laird G, Leptin M. Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish. Genome Biol. 2007;8(11):R251.
  195. 195. Wang T, Diaz-Rosales P, Martin SA, Secombes CJ. Cloning of a novel interleukin (IL)-20-like gene in rainbow trout Oncorhynchus mykiss gives an insight into the evolution of the IL-10 family. Dev Comp Immunol. 2010 Feb;34(2):158-67.
  196. 196. Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med. 2006 Oct 2;203(10):2271-9.
  197. 197. Cupedo T, Crellin NK, Papazian N, Rombouts EJ, Weijer K, Grogan JL, et al. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells. Nat Immunol. 2009 Jan;10(1):66-74.
  198. 198. Duhen T, Geiger R, Jarrossay D, Lanzavecchia A, Sallusto F. Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol. 2009 Aug;10(8):857-63.
  199. 199. Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest. 2009 Dec;119(12):3573-85.
  200. 200. Fujita H, Nograles KE, Kikuchi T, Gonzalez J, Carucci JA, Krueger JG. Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production. Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21795-800.
  201. 201. Wolk K, Haugen HS, Xu W, Witte E, Waggie K, Anderson M, et al. IL-22 and IL-20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-gamma are not. J Mol Med (Berl). 2009 May;87(5):523-36.
  202. 202. Feinen B, Russell MW. Contrasting Roles of IL-22 and IL-17 in Murine Genital Tract Infection by Neisseria gonorrhoeae. Front Immunol. 2012;3:11.
  203. 203. Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity. 2004 Aug;21(2):241-54.
  204. 204. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, Pociask DA, et al. IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med. 2008 Mar;14(3):275-81.
  205. 205. Andoh A, Zhang Z, Inatomi O, Fujino S, Deguchi Y, Araki Y, et al. Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology. 2005 Sep;129(3):969-84.
  206. 206. Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med. 2008 Mar;14(3):282-9.
  207. 207. Bird L. Mucosal immunology: IL-22 keeps commensals in their place. Nat Rev Immunol. 2012 Jul 6.
  208. 208. Wolk K, Kunz S, Asadullah K, Sabat R. Cutting edge: immune cells as sources and targets of the IL-10 family members? J Immunol. 2002 Jun 1;168(11):5397-402.
  209. 209. Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol. 2007 Sep;8(9):950-7.
  210. 210. Manel N, Unutmaz D, Littman DR. The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat. Nat Immunol. 2008 Jun;9(6):641-9.
  211. 211. Pene J, Chevalier S, Preisser L, Venereau E, Guilleux MH, Ghannam S, et al. Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes. J Immunol. 2008 Jun 1;180(11):7423-30.
  212. 212. Cella M, Fuchs A, Vermi W, Facchetti F, Otero K, Lennerz JK, et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature. 2009 Feb 5;457(7230):722-5.
  213. 213. Hughes T, Becknell B, McClory S, Briercheck E, Freud AG, Zhang X, et al. Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH 17 cytokine interleukin-22. Blood. 2009 Apr 23;113(17):4008-10.
  214. 214. Donnelly RP, Sheikh F, Dickensheets H, Savan R, Young HA, Walter MR. Interleukin-26: an IL-10-related cytokine produced by Th17 cells. Cytokine Growth Factor Rev. 2010 Oct;21(5):393-401.
  215. 215. Zou J, Yoshiura Y, Dijkstra JM, Sakai M, Ototake M, Secombes C. Identification of an interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol. 2004 Oct;17(4):403-9.
  216. 216. Igawa D, Sakai M, Savan R. An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. Mol Immunol. 2006 Mar;43(7):999-1009.
  217. 217. Corripio-Miyar Y, Zou J, Richmond H, Secombes CJ. Identification of interleukin-22 in gadoids and examination of its expression level in vaccinated fish. Mol Immunol. 2009 Jun;46(10):2098-106.
  218. 218. Kolls JK. Th17 cells in mucosal immunity and tissue inflammation. Semin Immunopathol. 2010 Mar;32(1):1-2.
  219. 219. Haugarvoll E, Bjerkas I, Nowak BF, Hordvik I, Koppang EO. Identification and characterization of a novel intraepithelial lymphoid tissue in the gills of Atlantic salmon. J Anat. 2008 Aug;213(2):202-9.
  220. 220. Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev. 2003 Apr;14(2):155-74.
  221. 221. Yao Z, Timour M, Painter S, Fanslow W, Spriggs M. Complete nucleotide sequence of the mouse CTLA8 gene. Gene. 1996 Feb 12;168(2):223-5.
  222. 222. Li H, Chen J, Huang A, Stinson J, Heldens S, Foster J, et al. Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family. Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):773-8.
  223. 223. Shi Y, Ullrich SJ, Zhang J, Connolly K, Grzegorzewski KJ, Barber MC, et al. A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity. J Biol Chem. 2000 Jun 23;275(25):19167-76.
  224. 224. Hymowitz SG, Filvaroff EH, Yin JP, Lee J, Cai L, Risser P, et al. IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBO J. 2001 Oct 1;20(19):5332-41.
  225. 225. Lee J, Ho WH, Maruoka M, Corpuz RT, Baldwin DT, Foster JS, et al. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. J Biol Chem. 2001 Jan 12;276(2):1660-4.
  226. 226. Starnes T, Robertson MJ, Sledge G, Kelich S, Nakshatri H, Broxmeyer HE, et al. Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cell cytokine production. J Immunol. 2001 Oct 15;167(8):4137-40.
  227. 227. Starnes T, Broxmeyer HE, Robertson MJ, Hromas R. Cutting edge: IL-17D, a novel member of the IL-17 family, stimulates cytokine production and inhibits hemopoiesis. J Immunol. 2002 Jul 15;169(2):642-6.
  228. 228. Gunimaladevi I, Savan R, Sakai M. Identification, cloning and characterization of interleukin-17 and its family from zebrafish. Fish Shellfish Immunol. 2006 Oct;21(4):393-403.
  229. 229. Wang T, Martin SA, Secombes CJ. Two interleukin-17C-like genes exist in rainbow trout Oncorhynchus mykiss that are differentially expressed and modulated. Dev Comp Immunol. 2010 May;34(5):491-500.
  230. 230. Kumari J, Larsen AN, Bogwald J, Dalmo RA. Interleukin-17D in Atlantic salmon (Salmo salar): molecular characterization, 3D modelling and promoter analysis. Fish Shellfish Immunol. 2009 Nov;27(5):647-59.
  231. 231. Korenaga H, Kono T, Sakai M. Isolation of seven IL-17 family genes from the Japanese pufferfish Takifugu rubripes. Fish Shellfish Immunol. 2010 May-Jun;28(5-6):809-18.
  232. 232. Kono T, Korenaga H, Sakai M. Genomics of fish IL-17 ligand and receptors: a review. Fish Shellfish Immunol. 2011 Nov;31(5):635-43.
  233. 233. Huang D, Cancilla MR, Morahan G. Complete primary structure, chromosomal localisation, and definition of polymorphisms of the gene encoding the human interleukin-12 p40 subunit. Genes Immun. 2000 Dec;1(8):515-20.
  234. 234. Yoshiura Y, Kiryu I, Fujiwara A, Suetake H, Suzuki Y, Nakanishi T, et al. Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits. Immunogenetics. 2003 Aug;55(5):296-306.
  235. 235. Nascimento DS, do Vale A, Tomas AM, Zou J, Secombes CJ, dos Santos NM. Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits. Mol Immunol. 2007 Mar;44(9):2277-91.
  236. 236. Li MO, Flavell RA. TGF-beta: a master of all T cell trades. Cell. 2008 Aug 8;134(3):392-404.
  237. 237. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol. 2006;24:99-146.
  238. 238. Saxena V, Lienesch DW, Zhou M, Bommireddy R, Azhar M, Doetschman T, et al. Dual roles of immunoregulatory cytokine TGF-beta in the pathogenesis of autoimmunity-mediated organ damage. J Immunol. 2008 Feb 1;180(3):1903-12.
  239. 239. Zhang L, Yi H, Xia XP, Zhao Y. Transforming growth factor-beta: an important role in CD4+CD25+ regulatory T cells and immune tolerance. Autoimmunity. 2006 Jun;39(4):269-76.
  240. 240. Li B, Samanta A, Song X, Furuuchi K, Iacono KT, Kennedy S, et al. FOXP3 ensembles in T-cell regulation. Immunol Rev. 2006 Aug;212:99-113.
  241. 241. Wan YY, Flavell RA. 'Yin-Yang' functions of transforming growth factor-beta and T regulatory cells in immune regulation. Immunol Rev. 2007 Dec;220:199-213.
  242. 242. Haddad G, Hanington PC, Wilson EC, Grayfer L, Belosevic M. Molecular and functional characterization of goldfish (Carassius auratus L.) transforming growth factor beta. Dev Comp Immunol. 2008;32(6):654-63.
  243. 243. Kadowaki T, Yasui Y, Takahashi Y, Kohchi C, Soma G, Inagawa H. Comparative immunological analysis of innate immunity activation after oral administration of wheat fermented extract to teleost fish. Anticancer Res. 2009 Nov;29(11):4871-7.
  244. 244. Yang M, Wang Y, Wang X, Chen C, Zhou H. Characterization of grass carp (Ctenopharyngodon idellus) Foxp1a/1b/2: evidence for their involvement in the activation of peripheral blood lymphocyte subpopulations. Fish Shellfish Immunol. 2010 Feb;28(2):289-95.
  245. 245. Cai Z, Gao C, Li L, Xing K. Bipolar properties of red seabream (Pagrus major) transforming growth factor-beta in induction of the leucocytes migration. Fish Shellfish Immunol. 2010 Apr;28(4):695-700.
  246. 246. Yang M, Wang X, Chen D, Wang Y, Zhang A, Zhou H. TGF-beta1 exerts opposing effects on grass carp leukocytes: implication in teleost immunity, receptor signaling and potential self-regulatory mechanisms. PLoS One. 2012;7(4):e35011.
  247. 247. Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev. 2001 Oct;14(4):778-809, table of contents.
  248. 248. Robertsen B. The interferon system of teleost fish. Fish Shellfish Immunol. 2006 Feb;20(2):172-91.
  249. 249. Bergan V, Steinsvik S, Xu H, Kileng O, Robertsen B. Promoters of type I interferon genes from Atlantic salmon contain two main regulatory regions. FEBS J. 2006 Sep;273(17):3893-906.
  250. 250. Kileng O, Brundtland MI, Robertsen B. Infectious salmon anemia virus is a powerful inducer of key genes of the type I interferon system of Atlantic salmon, but is not inhibited by interferon. Fish Shellfish Immunol. 2007 Aug;23(2):378-89.
  251. 251. Robertsen B, Bergan V, Rokenes T, Larsen R, Albuquerque A. Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity. J Interferon Cytokine Res. 2003 Oct;23(10):601-12.
  252. 252. Rokenes TP, Larsen R, Robertsen B. Atlantic salmon ISG15: Expression and conjugation to cellular proteins in response to interferon, double-stranded RNA and virus infections. Mol Immunol. 2007 Feb;44(5):950-9.
  253. 253. Zou J, Tafalla C, Truckle J, Secombes CJ. Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates. J Immunol. 2007 Sep 15;179(6):3859-71.
  254. 254. Robertsen B. Expression of interferon and interferon-induced genes in salmonids in response to virus infection, interferon-inducing compounds and vaccination. Fish Shellfish Immunol. 2008 Oct;25(4):351-7.
  255. 255. Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chinchar VG. Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics. 2006 Feb;58(1):70-80.
  256. 256. Stolte EH, Savelkoul HF, Wiegertjes G, Flik G, Lidy Verburg-van Kemenade BM. Differential expression of two interferon-gamma genes in common carp (Cyprinus carpio L.). Dev Comp Immunol. 2008;32(12):1467-81.
  257. 257. Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, Bols N, et al. Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J Immunol. 2005 Aug 15;175(4):2484-94.
  258. 258. Grayfer L, Garcia EG, Belosevic M. Comparison of macrophage antimicrobial responses induced by type II interferons of the goldfish (Carassius auratus L.). J Biol Chem. 2010 Jul 30;285(31):23537-47.
  259. 259. Arts JA, Tijhaar EJ, Chadzinska M, Savelkoul HF, Verburg-van Kemenade BM. Functional analysis of carp interferon-gamma: evolutionary conservation of classical phagocyte activation. Fish Shellfish Immunol. 2010 Nov;29(5):793-802.
  260. 260. Sun B, Skjaeveland I, Svingerud T, Zou J, Jorgensen J, Robertsen B. Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon. J Virol. 2011 Sep;85(17):9188-98.
  261. 261. Duguid JR, Dinauer MC. Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucleic Acids Res. 1990 May 11;18(9):2789-92.
  262. 262. Hara E, Kato T, Nakada S, Sekiya S, Oda K. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res. 1991 Dec;19(25):7097-104.
  263. 263. Hedrick SM, Cohen DI, Nielsen EA, Davis MM. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984 Mar 8-14;308(5955):149-53.
  264. 264. Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science. 1993 Feb 12;259(5097):946-51.
  265. 265. Hubank M, Schatz DG. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994 Dec 25;22(25):5640-8.
  266. 266. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967-71.
  267. 267. Welsh J, Chada K, Dalal SS, Cheng R, Ralph D, McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992 Oct 11;20(19):4965-70.
  268. 268. Sompayrac L, Jane S, Burn TC, Tenen DG, Danna KJ. Overcoming limitations of the mRNA differential display technique. Nucleic Acids Res. 1995 Nov 25;23(22):4738-9.
  269. 269. Bertioli DJ, Schlichter UH, Adams MJ, Burrows PR, Steinbiss HH, Antoniw JF. An analysis of differential display shows a strong bias towards high copy number mRNAs. Nucleic Acids Res. 1995 Nov 11;23(21):4520-3.
  270. 270. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 1995 Mar 25;23(6):1087-8.
  271. 271. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6025-30.
  272. 272. Wang L, Wu X. Identification of differentially expressed genes in lipopolysaccharide-stimulated yellow grouper Epinephelus awoara spleen. Fish Shellfish Immunol. 2007 Aug;23(2):354-63.
  273. 273. Xia JH, Yue GH. Identification and analysis of immune-related transcriptome in Asian seabass Lates calcarifer. BMC Genomics. 2010;11:356.
  274. 274. Feng CY, Johnson SC, Hori TS, Rise M, Hall JR, Gamperl AK, et al. Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida. Physiol Genomics. 2009 May 13;37(3):149-63.
  275. 275. Tsoi SC, Ewart KV, Penny S, Melville K, Liebscher RS, Brown LL, et al. Identification of immune-relevant genes from atlantic salmon using suppression subtractive hybridization. Mar Biotechnol (NY). 2004 May-Jun;6(3):199-214.
  276. 276. Li CH, Chen J, Shi YH, Lu XJ. Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection. Fish Shellfish Immunol. 2011 Sep;31(3):500-6.
  277. 277. Matsuyama T, Fujiwara A, Takano T, Nakayasu C. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection. Fish Shellfish Immunol. 2011 Oct;31(4):524-32.
  278. 278. Bo J, Giesy JP, Ye R, Wang KJ, Lee JS, Au DW. Identification of differentially expressed genes and quantitative expression of complement genes in the liver of marine medaka Oryzias melastigma challenged with Vibrio parahaemolyticus. Comp Biochem Physiol Part D Genomics Proteomics. 2012 Jun;7(2):191-200.
  279. 279. Purcell MK, Smith KD, Hood L, Winton JR, Roach JC. Conservation of Toll-Like Receptor Signaling Pathways in Teleost Fish. Comp Biochem Physiol Part D Genomics Proteomics. 2006 Mar;1(1):77-88.
  280. 280. Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol. 2007 Mar;7(3):179-90.
  281. 281. Bayne CJ, Gerwick L. The acute phase response and innate immunity of fish. Dev Comp Immunol. 2001 Oct-Dec;25(8-9):725-43.
  282. 282. Bayne CJ, Gerwick L, Fujiki K, Nakao M, Yano T. Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization. Dev Comp Immunol. 2001 Apr;25(3):205-17.
  283. 283. Holland MC, Lambris JD. The complement system in teleosts. Fish Shellfish Immunol. 2002 May;12(5):399-420.
  284. 284. Rise ML, Hall J, Rise M, Hori T, Gamperl AK, Kimball J, et al. Functional genomic analysis of the response of Atlantic cod (Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC). Dev Comp Immunol. 2008;32(8):916-31.
  285. 285. He W, Yinlt ZX, Li Y, Huo WL, Guan HJ, Weng SP, et al. Differential gene expression profile in spleen of mandarin fish Siniperca chuatsi infected with ISKNV, derived from suppression subtractive hybridization. Dis Aquat Organ. 2006 Dec 14;73(2):113-22.
  286. 286. Dios S, Poisa-Beiro L, Figueras A, Novoa B. Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus. Mol Immunol. 2007 Mar;44(9):2195-204.
  287. 287. Poisa-Beiro L, Dios S, Ahmed H, Vasta GR, Martinez-Lopez A, Estepa A, et al. Nodavirus infection of sea bass (Dicentrarchus labrax) induces up-regulation of galectin-1 expression with potential anti-inflammatory activity. J Immunol. 2009 Nov 15;183(10):6600-11.
  288. 288. Rise ML, Hall JR, Rise M, Hori TS, Browne MJ, Gamperl AK, et al. Impact of asymptomatic nodavirus carrier state and intraperitoneal viral mimic injection on brain transcript expression in Atlantic cod (Gadus morhua). Physiol Genomics. 2010 Jul 7;42(2):266-80.
  289. 289. Xu D, Wei J, Cui H, Gong J, Yan Y, Lai R, et al. Differential profiles of gene expression in grouper Epinephelus coioides, infected with Singapore grouper iridovirus, revealed by suppression subtractive hybridization and DNA microarray. J Fish Biol. 2010 Aug;77(2):341-60.
  290. 290. Fisicaro N, Aminian A, Hinchliffe SJ, Morgan BP, Pearse MJ, D'Apice AJ, et al. The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement. Transplantation. 2000 Sep 27;70(6):963-8.
  291. 291. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
  292. 292. Tasumi S, Ohira T, Kawazoe I, Suetake H, Suzuki Y, Aida K. Primary structure and characteristics of a lectin from skin mucus of the Japanese eel Anguilla japonica. J Biol Chem. 2002 Jul 26;277(30):27305-11.
  293. 293. Tsutsui S, Iwamoto K, Nakamura O, Watanabe T. Yeast-binding C-type lectin with opsonic activity from conger eel (Conger myriaster) skin mucus. Mol Immunol. 2007 Feb;44(5):691-702.
  294. 294. Zhang H, Robison B, Thorgaard GH, Ristow SS. Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss). Biochim Biophys Acta. 2000 Nov 15;1494(1-2):14-22.
  295. 295. Honda K, Yanai H, Takaoka A, Taniguchi T. Regulation of the type I IFN induction: a current view. Int Immunol. 2005 Nov;17(11):1367-78.
  296. 296. Krasnov A, Timmerhaus G, Afanasyev S, Jorgensen SM. Development and assessment of oligonucleotide microarrays for Atlantic salmon (Salmo salar L.). Comp Biochem Physiol Part D Genomics Proteomics. 2011 Mar;6(1):31-8.
  297. 297. von Schalburg KR, Rise ML, Cooper GA, Brown GD, Gibbs AR, Nelson CC, et al. Fish and chips: various methodologies demonstrate utility of a 16,006-gene salmonid microarray. BMC Genomics. 2005;6:126.
  298. 298. Koop BF, von Schalburg KR, Leong J, Walker N, Lieph R, Cooper GA, et al. A salmonid EST genomic study: genes, duplications, phylogeny and microarrays. BMC Genomics. 2008;9:545.
  299. 299. Krasnov A, Koskinen H, Pehkonen P, Rexroad CE, 3rd, Afanasyev S, Molsa H. Gene expression in the brain and kidney of rainbow trout in response to handling stress. BMC Genomics. 2005;6:3.
  300. 300. Schiotz BL, Jorgensen SM, Rexroad C, Gjoen T, Krasnov A. Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells. Virus Res. 2008 Sep;136(1-2):65-74.
  301. 301. Kurobe T, Yasuike M, Kimura T, Hirono I, Aoki T. Expression profiling of immune-related genes from Japanese flounder Paralichthys olivaceus kidney cells using cDNA microarrays. Dev Comp Immunol. 2005;29(6):515-23.
  302. 302. Williams TD, Diab AM, George SG, Godfrey RE, Sabine V, Conesa A, et al. Development of the GENIPOL European flounder (Platichthys flesus) microarray and determination of temporal transcriptional responses to cadmium at low dose. Environ Sci Technol. 2006 Oct 15;40(20):6479-88.
  303. 303. Park KC, Osborne JA, Montes A, Dios S, Nerland AH, Novoa B, et al. Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays. Fish Shellfish Immunol. 2009 Jan;26(1):91-108.
  304. 304. Cerda J, Mercade J, Lozano JJ, Manchado M, Tingaud-Sequeira A, Astola A, et al. Genomic resources for a commercial flatfish, the Senegalese sole (Solea senegalensis): EST sequencing, oligo microarray design, and development of the Soleamold bioinformatic platform. BMC Genomics. 2008;9:508.
  305. 305. Skugor S, Jorgensen SM, Gjerde B, Krasnov A. Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis. BMC Genomics. 2009;10:503.
  306. 306. Tilton SC, Gerwick LG, Hendricks JD, Rosato CS, Corley-Smith G, Givan SA, et al. Use of a rainbow trout oligonucleotide microarray to determine transcriptional patterns in aflatoxin B1-induced hepatocellular carcinoma compared to adjacent liver. Toxicol Sci. 2005 Dec;88(2):319-30.
  307. 307. Millan A, Gomez-Tato A, Fernandez C, Pardo BG, Alvarez-Dios JA, Calaza M, et al. Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues. Mar Biotechnol (NY). 2010 Aug;12(4):452-65.
  308. 308. Peatman E, Baoprasertkul P, Terhune J, Xu P, Nandi S, Kucuktas H, et al. Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) after infection with a Gram-negative bacterium. Dev Comp Immunol. 2007;31(11):1183-96.
  309. 309. MacKenzie S, Balasch JC, Novoa B, Ribas L, Roher N, Krasnov A, et al. Comparative analysis of the acute response of the trout, O. mykiss, head kidney to in vivo challenge with virulent and attenuated infectious hematopoietic necrosis virus and LPS-induced inflammation. BMC Genomics. 2008;9:141.
  310. 310. Prieto-Alamo MJ, Abril N, Osuna-Jimenez I, Pueyo C. Solea senegalensis genes responding to lipopolysaccharide and copper sulphate challenges: large-scale identification by suppression subtractive hybridization and absolute quantification of transcriptional profiles by real-time RT-PCR. Aquat Toxicol. 2009 Mar 9;91(4):312-9.
  311. 311. Li RW, Waldbieser GC. Production and utilization of a high-density oligonucleotide microarray in channel catfish, Ictalurus punctatus. BMC Genomics. 2006;7:134.
  312. 312. Fittipaldi N, Gottschalk M, Vanier G, Daigle F, Harel J. Use of selective capture of transcribed sequences to identify genes preferentially expressed by Streptococcus suis upon interaction with porcine brain microvascular endothelial cells. Appl Environ Microbiol. 2007 Jul;73(13):4359-64.
  313. 313. Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ. Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis. 2007 Mar;7(3):201-9.
  314. 314. Rise ML, von Schalburg KR, Brown GD, Mawer MA, Devlin RH, Kuipers N, et al. Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics. Genome Res. 2004 Mar;14(3):478-90.
  315. 315. Matsumoto M, Tanaka T, Kaisho T, Sanjo H, Copeland NG, Gilbert DJ, et al. A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages. J Immunol. 1999 Nov 1;163(9):5039-48.
  316. 316. MacKenzie S, Iliev D, Liarte C, Koskinen H, Planas JV, Goetz FW, et al. Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol. Mol Immunol. 2006 Mar;43(9):1340-8.
  317. 317. Martin SA, Blaney SC, Houlihan DF, Secombes CJ. Transcriptome response following administration of a live bacterial vaccine in Atlantic salmon (Salmo salar). Mol Immunol. 2006 Apr;43(11):1900-11.
  318. 318. Ching B, Jamieson S, Heath JW, Heath DD, Hubberstey A. Transcriptional differences between triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) during live Vibrio anguillarum challenge. Heredity (Edinb). 2010 Feb;104(2):224-34.
  319. 319. Dumrongphol Y, Hirota T, Kondo H, Aoki T, Hirono I. Identification of novel genes in Japanese flounder (Paralichthys olivaceus) head kidney up-regulated after vaccination with Streptococcus iniae formalin-killed cells. Fish Shellfish Immunol. 2009 Jan;26(1):197-200.
  320. 320. Gerwick L, Corley-Smith G, Bayne CJ. Gene transcript changes in individual rainbow trout livers following an inflammatory stimulus. Fish Shellfish Immunol. 2007 Mar;22(3):157-71.
  321. 321. Meijer AH, Verbeek FJ, Salas-Vidal E, Corredor-Adamez M, Bussman J, van der Sar AM, et al. Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection. Mol Immunol. 2005 Jun;42(10):1185-203.
  322. 322. van der Sar AM, Spaink HP, Zakrzewska A, Bitter W, Meijer AH. Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection and the role of innate and adaptive immune components. Mol Immunol. 2009 Jul;46(11-12):2317-32.
  323. 323. Purcell MK, Nichols KM, Winton JR, Kurath G, Thorgaard GH, Wheeler P, et al. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus. Mol Immunol. 2006 May;43(13):2089-106.
  324. 324. Byon JY, Ohira T, Hirono I, Aoki T. Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination. Fish Shellfish Immunol. 2005 Feb;18(2):135-47.
  325. 325. Jorgensen SM, Afanasyev S, Krasnov A. Gene expression analyses in Atlantic salmon challenged with infectious salmon anemia virus reveal differences between individuals with early, intermediate and late mortality. BMC Genomics. 2008;9:179.
  326. 326. Encinas P, Rodriguez-Milla MA, Novoa B, Estepa A, Figueras A, Coll J. Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach. BMC Genomics. 2010;11:518.

Written By

Sebastián Reyes-Cerpa, Kevin Maisey, Felipe Reyes-López, Daniela Toro-Ascuy, Ana María Sandino and Mónica Imarai

Submitted: March 22nd, 2012 Published: November 21st, 2012