Fish Cytokines and Immune Response

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 for‐ eign structures and trigger different molecular and cellular mechanisms for antigen elimina‐ tion. The immune response is critical to all individuals; therefore numerous changes have taken place during evolution to generate variability and specialization, although the im‐ mune 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 coincid‐ ed with the development of a novel type of immune response. Apparently, vertebrates in‐ herited innate immunity from their invertebrate ancestors [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 genera-tive 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][13][14][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].

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].

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][25][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][35][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][39][40][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]. cytes, 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 Creactive 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.

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] . 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].

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].

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][86][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][91][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].

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][103][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][107][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][118][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].

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 CX 3 C (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].

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].

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].

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 New Advances and Contributions to Fish Biology 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][134][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].

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 con-taining 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].

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][165][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].

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][169][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].

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][173][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].

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 Fish Cytokines and Immune Response http://dx.doi.org/10.5772/53504 different type-2 cytokine receptors. This leads to diverse biological effects through the activation of signal transducer and activator of transcription (STAT) factors [184].

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].

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].

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][210][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].

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][223][224][225][226][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].

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].

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 TGFβ 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 downregulates 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 regula-tor in teleost, but also indicate that TGF-β1 may have retained similar functions in immunity during the evolution of vertebrates [246].

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][250][251][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][256][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][258][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]

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.

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][262][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.
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][282][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]. 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][287][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][292][293][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 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].

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 stud-ies 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).

Studies with bacterial pathogens, PAMPs and cytokine network interactions
One of the most commonly studied bacterial pathogens is Aeromonas salmonicida, a gramnegative 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 (GENE-FILTERS 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 Fish Cytokines and Immune Response http://dx.doi.org/10.5772/53504 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.  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 nonlethal 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 descrip-tion 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.

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.

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 immuni-Fish Cytokines and Immune Response http://dx.doi.org/10.5772/53504 31 ty. 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.  [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.