**3. Cytokine regulation of inflammatory responses**

The onset, progression and resolution of the inflammatory responses are tightly regultaed through soluble mediators (cytokines, monokines, chemokines) that orchestrate inflamma‐ tion. Pro-inflammatory cytokines such as TNFα, IFNγ, and IL-1β enhance antimicrobial functions of immune cells and facilitate the pathogen clearance, while anti-inflammatory cy‐ tokines such as TGFβ and IL-10 down-regulate inflammatory processes and skew cell func‐ tions towards tissue repair mechanisms. It is important to note that in addition to the hallmark cytokines discussed in this review, other cytokines such as chemokines and growth factors also participate in the regulation of an inflammatory response.

The last decade has yeilded significant advances in the understanding of inflammatory re‐ sponses of lower vertebrates, such as bony fish. The genes encoding hallmark cytokines have been identified and characterized in a number of fish species. Interestingly, many of these exhibit structural similarities and gene synteny organization comparable to their high‐ er vertebrate counterparts. Conversely, multiple isoforms of certain cytokines are present in distinct fish species.

#### **3.1. Tumor necrosis factor alpha (TNFα)**

**2. Antimicrobial responses of fish phagocytes**

60 New Advances and Contributions to Fish Biology

sponse [58, 59, 136, 171].

reviewed in references [2, 111, 126].

and NO production is described below.

**3. Cytokine regulation of inflammatory responses**

It is well established that fish phagocytes possess oxidative burst responses, comparable to those of mammals. Absence of readily available fish cytokines limited the early research of fish phagocytes to employing pathogen products and/or crude activated cell supernatants, presumed to contain hallmark "activating" agents. This fundemental work of fish phago‐ cyte-mediated inflammatory processes has been comprehensively reviewed [138]. Since then the specific genes encoding the components of the fish NADPH oxidase complex (Fig. 1) have been cloned in various fish species [13, 84, 119] and their expression correlated with reactive oxygen radical production [13, 63, 141]. The priming of the fish phagocyte ROI re‐ sponses by recombinant fish cytokines such as TNFα [67, 133, 217], IFNγ [62, 63, 215] and IL-1β [54, 97, 144] has also been reported. Similar to the mammalian monocyte/macrophage paradigm [22], fish monocytes have greater ability to generate reactive oxygen intermediates (ROI) following short stimulation [67, 136, 152], whereas mature fish macrophages require relatively prolonged immune stimulations to achieve comparable magnitudes of this re‐

Phagocytes (primarily mature macrophages) also produce microbicidal/tumoricidal reactive nitrogen intermediates in a stimulus-specific manner. This response, catalyzed by the indu‐ cible nitric oxide synthase enzyme (iNOS, Fig. 2), involves the conversion of arginine to cit‐ ruline and results in the production of nitric oxide (NO) and other products including nitrite, nitrate, and nitrosamines [86, 134, 184]. The NADPH oxidase produced superoxide

potent microbiacidal activity [35, 156, 191, 213]. The biology of the iNOS enzyme has been

The ability of fish phagocytes to produce microbicidal NOs has been well established (re‐ viewed in reference [138]). The iNOS gene transcript has been identified in several fish spe‐ cies [101, 102, 163, 200] and fish macrophages have been demonstrated to up-regulate iNOS expression and produce copious amounts of NO in response to a plethora of immune stimu‐ li [62-64, 66, 67, 85, 88, 140, 158, 180, 210]. The inflammatory cytokine regulation of fish iNOS

The onset, progression and resolution of the inflammatory responses are tightly regultaed through soluble mediators (cytokines, monokines, chemokines) that orchestrate inflamma‐ tion. Pro-inflammatory cytokines such as TNFα, IFNγ, and IL-1β enhance antimicrobial functions of immune cells and facilitate the pathogen clearance, while anti-inflammatory cy‐ tokines such as TGFβ and IL-10 down-regulate inflammatory processes and skew cell func‐ tions towards tissue repair mechanisms. It is important to note that in addition to the hallmark cytokines discussed in this review, other cytokines such as chemokines and

growth factors also participate in the regulation of an inflammatory response.

] that also has

anion may also react with NO to form the peroxynitrite intermediate [ONOO<sup>−</sup>

Tumor necrosis factor alpha is a central inflammatory mediator, initially identifed as a se‐ rum component capable of eliciting "hemorrhagic necrosis" of certain tumors [20]. Since dis‐ covery, TNFα has been found to be produced by many cell-types and confer an increadible range of immune processess [44, 203, 209]. In the context of inflammatory responses, TNFα promotes the chemotaxis of neutrophils and monocytes/macrophages [123, 207], enhances their phagocitic capacity [95, 105, 194], primes ROI and NO reposnses [42, 135], chemoat‐ tracts fibroblasts [168] and elicits platelet activating factor production [19, 73, 104].

The mammaian TNFα functions as a 26 kDa type II trans-membrane protein as well as a 17 kDa soluble moiety, released by the TNFα cleaving metalloproteinase enzyme (TACE) mediated cleavage [98, 128, 148]. A homotrimerized TNFα (soluble or membrane bound) en‐ gages one of two cognate receptors, TNF-R1 or TNF-R2, which in turn trimerize around the ligand [7, 45]. Currently, there is no consensus as to the respective contribution of these re‐ ceptors to the biological effects caused by TNFα. Some evidence suggests that TNF-R1 prop‐ agates the signal from the soluble TNFα, while the membrane-bound TNFα acts exclusively through TNF-R2 [70]. Other evidence suggest that TNF-R1 is primarily involved in induc‐ tion of apoptosis while the TNF-R2 functions in proliferation and cell survival [129], while other contributions suggest cooperation between the two receptors [204]. The prevailing theory proposes that TNF-R1 confers signal propagation, while TNF-R2 binds and redistrib‐ utes TNFα to TNF-R1 in a process coined "ligand passing" [28, 43, 197]. Despite this, more recent literature suggests that TNF-R2 is directly involved in many inflammatory processes including the activation of T lymphocytes [93, 94], stimulation of myofibroblasts [190], as well as tumor suppression [212]. The TNF-R1 and TNF-R2 utilize largely non-overlapping signaling mechanisms, relying on recruitment of numerous downstream signaling mole‐ cules, the relative abundance of which ultimately dictates signaling outcomes (for a current review of TNFα signal transduction see recent review [143]). It is likely that the roles of re‐ spective TNF receptors in the biological outcomes of TNFα stimulation are cell type and cell activation state dependent.

#### *3.1.1. Identification of TNFα in fish*

Presence of an endogenous bony fish TNF system was first suggested in the early 1990s where the human recombinant TNFα elicited ROI production in trout leukocytes while ad‐ ministration of a monoclonal anti-TNF-R1 antibody blocked this response [75, 87]. Hirono *et al.* [76] identified and characterized the first cDNA transcript encoding a Japanese flounder TNFα, which had only 20-30% amino acid identity with the mammalian TNFs, but had very similar intron/exon organization. The expression of this flounder TNFα gene increased in PBLs following LPS, ConA or PMA stimulation, suggesting a conserved role for this cyto‐ kine in fish inflammatory responses. Shortly thereafter, the rainbow trout TNFα was identi‐ fied, shown to be constitutively expressed in the gill and kidney tissues, and upregulated in LPS-stimulated or IL-1β-treated kidney leukocytes and in the trout macrophage cell line, RTS11 [103]. The catfish TNFα was also constitutively expressed in healthy fish and several catfish immune cell lines including macrophage and T cell lines, but not in B cell or fibro‐ blast cell lines [218]. Notably, all fish TNFα proteins possess the TNF family signature, [LV] x-[LIVM]-x3-G-[LIVMF]-Y-[LIMVMFY]2-x2-[QEKHL] [103], underlining the evolutionary conservation of this cytokine.

more, the mammalian p75/NTR neurotrophin receptor, a member of the TNF superfamily of proteins with many structural similarities to teleost and mammalian TNF-R1, binds to its

Cytokine Regulation of Teleost Inflammatory Responses

http://dx.doi.org/10.5772/53505

63

By examining the TNF system in lower vertebrates such as teleost fish, we can gain insight into the evolutionary origins of our own immune systems and the selective pressures that shaped them. As in mammals, TNFα appears to be central to the regulation of inflammatory responses of bony fish. Further examination of biological effects of this molecule using dif‐ ferent lineages of fish immune cells will yield a more concrete understanding of this system

The first functional characterization of a fish TNFα was reported in 2003 when Zou et al. [217] demonstrated that recombinant trout TNFα1 and TNFα2 (isoforms) both induced the IL1β, TNFα1, TNFα2, IL-8 and COX-2 gene expression in primary kidney leukocytes and in the RTS11 trout macrophage cell line [217]. These recombinant TNFα isoforms also elicited dose-dependent chemotaxis of trout kidney leukocytes and phagocytosis of yeast particles. SDS-PAGE analysis of the recombinant TNFα1 and TNFα2 suggested that both of the re‐ combinant molecules existed in monomeric, dimeric and trimeric states. Together, these

Intraperitoneal administration of a mature recombinant (cleaved) form of gilthead sea bream TNFα to fish resulted in rapid recruitment of phagocytic granulocytes to the sites of injection, granulopoeisis and the priming for enhanced ROI of peritoneal and primary kid‐ ney leukocytes [56]. Intriguingly, size exclusion chromatography of this recombinant sea bream TNFα suggested that this protein existed primarily in a dimeric state [56], in contrast

Subsequent reports indicated that the pro-inflammatory effects of sea bream and zebrafish TNFαs were not direct but resulted from the stimulation of endothelial cells [155]. When sea bream peritoneal and head kidney leukocytes were primed with recombinant TNFα (100 ng/mL) or bacterial DNA (*Vibrio anguillarum*, 50 μg/mL) for 16 hours, TNFα elicited signifi‐ cant ROI responses, albeit modest when compared to those induced by *V. anguillarum* DNA. Intraperitoneal injections of the recombinant TNFα resulted in increased expression of sev‐ eral pro-inflammatory genes of sea bream peritoneal leukocytes, while TNFα treatments of sea bream endocardium endothelial cells (EECs) *in vitro* also increased their immune gene expression. Notably, while the *in vitro* TNFα stimulation of EECs and macrophages resulted in substantially elevated pro-inflammatory gene expression in both cell types, the stimulat‐ ed macrophages exhibited significantly more robust transcriptional responses. Although this TNFα failed to chemoattract leukocytes, TNFα-stimulated EEC-conditioned medium and supernatants from TNFα-injected peritoneal exudate cells elicited leukocyte chemoat‐ traction. Furthermore, the zebrafish TNFα conferred neutrophil recruitment but also in‐ creased fish susceptibility to bacterial and viral infections. Accordingly, the authors of these studies proposed that unlike the mammalian cytokine, the fish TNFα elicits inflammatory

findings suggested conservation in the pro-inflammatory roles for teleost TNFα.

cognate NTR ligand as a dimer [26, 57].

*3.1.4. Inflammatory roles of the fish TNFα*

to the trimeric state of the mammalian TNFα.

functions indirectly, through non-immune cells.

in teleosts.

#### *3.1.2. Isoforms of TNFα in fish*

Trout were reported to possess an additional TNFα isoform [219] and the presence of multi‐ ple bony fish TNFs was confirmed in common carp, in which first two [162], and soon there‐ after a third carp TNFα isoform [167] were identified. Interestingly, polymorphisms in the gene encoding the carp TNFα2 isoform were linked to carp resistance to the protozoan para‐ site, *T. borreli* [162].

#### *3.1.3. TNFα receptors of fish*

Tumor necrosis factor alpha has now been identified in a number of fish species including flounder [76], trout [103, 114], catfish [218], carp [162, 167], sea bream [18, 56], tilapia [151], turbot [142], ayu [193], fugu [165], zebrafish [165], sea bass [133], goldfish [67] and tuna [89]. Additionally, novel TNF-like molecules with unique intron/exon organization have been identified in zebrafish and flounder [165]. Despite this, knowledge regarding the cognate re‐ ceptors for the teleost TNF proteins has been relatively limited.

A death domain containing TNF receptor was identified in zebrafish ovarian tissues and coined the ovarian TNF receptor (OTR) [12]. Predicted zebrafish TNF-R1 and TNF-R2 se‐ quences are in the NCBI database, with zebrafish TNF-R1 showing high sequence homology to the OTR. The goldfish TNF-R1 and TNF-R2 cDNAs were identified based on the zebra‐ fish sequences [61] and display conserved regions included cysteine residues and predicted docking sites for downstream signaling. Also, the goldfish TNF-R1 possesses a conserved death domain including the highly conserved motif (W/E)-X31-L-X2-W-X12-L-X3-L (with R in the W/E position) and six conserved or semi-conserved residues, crucial for the mammalian TNF-R1-mediated cytotoxicity [189]. Interestingly, while the mammalian TNF-R1 and TNF-R2 and the fish TNF-R2 contain four TNF homology domains (THD, defined by specific cys‐ teine residues), the fish TNF-R1 proteins exhibit only 3 full THDs [61].

Recombinant extracellular domains of goldfish TNF-R1 and TNF-R2 bound to both goldfish recombinant TNFα1 or TNFα2 in *in vitro* binding assays [61]. As with the sea bream TNFα [56], the goldfish recombinant TNFα ligands and receptors all adopted dimeric conforma‐ tions and interacted as dimers rather than trimers *in vitro*. Interetsingly, there have also been several reports indicateding dimerizion of the mammalian TNF-R1 [131, 132, 147]. Further‐ more, the mammalian p75/NTR neurotrophin receptor, a member of the TNF superfamily of proteins with many structural similarities to teleost and mammalian TNF-R1, binds to its cognate NTR ligand as a dimer [26, 57].

By examining the TNF system in lower vertebrates such as teleost fish, we can gain insight into the evolutionary origins of our own immune systems and the selective pressures that shaped them. As in mammals, TNFα appears to be central to the regulation of inflammatory responses of bony fish. Further examination of biological effects of this molecule using dif‐ ferent lineages of fish immune cells will yield a more concrete understanding of this system in teleosts.

#### *3.1.4. Inflammatory roles of the fish TNFα*

similar intron/exon organization. The expression of this flounder TNFα gene increased in PBLs following LPS, ConA or PMA stimulation, suggesting a conserved role for this cyto‐ kine in fish inflammatory responses. Shortly thereafter, the rainbow trout TNFα was identi‐ fied, shown to be constitutively expressed in the gill and kidney tissues, and upregulated in LPS-stimulated or IL-1β-treated kidney leukocytes and in the trout macrophage cell line, RTS11 [103]. The catfish TNFα was also constitutively expressed in healthy fish and several catfish immune cell lines including macrophage and T cell lines, but not in B cell or fibro‐ blast cell lines [218]. Notably, all fish TNFα proteins possess the TNF family signature, [LV] x-[LIVM]-x3-G-[LIVMF]-Y-[LIMVMFY]2-x2-[QEKHL] [103], underlining the evolutionary

Trout were reported to possess an additional TNFα isoform [219] and the presence of multi‐ ple bony fish TNFs was confirmed in common carp, in which first two [162], and soon there‐ after a third carp TNFα isoform [167] were identified. Interestingly, polymorphisms in the gene encoding the carp TNFα2 isoform were linked to carp resistance to the protozoan para‐

Tumor necrosis factor alpha has now been identified in a number of fish species including flounder [76], trout [103, 114], catfish [218], carp [162, 167], sea bream [18, 56], tilapia [151], turbot [142], ayu [193], fugu [165], zebrafish [165], sea bass [133], goldfish [67] and tuna [89]. Additionally, novel TNF-like molecules with unique intron/exon organization have been identified in zebrafish and flounder [165]. Despite this, knowledge regarding the cognate re‐

A death domain containing TNF receptor was identified in zebrafish ovarian tissues and coined the ovarian TNF receptor (OTR) [12]. Predicted zebrafish TNF-R1 and TNF-R2 se‐ quences are in the NCBI database, with zebrafish TNF-R1 showing high sequence homology to the OTR. The goldfish TNF-R1 and TNF-R2 cDNAs were identified based on the zebra‐ fish sequences [61] and display conserved regions included cysteine residues and predicted docking sites for downstream signaling. Also, the goldfish TNF-R1 possesses a conserved death domain including the highly conserved motif (W/E)-X31-L-X2-W-X12-L-X3-L (with R in the W/E position) and six conserved or semi-conserved residues, crucial for the mammalian TNF-R1-mediated cytotoxicity [189]. Interestingly, while the mammalian TNF-R1 and TNF-R2 and the fish TNF-R2 contain four TNF homology domains (THD, defined by specific cys‐

Recombinant extracellular domains of goldfish TNF-R1 and TNF-R2 bound to both goldfish recombinant TNFα1 or TNFα2 in *in vitro* binding assays [61]. As with the sea bream TNFα [56], the goldfish recombinant TNFα ligands and receptors all adopted dimeric conforma‐ tions and interacted as dimers rather than trimers *in vitro*. Interetsingly, there have also been several reports indicateding dimerizion of the mammalian TNF-R1 [131, 132, 147]. Further‐

ceptors for the teleost TNF proteins has been relatively limited.

teine residues), the fish TNF-R1 proteins exhibit only 3 full THDs [61].

conservation of this cytokine.

62 New Advances and Contributions to Fish Biology

*3.1.2. Isoforms of TNFα in fish*

site, *T. borreli* [162].

*3.1.3. TNFα receptors of fish*

The first functional characterization of a fish TNFα was reported in 2003 when Zou et al. [217] demonstrated that recombinant trout TNFα1 and TNFα2 (isoforms) both induced the IL1β, TNFα1, TNFα2, IL-8 and COX-2 gene expression in primary kidney leukocytes and in the RTS11 trout macrophage cell line [217]. These recombinant TNFα isoforms also elicited dose-dependent chemotaxis of trout kidney leukocytes and phagocytosis of yeast particles. SDS-PAGE analysis of the recombinant TNFα1 and TNFα2 suggested that both of the re‐ combinant molecules existed in monomeric, dimeric and trimeric states. Together, these findings suggested conservation in the pro-inflammatory roles for teleost TNFα.

Intraperitoneal administration of a mature recombinant (cleaved) form of gilthead sea bream TNFα to fish resulted in rapid recruitment of phagocytic granulocytes to the sites of injection, granulopoeisis and the priming for enhanced ROI of peritoneal and primary kid‐ ney leukocytes [56]. Intriguingly, size exclusion chromatography of this recombinant sea bream TNFα suggested that this protein existed primarily in a dimeric state [56], in contrast to the trimeric state of the mammalian TNFα.

Subsequent reports indicated that the pro-inflammatory effects of sea bream and zebrafish TNFαs were not direct but resulted from the stimulation of endothelial cells [155]. When sea bream peritoneal and head kidney leukocytes were primed with recombinant TNFα (100 ng/mL) or bacterial DNA (*Vibrio anguillarum*, 50 μg/mL) for 16 hours, TNFα elicited signifi‐ cant ROI responses, albeit modest when compared to those induced by *V. anguillarum* DNA. Intraperitoneal injections of the recombinant TNFα resulted in increased expression of sev‐ eral pro-inflammatory genes of sea bream peritoneal leukocytes, while TNFα treatments of sea bream endocardium endothelial cells (EECs) *in vitro* also increased their immune gene expression. Notably, while the *in vitro* TNFα stimulation of EECs and macrophages resulted in substantially elevated pro-inflammatory gene expression in both cell types, the stimulat‐ ed macrophages exhibited significantly more robust transcriptional responses. Although this TNFα failed to chemoattract leukocytes, TNFα-stimulated EEC-conditioned medium and supernatants from TNFα-injected peritoneal exudate cells elicited leukocyte chemoat‐ traction. Furthermore, the zebrafish TNFα conferred neutrophil recruitment but also in‐ creased fish susceptibility to bacterial and viral infections. Accordingly, the authors of these studies proposed that unlike the mammalian cytokine, the fish TNFα elicits inflammatory functions indirectly, through non-immune cells.

In similar studies, supernatants from recombinant carp TNFα-stimulated cardiac endothelial cells primed kidney phagocyte ROI responses while recombinant carp TNFα1 and 2 did not enhance phagocyte antimicrobial functions [155]. Notably, the recombinant forms of the ze‐ brafish, trout, sea bream and carp TNFα proteins exhibited lytic activity towards *Trypano‐ plasma borreli*, akin to the trypanolytic capacity of the mammalian TNFs [52], where the membrane form of the fish TNFα was thought to be responsible for these effects. In summa‐ tion of the above observations, Florenza *et al.* (2009) suggested that while the trypanolyitic roles of TNFα are evolutionarily conserved, the pro-inflammatory mechanisms elicited by this molecule were not [155].

ties after prolonged cvultivation [67, 136]. Specifically, ROI is primarily mediated by PKM-

Cytokine Regulation of Teleost Inflammatory Responses

http://dx.doi.org/10.5772/53505

65

Interferon gamma is a pleiotropic, pro-inflammatory and anti-viral cytokine, identified in the supernatants of PHA-activated lymphocyes for its unique anti-viral properties [205]. IFNγ is primarily produced by activated Th1 phenotype CD4+ cells [127], CD8+ cells [161] and natural killer (NK) cells [149] and is of central importance in host defense against intra‐ cellular and extracellular pathogens [8, 91, 99, 179, 182]. For example, IFNγ gene knock-out mice are incapable of controlling infections with *Leishmania major* [202], *Listeria monocyto‐ genes* [81], and *Mycobacterium* [32], underlining the importance of this cytokine in the regula‐

The mammalian IFNγ dimer ligates the interferon gamma receptor 1 (IFNGR1), which then as‐ sociates with IFNGR2, forming a signaling complex and activating the Janus kinases (Jak) 1 and 2, associated with the receptor chains 1 and 2, respectively [83]. Jak1 and Jak2 in turn acti‐ vate the IFNGR1-associated signal transducer of activation-1 (Stat1) transcription factor [33]. The IFNGR ligation may also activate and utilize Stat2 [187], albeit to lesser extent than Stat1. Subsequent transcriptional regulation of several other genes then ensues through homodimer‐ ic Stat1, heterodimeric Stat1: Stat2, through the transcription factor complexes ISGF3 and Stat1-p48, composed of Stat1: Stat2:IRF-9 and Stat1: Stat1:IRF-9, respectively [11, 118, 187, 188]. These confer transcriptional changes through recognition of IFNγ-activated sequences (GAS) in the promoter regions of target genes [188]. Within 30 minutes of IFNγ receptor ligation, there are increased transcript levels for several interferon regulatory factors (IRFs), which then

modulate subsequent waves of gene expression in the IFNγ signaling cascade [208].

like factor was shown to bind the mammalian IFNγ promoter [160].

Trout mitogen-simulated leukocyte supernatants possess macrophage activating capabilities (MAFs) akin to the mammalian IFNγ [58, 59], suggesting the existence of an IFNγ counter‐ part(s) in fish. It was also established that downstream signaling factors employed by the mammalian IFNγ (Stats), were present in fish [160] where the antibody-purified fish Stat-

The initial fish IFNγ homolog discovery came from examination of fugu gene scaffolds [220]. Fugu homologs of mammalian genes syntenic to IFNγ were also present on the same fugu gene scaffold. This fugu IFNγ had 4 exon / 3 intron organization similar to its mamma‐ lian counterpart and shared 32.3-32.7 % and 34.9-43.3% identities with bird and mammalian IFNγ sequences, respectively. An identified trout IFNγ sequence exhibited low sequence identity with other vertebrate IFNγ proteins, but possessed the conserved signature motif ([IV]-Q-X-[KQ]-A-X2-E-[LF]-X2-[IV]) and C-terminal nuclear localization signal (NLS), char‐

monocytes while NO is predominantly produced by mature macrophages [67, 136].

**3.2. Interferon-gamma (IFNγ)**

tion of antimicrobial responses [8, 10, 21, 51, 115].

*3.2.1. Identification of IFNγ in bony fish*

acteristic of the mammalian IFNγ proteins [215].

Contrary to the sea bream and carp studies, other literature suggests that akin to the mam‐ malian cytokine, teleost TNFα also directly elicits pro-inflammatory functions. The tilapia non-specific cytotoxic cells (NCCs) constitutively express membrane bound as well as solu‐ ble forms of TNFα (in addition to granzymes and Fas ligand) to confer cytolytic activity to‐ wards target cells, and when stimulated with recombinant tilapia TNFα, become protected from activation-induced apoptosis [151]. While the turbot recombinant TNFα did not en‐ hance macrophage ROI, it elicited *in vitro* NO production and *in vivo* inflammatory cell re‐ cruitment and activation [142]. The Ayu fish, recombinant TNFα induced ROI production by kidney cells [193] and the blue fin tuna recombinant TNFα1 and 2 enhanced the phago‐ cytic responses of tuna PBLs [89].

Zebrafish TNFα elicited cell signaling and conferred increased resistance to *Mycobacterium marinum* [30] while knockdown of the TNF-R1 led to enhanced mycobacterial disease pro‐ gression, increased fish mortality, accelerated bacterial growth, granuloma breakdown and necrotic macrophage cell death [30]. Thus, it appears that the zebrafish TNFα is pivotal in the maintenance of encapsulated *M. marinum* granulomas and the restriction of the growth of this pathogen. Our recent work supports these findings, where the pre-treatment of gold‐ fish macrophages with recombinant goldfish TNFα2 ablated the *M. marinum*-mediated down-regulation of NO production by these cells and reduced the survival of intracellular bacteria [65].

We recently identified two isoforms of the goldfish TNFα and functionally characterized a recombinant goldfish TNFα2 (rgTNFα2) in the context of primary kidney-derived goldfish macrophage cultures (PKMs) [67]. This rgTNFα2 induced dose-dependent chemotaxis of goldfish macrophages, enhanced their phagocytic abilities, NO production and primed the ROI responses of PKMs.

The extent of the conservation in the biology of teleost TNFα will become more evident with increased availability of tools, reagents and cell culture systems. While some literature pro‐ poses a lack of conservation of the inflammatory roles of teleost TNFα, others strongly im‐ plicate this molecule in the regulation of fish antimicrobial functions. It is also possible that the discrepancies in the above findings stem from the culture systems employed. For exam‐ ple, both the sea bream and carp studies that failed to observe direct effects of fish TNFα utilized freshly isolated adherent kidney phagocyte populations [52, 155]. Notably, freshly isolated mammalian myeloid populations are highly variable in their antimicrobial capabili‐ ties [116]. Similarly, goldfish PKMs exhibit temporal gain and loss of antimicrobial capabili‐ ties after prolonged cvultivation [67, 136]. Specifically, ROI is primarily mediated by PKMmonocytes while NO is predominantly produced by mature macrophages [67, 136].

#### **3.2. Interferon-gamma (IFNγ)**

In similar studies, supernatants from recombinant carp TNFα-stimulated cardiac endothelial cells primed kidney phagocyte ROI responses while recombinant carp TNFα1 and 2 did not enhance phagocyte antimicrobial functions [155]. Notably, the recombinant forms of the ze‐ brafish, trout, sea bream and carp TNFα proteins exhibited lytic activity towards *Trypano‐ plasma borreli*, akin to the trypanolytic capacity of the mammalian TNFs [52], where the membrane form of the fish TNFα was thought to be responsible for these effects. In summa‐ tion of the above observations, Florenza *et al.* (2009) suggested that while the trypanolyitic roles of TNFα are evolutionarily conserved, the pro-inflammatory mechanisms elicited by

Contrary to the sea bream and carp studies, other literature suggests that akin to the mam‐ malian cytokine, teleost TNFα also directly elicits pro-inflammatory functions. The tilapia non-specific cytotoxic cells (NCCs) constitutively express membrane bound as well as solu‐ ble forms of TNFα (in addition to granzymes and Fas ligand) to confer cytolytic activity to‐ wards target cells, and when stimulated with recombinant tilapia TNFα, become protected from activation-induced apoptosis [151]. While the turbot recombinant TNFα did not en‐ hance macrophage ROI, it elicited *in vitro* NO production and *in vivo* inflammatory cell re‐ cruitment and activation [142]. The Ayu fish, recombinant TNFα induced ROI production by kidney cells [193] and the blue fin tuna recombinant TNFα1 and 2 enhanced the phago‐

Zebrafish TNFα elicited cell signaling and conferred increased resistance to *Mycobacterium marinum* [30] while knockdown of the TNF-R1 led to enhanced mycobacterial disease pro‐ gression, increased fish mortality, accelerated bacterial growth, granuloma breakdown and necrotic macrophage cell death [30]. Thus, it appears that the zebrafish TNFα is pivotal in the maintenance of encapsulated *M. marinum* granulomas and the restriction of the growth of this pathogen. Our recent work supports these findings, where the pre-treatment of gold‐ fish macrophages with recombinant goldfish TNFα2 ablated the *M. marinum*-mediated down-regulation of NO production by these cells and reduced the survival of intracellular

We recently identified two isoforms of the goldfish TNFα and functionally characterized a recombinant goldfish TNFα2 (rgTNFα2) in the context of primary kidney-derived goldfish macrophage cultures (PKMs) [67]. This rgTNFα2 induced dose-dependent chemotaxis of goldfish macrophages, enhanced their phagocytic abilities, NO production and primed the

The extent of the conservation in the biology of teleost TNFα will become more evident with increased availability of tools, reagents and cell culture systems. While some literature pro‐ poses a lack of conservation of the inflammatory roles of teleost TNFα, others strongly im‐ plicate this molecule in the regulation of fish antimicrobial functions. It is also possible that the discrepancies in the above findings stem from the culture systems employed. For exam‐ ple, both the sea bream and carp studies that failed to observe direct effects of fish TNFα utilized freshly isolated adherent kidney phagocyte populations [52, 155]. Notably, freshly isolated mammalian myeloid populations are highly variable in their antimicrobial capabili‐ ties [116]. Similarly, goldfish PKMs exhibit temporal gain and loss of antimicrobial capabili‐

this molecule were not [155].

64 New Advances and Contributions to Fish Biology

cytic responses of tuna PBLs [89].

bacteria [65].

ROI responses of PKMs.

Interferon gamma is a pleiotropic, pro-inflammatory and anti-viral cytokine, identified in the supernatants of PHA-activated lymphocyes for its unique anti-viral properties [205]. IFNγ is primarily produced by activated Th1 phenotype CD4+ cells [127], CD8+ cells [161] and natural killer (NK) cells [149] and is of central importance in host defense against intra‐ cellular and extracellular pathogens [8, 91, 99, 179, 182]. For example, IFNγ gene knock-out mice are incapable of controlling infections with *Leishmania major* [202], *Listeria monocyto‐ genes* [81], and *Mycobacterium* [32], underlining the importance of this cytokine in the regula‐ tion of antimicrobial responses [8, 10, 21, 51, 115].

The mammalian IFNγ dimer ligates the interferon gamma receptor 1 (IFNGR1), which then as‐ sociates with IFNGR2, forming a signaling complex and activating the Janus kinases (Jak) 1 and 2, associated with the receptor chains 1 and 2, respectively [83]. Jak1 and Jak2 in turn acti‐ vate the IFNGR1-associated signal transducer of activation-1 (Stat1) transcription factor [33]. The IFNGR ligation may also activate and utilize Stat2 [187], albeit to lesser extent than Stat1. Subsequent transcriptional regulation of several other genes then ensues through homodimer‐ ic Stat1, heterodimeric Stat1: Stat2, through the transcription factor complexes ISGF3 and Stat1-p48, composed of Stat1: Stat2:IRF-9 and Stat1: Stat1:IRF-9, respectively [11, 118, 187, 188]. These confer transcriptional changes through recognition of IFNγ-activated sequences (GAS) in the promoter regions of target genes [188]. Within 30 minutes of IFNγ receptor ligation, there are increased transcript levels for several interferon regulatory factors (IRFs), which then modulate subsequent waves of gene expression in the IFNγ signaling cascade [208].

#### *3.2.1. Identification of IFNγ in bony fish*

Trout mitogen-simulated leukocyte supernatants possess macrophage activating capabilities (MAFs) akin to the mammalian IFNγ [58, 59], suggesting the existence of an IFNγ counter‐ part(s) in fish. It was also established that downstream signaling factors employed by the mammalian IFNγ (Stats), were present in fish [160] where the antibody-purified fish Statlike factor was shown to bind the mammalian IFNγ promoter [160].

The initial fish IFNγ homolog discovery came from examination of fugu gene scaffolds [220]. Fugu homologs of mammalian genes syntenic to IFNγ were also present on the same fugu gene scaffold. This fugu IFNγ had 4 exon / 3 intron organization similar to its mamma‐ lian counterpart and shared 32.3-32.7 % and 34.9-43.3% identities with bird and mammalian IFNγ sequences, respectively. An identified trout IFNγ sequence exhibited low sequence identity with other vertebrate IFNγ proteins, but possessed the conserved signature motif ([IV]-Q-X-[KQ]-A-X2-E-[LF]-X2-[IV]) and C-terminal nuclear localization signal (NLS), char‐ acteristic of the mammalian IFNγ proteins [215].

### *3.2.2. Inflammatory roles of the fish IFNγ*

The recombinant trout IFNγ (rtIFNγ) elicited increased immune gene expression in the RTS-11 monocyte-macrophage cell line [199], while this response was pharmacologically ab‐ rogated with ERK (transcription factor) or protein kinase C (PKC) inhibitors as well as by deleting the C-terminal NLS on the rtIFNγ [215]. Also, rtIFNγ stimulation of trout kidney leukocytes primed their ROI production, suggesting functional similarities between the trout and mammalian type II IFNs [215].

CXCL-8\_L1 and CXCL-8\_L2 [3]. The latter is reminiscent of the mammalian IFNγ and CXCL-8 relationship, where shorter treatments of mammalian granulocytes with IFNγ caus‐ es decreased CXCL-8 production [23, 90, 120], while prolonged treatments increase the CXCL-8 mRNA and protein levels [90]. Additionally, mammalian blood monocytes and macrophage cell lines stimulated with IFNγ up-regulate CXCL-8 mRNA transcripts and protein levels [14, 36] due to post-transcriptional stabilization of the CXCL-8 mRNAs rather than gene expression [14]. Together, these findings suggest that the pro-inflammatory roles of IFNγ, including synergism with LPS, are conserved in cyprinid fish, while it is probable that the discrepancies in gene expression profiles may be due to distinct immune cell model

Using gene synteny analysis, Igawa *et al*. (2006) discovered two tandem IFNγ isoforms next to the fish IL-22 and IL-26 genes [82]. The corresponding IFNγ sequences, coined IFNγ1 and IFNγ2, were later renamed IFNγ-related (rel) and FNγ respectively [166]. These proteins share only 17 % amino acid identity, but exhibit exon/intron organization similar to that of mammalian and fugu IFNγ, and have the IFNγ signature motif ([IV]-Q-X-[KQ]-A-X2-E-[LF]- X2-[IV]). Interestingly, only IFNγ but not IFNγrel has a C-terminal nuclear localization sig‐

The existence of two IFNγ isoforms, sharing all of the above characteristics was soon con‐ firmed in siluriformes and other cypriniformes when IFNγ and IFNγrel were identified in the catfish [122] and common carp [183], respectively. Catfish and carp were both reported to possess two distinct, alternatively spliced IFNγ transcripts, as well as a single transcript of IFNγrel [122, 183]. The pattern of tissue gene expression of catfish IFNγ and IFNγrel dif‐ fered while both cytokines were expressed in various cell lines and immune cells, with high‐ est expression observed in macrophages, T cells and NK cells [122]. Interestingly, low transcript levels of IFNγrel but not IFNγ were also reported in a catfish B-cell line. Expres‐ sion of the carp IFNγ increased in T cells after stimulation with PHA, while the mRNA lev‐

stimulated with high doses of LPS [183]. Furthermore, increased transcript levels of the carp IFNγ, but not IFNγrel, were observed in the head kidneys of fish infected with *T. borreli*. The goldfish IFNγ and IFNγrel exhibited similar mRNA levels across tissues and immune cell

While the siluriforme IFNγrel has yet to be functionally characterized, there has been some insight into the functions of cyprinid IFNγrel. Zebrafish IFNγrel mRNA is detectable in freshly laid eggs, suggesting maternal supply of this transcript [174]. The IFNγrel mRNA levels persist throughout embryonic development, while the expression of IFNγ is not de‐ tected until later stages of development. Embryos injected with *in vitro* transcribed IFNγ or

, B lymphocyte-enriched immune cell fractions,

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67

systems used (primary kidney phagocytes versus cultured mature macrophages).

*3.2.3. Interferon-gamma-related (IFNγrel) cytokine*

els of carp IFNγrel increased in IgM+

*3.2.4. Inflammatory roles of fish IFNγrel*

nal (NLS).

types [63].

Adult zebrafish microinjected with recombinant IFNγ did not exhibit increased immune gene expression or enhanced protection against *Streptococcus iniae* and the spring viremia carp virus (SVCV) [110]. However, this lack of response could stem from IFNγ being bound up by cells expressing only the IFNGR1 without eliciting a detectable level of response from the relatively few cells expressing both IFNGR1 and IFNGR2. If this is the case, these results underline the localized nature of the zebrafish IFNγ function rather then the efficacy of reg‐ ulation of antimicrobial functions by this cytokine.

We [62] and others [3] have functionally characterized the cyprinid IFNγ. A recombinant goldfish IFNγ (rgIFNγ) primed goldfish monocytes ROI response in a concentration de‐ pendent manner [62] and, as in mammals [72, 157], at lower concentrations rgIFNγ confer‐ red additive ROI priming effects. The rgIFNγ also enhanced the expression of the ROI enzyme, NADPH oxidase, catalytic subunits, p67phox and gp91phox. Similarly, the recombinant carp IFNγ (rcIFNγ) also primed carp kidney phagocytes for enhanced ROI production [3].

The goldfish rIFNγ elicited modest but significant enhancement of phagocytosis and NO production by goldfish monocytes and macrophages, respectively [62]. This was paralled with increased iNOS gene expression in rgIFNγ-stimulated macrophages. In contarst, carp kidney phagocytes only displayed significant iNOS gene expression and NO production when treated with a combination of carp recombinant IFNγ and 30 μg/mL LPS, but not fol‐ lowing rcIFNγ treatments alone [3].

Since carp and goldfish are closely related species, the above discrepancies presumably stem from differences in experimental systems. Akin to bone-marrow-derived macrophages, which acquire antimicrobial capabilities with culture time, we have observed that the anti‐ microbial capabilities of goldfish kidney-derived phagocytes are dynamic [136, 137]. For this reason, we used culture-derived cells, while the carp studies used freshly isolated phago‐ cytes. Additionally, it is well established that in mammals the production of ROIs is seen as early as 1 hour after immune stimulation, whereas the production RNIs is first detected ap‐ proximately 24 hours post stimulation [169]. This is very similar to what we have observed with rgIFNγ-elicited iNOS expression and NO responses.

The treatment of mature goldfish macrophages with rgIFNγ resulted in increased expres‐ sion of TNFα and IL-1β isoforms; IL-12 p35 and p40; IFNγ; IL-8 (CXCL-8]; CCL-1; and viper‐ in (an anti-viral molecule) [62]. The treatment of carp phagocytes with a combination of carp IFNγ and LPS resulted in increased expression of TNFα; IL-1β; IL-12 subunits p35 and iso‐ forms of IL-12 p40 subunit [3]. The carp IFNγ also induced the expression of the CXCL-10 like chemokine, CXCLb, while inhibiting the LPS-induced expression of CXCL-8 isoforms, CXCL-8\_L1 and CXCL-8\_L2 [3]. The latter is reminiscent of the mammalian IFNγ and CXCL-8 relationship, where shorter treatments of mammalian granulocytes with IFNγ caus‐ es decreased CXCL-8 production [23, 90, 120], while prolonged treatments increase the CXCL-8 mRNA and protein levels [90]. Additionally, mammalian blood monocytes and macrophage cell lines stimulated with IFNγ up-regulate CXCL-8 mRNA transcripts and protein levels [14, 36] due to post-transcriptional stabilization of the CXCL-8 mRNAs rather than gene expression [14]. Together, these findings suggest that the pro-inflammatory roles of IFNγ, including synergism with LPS, are conserved in cyprinid fish, while it is probable that the discrepancies in gene expression profiles may be due to distinct immune cell model systems used (primary kidney phagocytes versus cultured mature macrophages).

#### *3.2.3. Interferon-gamma-related (IFNγrel) cytokine*

*3.2.2. Inflammatory roles of the fish IFNγ*

66 New Advances and Contributions to Fish Biology

trout and mammalian type II IFNs [215].

lowing rcIFNγ treatments alone [3].

with rgIFNγ-elicited iNOS expression and NO responses.

ulation of antimicrobial functions by this cytokine.

The recombinant trout IFNγ (rtIFNγ) elicited increased immune gene expression in the RTS-11 monocyte-macrophage cell line [199], while this response was pharmacologically ab‐ rogated with ERK (transcription factor) or protein kinase C (PKC) inhibitors as well as by deleting the C-terminal NLS on the rtIFNγ [215]. Also, rtIFNγ stimulation of trout kidney leukocytes primed their ROI production, suggesting functional similarities between the

Adult zebrafish microinjected with recombinant IFNγ did not exhibit increased immune gene expression or enhanced protection against *Streptococcus iniae* and the spring viremia carp virus (SVCV) [110]. However, this lack of response could stem from IFNγ being bound up by cells expressing only the IFNGR1 without eliciting a detectable level of response from the relatively few cells expressing both IFNGR1 and IFNGR2. If this is the case, these results underline the localized nature of the zebrafish IFNγ function rather then the efficacy of reg‐

We [62] and others [3] have functionally characterized the cyprinid IFNγ. A recombinant goldfish IFNγ (rgIFNγ) primed goldfish monocytes ROI response in a concentration de‐ pendent manner [62] and, as in mammals [72, 157], at lower concentrations rgIFNγ confer‐ red additive ROI priming effects. The rgIFNγ also enhanced the expression of the ROI enzyme, NADPH oxidase, catalytic subunits, p67phox and gp91phox. Similarly, the recombinant carp IFNγ (rcIFNγ) also primed carp kidney phagocytes for enhanced ROI production [3]. The goldfish rIFNγ elicited modest but significant enhancement of phagocytosis and NO production by goldfish monocytes and macrophages, respectively [62]. This was paralled with increased iNOS gene expression in rgIFNγ-stimulated macrophages. In contarst, carp kidney phagocytes only displayed significant iNOS gene expression and NO production when treated with a combination of carp recombinant IFNγ and 30 μg/mL LPS, but not fol‐

Since carp and goldfish are closely related species, the above discrepancies presumably stem from differences in experimental systems. Akin to bone-marrow-derived macrophages, which acquire antimicrobial capabilities with culture time, we have observed that the anti‐ microbial capabilities of goldfish kidney-derived phagocytes are dynamic [136, 137]. For this reason, we used culture-derived cells, while the carp studies used freshly isolated phago‐ cytes. Additionally, it is well established that in mammals the production of ROIs is seen as early as 1 hour after immune stimulation, whereas the production RNIs is first detected ap‐ proximately 24 hours post stimulation [169]. This is very similar to what we have observed

The treatment of mature goldfish macrophages with rgIFNγ resulted in increased expres‐ sion of TNFα and IL-1β isoforms; IL-12 p35 and p40; IFNγ; IL-8 (CXCL-8]; CCL-1; and viper‐ in (an anti-viral molecule) [62]. The treatment of carp phagocytes with a combination of carp IFNγ and LPS resulted in increased expression of TNFα; IL-1β; IL-12 subunits p35 and iso‐ forms of IL-12 p40 subunit [3]. The carp IFNγ also induced the expression of the CXCL-10 like chemokine, CXCLb, while inhibiting the LPS-induced expression of CXCL-8 isoforms, Using gene synteny analysis, Igawa *et al*. (2006) discovered two tandem IFNγ isoforms next to the fish IL-22 and IL-26 genes [82]. The corresponding IFNγ sequences, coined IFNγ1 and IFNγ2, were later renamed IFNγ-related (rel) and FNγ respectively [166]. These proteins share only 17 % amino acid identity, but exhibit exon/intron organization similar to that of mammalian and fugu IFNγ, and have the IFNγ signature motif ([IV]-Q-X-[KQ]-A-X2-E-[LF]- X2-[IV]). Interestingly, only IFNγ but not IFNγrel has a C-terminal nuclear localization sig‐ nal (NLS).

The existence of two IFNγ isoforms, sharing all of the above characteristics was soon con‐ firmed in siluriformes and other cypriniformes when IFNγ and IFNγrel were identified in the catfish [122] and common carp [183], respectively. Catfish and carp were both reported to possess two distinct, alternatively spliced IFNγ transcripts, as well as a single transcript of IFNγrel [122, 183]. The pattern of tissue gene expression of catfish IFNγ and IFNγrel dif‐ fered while both cytokines were expressed in various cell lines and immune cells, with high‐ est expression observed in macrophages, T cells and NK cells [122]. Interestingly, low transcript levels of IFNγrel but not IFNγ were also reported in a catfish B-cell line. Expres‐ sion of the carp IFNγ increased in T cells after stimulation with PHA, while the mRNA lev‐ els of carp IFNγrel increased in IgM+ , B lymphocyte-enriched immune cell fractions, stimulated with high doses of LPS [183]. Furthermore, increased transcript levels of the carp IFNγ, but not IFNγrel, were observed in the head kidneys of fish infected with *T. borreli*. The goldfish IFNγ and IFNγrel exhibited similar mRNA levels across tissues and immune cell types [63].

#### *3.2.4. Inflammatory roles of fish IFNγrel*

While the siluriforme IFNγrel has yet to be functionally characterized, there has been some insight into the functions of cyprinid IFNγrel. Zebrafish IFNγrel mRNA is detectable in freshly laid eggs, suggesting maternal supply of this transcript [174]. The IFNγrel mRNA levels persist throughout embryonic development, while the expression of IFNγ is not de‐ tected until later stages of development. Embryos injected with *in vitro* transcribed IFNγ or IFNγrel mRNAs individually, exhibited similar immune gene expression changes, while combined injections further increased certain expression profiles, suggesting non-overlap‐ ping roles for the respective IFNγ proteins.

lished and was effectively demonstrated to specifically increase luciferase reporter expression following IFNγ stimulation but not in response to a range of other stimuli [24, 25]. Hence, a system is now in place to elucidate the distinct binding and signaling mecha‐

Cytokine Regulation of Teleost Inflammatory Responses

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69

In light of the functional differences between IFNγ and IFNγrel, we postulated that these cytokines might function through distinct receptors. When we performed gene synteny analysis of IFNGR1, we observed that while some genes were localized to the chromo‐ some bearing the known zebrafish IFNGR1 gene, other syntenic neighbours of the mam‐ malian IFNGR1 were present on a distinct zebrafish chromosome. Further analysis of the chromosomal region flanked by these genes revealed a second gene, encoding a distinct IFNGR1 protein [60], (these genes were denoted IFNGR1-1 and IFNGR1-2). The corre‐ sponding goldfish receptor cDNA transcripts were identified. The fish IFNGR1 sequences displayed putative Jak1 and Stat1 binding sites, pivotal for the biological functions of the mammalian IFNγ [49, 68, 69]. While the zebrafish receptors displayed comparable tissue expression, the goldfish IFNGR1-1 exhibited substantially greater mRNA levels than the IFNGR1-2 in all tissues and immune cell types examined. In order to elucidate possible binding partners for the goldfish IFNGR1-1 and IFNGR1-2, recombinant forms of their ex‐ tracellular domains were produced and *in vitro* binding assays were performed. While IFNGR1-1 bound exclusively to IFNγrel (IFNγ1), IFNGR1-2 bound strictly to IFNγ (IFNγ2, receptors were named after the fact). It has recently been reported that morpholi‐ no knockdowns of IFNGR1-1, IFNGR1-2 or a putative IFNGR2 abolished zebrafish IFNγinduced gene expression [1]. In contrast, only the knockdown of IFNGR1-1, but not the knockdown of IFNGR1-2 or IFNGR2, abrogated gene expression elicited by IFNγrel. It was suggested that IFNγ might signal through a heterodimer of IFNGR1-1 and IFNGR1-2 and a homodimer of IFNGR2 while the IFNγrel would ligate with a homodimeric IFNGR1-1 and an as of yet unidentified receptor 2 chains. Alternatively, since IFNγrel is present in cyprinids early in development, the knockdown of its putative receptor, IFNGR1-1 in embryos might effect development of the components required for IFNγ function. A biological relationship of such nature would be phenotypically manifest as a

nisms involved in the salmonid IFNγ biological processes.

loss of IFNγ function (as seen in the above zebrafish study).

an NLS, IFNγrel may have evolved to utilize distinct signaling mechanism.

It would appear that certain teleost species possess receptor signaling systems to facilitate a dichotomy of type II IFN functions. Presumably, the cyprinid IFNGR1 genes arose from du‐ plications of an ancestral IFNGR1 and subsequently diverged in respective signaling mecha‐ nisms used, where the IFNγrel-induced Stat1-phosphorylation might be an artifact, remnant of the ancestral gene. Indeed, the importance of the C-terminal NLS of fish (and mammali‐ an) IFNγ has been demonstrated [217] while the lack of this NLS on IFNγrel proteins is the key distinguishing feature of the latter cytokine. The leading model for mammalian IFNγ signaling [185] suggests that after IFNγ receptor ligation, Stat1 is delivered into the nucleus via the IFNγ NLS in a complex consisting of Stat1:IFNGR1:IFNγ. Therefore, due to a lack of

Morpholino knock-downs of either IFNγ or IFNγrel alone had negligible effects on zebra‐ fish embryo survival following *Escherichia coli* challenge, while knock-down of both IFNγs resulted in substantially diminished survival following infection [174]. Interestingly, indi‐ vidual morpholino knockdowns of IFNγ or IFNγrel caused decreased survival rates of em‐ bryos infected with *Yersinia ruckeri,* while double knockdowns had a further deleterious effect on embryo survival. Presumably IFNγ and IFNγrel elicit some overlapping and some distinct antimicrobial mechanisms such that the presence of one cytokine may be sufficient for dealing with certain pathogens but not others. It is noteworthy that while the *E. coli* (strain DH5α) is not a natural fish pathogen, the *Y. ruckeri* is [159].

A comprehensive functional investigation of recombinant goldfish (rg) IFNγrel and rgIFNγ revealed that these molecules differed in respective capacities to modulate pro-inflammato‐ ry responses of goldfish PKMs [63]. While rgIFNγ conferred long-lasting ROI priming ef‐ fects, rgIFNγrel induced short-lived ROI priming, causing subsequent unresponsiveness to ROI priming by other recombinant cytokines (rgIFNγ or rgTNFα2). While rgIFNγ elicited modest phagocytosis and nitric oxide responses in goldfish monocytes and macrophages, respectively [62, 63], rgIFNγrel was a highly potent inducer of both responses. Interestingly, rgIFNγ and rgIFNγrel induced different gene expression profiles in goldfish monocytes, where rgIFNγrel elicited significantly greater expression of key inflammatory genes. Nota‐ bly, while both cytokines induced the phosphorylation of Stat1, its nuclear translocation was only observed following rgIFNγ treatment. Together, these findings suggest a functional segregation of the goldfish type II interferons in the regulation macrophage antimicrobial functions.

Further confirmation of this functional dichotomy between the fish type II IFNs is warrant‐ ed using *in vivo* and other *in vitro* fish models. Notably, the zebrafish IFNγrel has recently also been demonstrated to elicit more robust pro-infalmmatory gene expression than IFNγ in larvae microinjected with respective IFN expression constructs [110]. Furthermore, these zebrafish IFNγrel-mediated effects were dependent on the myeloid transcription factor SP1, underlying the specificity of this cytokine for macrophag-lineage cells.

#### *3.2.5. IFNγ receptors in fish*

Despite the growing knowledge regarding the teleost type II IFNs, the receptor systems em‐ ployed by these fish molecules remain poorly understood. The IFNGR1 and IFNGR2 chains were recently identified in the rainbow trout [55]. The expression of the IFNGR1 was gener‐ ally greater than that of IFNGR2 and was subject to decrease following rIFNγ or rIL1β stim‐ ulation. Furthermore RTG-2 trout fibroblast cells transfected with an IFNGR1 construct, or CHO cells transfected with constructs expressing IFNGR1 and IFNGR2, both bound rtIFNγ, where, as in mammals, the expression of the IFNGR2 chain was essential for the IFNγ-in‐ duced activity. It should be noted that a reliable trout IFNγ reporter cell line has been estab‐ lished and was effectively demonstrated to specifically increase luciferase reporter expression following IFNγ stimulation but not in response to a range of other stimuli [24, 25]. Hence, a system is now in place to elucidate the distinct binding and signaling mecha‐ nisms involved in the salmonid IFNγ biological processes.

IFNγrel mRNAs individually, exhibited similar immune gene expression changes, while combined injections further increased certain expression profiles, suggesting non-overlap‐

Morpholino knock-downs of either IFNγ or IFNγrel alone had negligible effects on zebra‐ fish embryo survival following *Escherichia coli* challenge, while knock-down of both IFNγs resulted in substantially diminished survival following infection [174]. Interestingly, indi‐ vidual morpholino knockdowns of IFNγ or IFNγrel caused decreased survival rates of em‐ bryos infected with *Yersinia ruckeri,* while double knockdowns had a further deleterious effect on embryo survival. Presumably IFNγ and IFNγrel elicit some overlapping and some distinct antimicrobial mechanisms such that the presence of one cytokine may be sufficient for dealing with certain pathogens but not others. It is noteworthy that while the *E. coli*

A comprehensive functional investigation of recombinant goldfish (rg) IFNγrel and rgIFNγ revealed that these molecules differed in respective capacities to modulate pro-inflammato‐ ry responses of goldfish PKMs [63]. While rgIFNγ conferred long-lasting ROI priming ef‐ fects, rgIFNγrel induced short-lived ROI priming, causing subsequent unresponsiveness to ROI priming by other recombinant cytokines (rgIFNγ or rgTNFα2). While rgIFNγ elicited modest phagocytosis and nitric oxide responses in goldfish monocytes and macrophages, respectively [62, 63], rgIFNγrel was a highly potent inducer of both responses. Interestingly, rgIFNγ and rgIFNγrel induced different gene expression profiles in goldfish monocytes, where rgIFNγrel elicited significantly greater expression of key inflammatory genes. Nota‐ bly, while both cytokines induced the phosphorylation of Stat1, its nuclear translocation was only observed following rgIFNγ treatment. Together, these findings suggest a functional segregation of the goldfish type II interferons in the regulation macrophage antimicrobial

Further confirmation of this functional dichotomy between the fish type II IFNs is warrant‐ ed using *in vivo* and other *in vitro* fish models. Notably, the zebrafish IFNγrel has recently also been demonstrated to elicit more robust pro-infalmmatory gene expression than IFNγ in larvae microinjected with respective IFN expression constructs [110]. Furthermore, these zebrafish IFNγrel-mediated effects were dependent on the myeloid transcription factor SP1,

Despite the growing knowledge regarding the teleost type II IFNs, the receptor systems em‐ ployed by these fish molecules remain poorly understood. The IFNGR1 and IFNGR2 chains were recently identified in the rainbow trout [55]. The expression of the IFNGR1 was gener‐ ally greater than that of IFNGR2 and was subject to decrease following rIFNγ or rIL1β stim‐ ulation. Furthermore RTG-2 trout fibroblast cells transfected with an IFNGR1 construct, or CHO cells transfected with constructs expressing IFNGR1 and IFNGR2, both bound rtIFNγ, where, as in mammals, the expression of the IFNGR2 chain was essential for the IFNγ-in‐ duced activity. It should be noted that a reliable trout IFNγ reporter cell line has been estab‐

underlying the specificity of this cytokine for macrophag-lineage cells.

ping roles for the respective IFNγ proteins.

68 New Advances and Contributions to Fish Biology

functions.

*3.2.5. IFNγ receptors in fish*

(strain DH5α) is not a natural fish pathogen, the *Y. ruckeri* is [159].

In light of the functional differences between IFNγ and IFNγrel, we postulated that these cytokines might function through distinct receptors. When we performed gene synteny analysis of IFNGR1, we observed that while some genes were localized to the chromo‐ some bearing the known zebrafish IFNGR1 gene, other syntenic neighbours of the mam‐ malian IFNGR1 were present on a distinct zebrafish chromosome. Further analysis of the chromosomal region flanked by these genes revealed a second gene, encoding a distinct IFNGR1 protein [60], (these genes were denoted IFNGR1-1 and IFNGR1-2). The corre‐ sponding goldfish receptor cDNA transcripts were identified. The fish IFNGR1 sequences displayed putative Jak1 and Stat1 binding sites, pivotal for the biological functions of the mammalian IFNγ [49, 68, 69]. While the zebrafish receptors displayed comparable tissue expression, the goldfish IFNGR1-1 exhibited substantially greater mRNA levels than the IFNGR1-2 in all tissues and immune cell types examined. In order to elucidate possible binding partners for the goldfish IFNGR1-1 and IFNGR1-2, recombinant forms of their ex‐ tracellular domains were produced and *in vitro* binding assays were performed. While IFNGR1-1 bound exclusively to IFNγrel (IFNγ1), IFNGR1-2 bound strictly to IFNγ (IFNγ2, receptors were named after the fact). It has recently been reported that morpholi‐ no knockdowns of IFNGR1-1, IFNGR1-2 or a putative IFNGR2 abolished zebrafish IFNγinduced gene expression [1]. In contrast, only the knockdown of IFNGR1-1, but not the knockdown of IFNGR1-2 or IFNGR2, abrogated gene expression elicited by IFNγrel. It was suggested that IFNγ might signal through a heterodimer of IFNGR1-1 and IFNGR1-2 and a homodimer of IFNGR2 while the IFNγrel would ligate with a homodimeric IFNGR1-1 and an as of yet unidentified receptor 2 chains. Alternatively, since IFNγrel is present in cyprinids early in development, the knockdown of its putative receptor, IFNGR1-1 in embryos might effect development of the components required for IFNγ function. A biological relationship of such nature would be phenotypically manifest as a loss of IFNγ function (as seen in the above zebrafish study).

It would appear that certain teleost species possess receptor signaling systems to facilitate a dichotomy of type II IFN functions. Presumably, the cyprinid IFNGR1 genes arose from du‐ plications of an ancestral IFNGR1 and subsequently diverged in respective signaling mecha‐ nisms used, where the IFNγrel-induced Stat1-phosphorylation might be an artifact, remnant of the ancestral gene. Indeed, the importance of the C-terminal NLS of fish (and mammali‐ an) IFNγ has been demonstrated [217] while the lack of this NLS on IFNγrel proteins is the key distinguishing feature of the latter cytokine. The leading model for mammalian IFNγ signaling [185] suggests that after IFNγ receptor ligation, Stat1 is delivered into the nucleus via the IFNγ NLS in a complex consisting of Stat1:IFNGR1:IFNγ. Therefore, due to a lack of an NLS, IFNγrel may have evolved to utilize distinct signaling mechanism.

#### **3.3. Interleukin-1 beta (IL-1β)**

#### *3.3.1. Interleukin-1 cytokine family*

The interleukin-1 cytokine family is becoming increasingly diverse, with new family mem‐ bers being discovered and/or assigned to this family. In addition to the well characterized IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra) and IL-18 the new members of the family now include the IL-1F5-10, and IL-1F11 [34, 192]. Accordingly, it has been proposed that IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra) should be renamed IL-1F1, IL-1F2, IL-1F3 and IL-1F4, respectively [177]. The exact biological roles of these new IL-1 cytokines (IL-1F5-11) have not been fully elucidated, and it was reported that concentrations of 100-1000 fold greater than those of IL-1β are required to them to induce biological effects [177, 178]. These new family members will not be discussed further, instead the remainder of this section will deal with the classical IL-1 cytokines and primarily IL-1β.

clusive and/or cell type dependent. Further research in multiple model organism and cell

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Both IL-1α and IL-1β bind to IL-1RI, resulting in recruitment of the IL-1R associated protein (IL-1RAcP), which amplifies the signal transduction [176]. The IL-1RI is structurally related to the toll-like pattern recognition receptors where the signal propagation through the IL-1RI involves many of the same downstream signaling components (MyD88, IL-1R associ‐ ated kinase (IRAK), and NFκB) as those employed in TLR signaling [139]. IL-1α and IL-1β also bind to the IL-1RII, but because this receptor lacks the intracellular signaling compo‐ nents of IL-1RI, it functionally serves as a "decoy" receptor by dampening the IL-1α/β signal transduction [31]. Additionally, the IL-1 receptor antagonist (IL-1Ra) exhibits competitive in‐ hibition of the IL-1α/β signaling by interacting with the IL-1RI without eliciting the down‐

Unlike most other cyokines and growth factors IL-1α and IL-1β (primarily IL-1β) target nearly every cell type and induce a range of biological processes (reviewed in references [37, 38, 41]). Some of the numerous pro-inflammatory roles of IL-1β include increases in collagen and pro-collagenase synthesis; increase of chondrocyte protease and proteoglycan release; increase in osteoclast-activating factor release and hence bone resorption; induction of the synthesis of lipid mediators such as PGE2; and enhancement of the proliferation of fibro‐ blasts, keratinocytes, mesangial, glial cells and smooth muscle cells. IL-1β also induces che‐ motaxis of T and B cells, the synthesis of thromboxane by neutrophils and monocytes, basophil histamine release and eosinophil degranulation. Additionally, IL-1β induces syn‐ thesis of type I IFNs, endothelial plasminogen activator inhibitors, and expression of leuko‐ cyte adherence receptors on endothelial cell surfaces. Based on the above, it is not surprising that IL-1β has been implicated in numerous disorders including cardiac disease [15], rheu‐

Fish were first suspected of possessing an IL-1 homolog when the mammalian PBL-derived IL-1 was demonstrated to enhance the proliferation of catfish T-lymphocytes in response to ConA [74], while carp epithelial cells [175], carp macrophages and granulocytes [195] and cat‐ fish monocytes [47], were shown to produce factor(s) with properties similar to those of the mammalian IL-1. Gel filtration analysis of catfish monocyte revealed two distinct bands of ap‐ proximately 70 kDa and 15 kDa, recognized by polyclonal antibodies raised against mammali‐ an IL-1α and IL-1β. Paradoxically, the catfish 70 kDa molecule activated catfish (but not

mouse) T cells, while the 15 kDa molecule activated mouse (but not catfish) T cells [47].

cytes following LPS stimulation [170, 216], suggesting its pro-inflammatory nature.

The first fish IL-1β cDNA sequence was identified in trout and exhibited 49-56% amino acid identity to the mammalian IL-1β [170]. Notably, the trout IL-1β did not possess a putative ICE cleavage site required for the maturation-cleavage of the mammalian IL-1β, while the gene expression of this trout cytokine could be induced in tissues and head kidney leuko‐

types is warranted before specific mechanism(s) can be defined.

matoid arthritis [92], and neurodegenerative diseases [71].

stream signaling events [31].

*3.3.2. Identification of IL-1 in fish*

The IL-1α [106] and IL-1β [6] were initially identified as monocyte transcripts with only 23% amino acid identitiy but with structurally similarities. Both of these cytokines are produced as leaderless 31 kDa pro-peptides that are cleaved to generate mature 17 kDa molecules, which mediated their respective effects by binding to the IL-1RI [37, 38, 40]. The synthesis of IL-1 has been reported in several cell types, including keratinocytes, Langerhan's cells, syno‐ vial fibroblasts, mesangial cells, astrocytes, microglia, corneal cells, gingival cells, thymic ep‐ ithelial cells, in addition to myeloid and some lymphoid cell types (reviewed in references [38, 40]). Although IL-1α and IL-1β signal through the same receptor, they induce different biological functions. It has been suggested that IL-1α is produced primarily by epithelial cells and keratinocytes [125] and is involved in mediating local inflammatory processes. In contrast, IL-1β is synthesized by cells such as monocytes, macrophages, Langerhan's cells and dendritic cells [38], and mediates systemic inflammatory responses [37]. This is corrobo‐ rated by the fact that while IL-1α may act as a membrane-bound pro-IL-1α through myristo‐ lation of the protein [100, 181], IL-1β requires intracellular processing for activation.

As mentioned, IL-1α and IL-1β are both produced as pro-peptides and while IL-1α can me‐ diate biological effects as pro-IL-1α or as a mature IL-1α following enzymatic cleavage (e.g. with calpain) [96], only the proteolytically cleaved mature IL-1β, but not the pro-IL-1β can elicit immune functions. In fact, the release of IL-1β appears to be a two-step process, where‐ by the first stimulus such myeloid cell encounter of a pathogen results in increased synthesis and cytosolic accumulation of pro-IL-1β, while a second (as of yet poorly defined) stimulus induces the proteolytic processing of pro-IL-1β by caspase-1/IL-1β-converting enzyme (ICE), and the subsequent release of the biologically active mature IL-1β [39]. This processing event is further enhanced by the presence of extracellular ATP, which is recognized by the ATP receptor P2X7R, causing an efflux of K<sup>+</sup> and a concomitant activation of ICE [50]. A number of mechanisms have been proposed for the release of the mature IL-1β into the ex‐ tracellular milieu. These include: exocytosis of IL-1β containing lysosomes; through micro‐ vesicular budding of the plasma membrane; release in exosomes by fusion of multivesicular bodies with the plasma membrane; export through specific membrane transporters; and cell-lysis-mediated release (reviewed in [46]). Possibly, these mechanisms are mutually ex‐ clusive and/or cell type dependent. Further research in multiple model organism and cell types is warranted before specific mechanism(s) can be defined.

Both IL-1α and IL-1β bind to IL-1RI, resulting in recruitment of the IL-1R associated protein (IL-1RAcP), which amplifies the signal transduction [176]. The IL-1RI is structurally related to the toll-like pattern recognition receptors where the signal propagation through the IL-1RI involves many of the same downstream signaling components (MyD88, IL-1R associ‐ ated kinase (IRAK), and NFκB) as those employed in TLR signaling [139]. IL-1α and IL-1β also bind to the IL-1RII, but because this receptor lacks the intracellular signaling compo‐ nents of IL-1RI, it functionally serves as a "decoy" receptor by dampening the IL-1α/β signal transduction [31]. Additionally, the IL-1 receptor antagonist (IL-1Ra) exhibits competitive in‐ hibition of the IL-1α/β signaling by interacting with the IL-1RI without eliciting the down‐ stream signaling events [31].

Unlike most other cyokines and growth factors IL-1α and IL-1β (primarily IL-1β) target nearly every cell type and induce a range of biological processes (reviewed in references [37, 38, 41]). Some of the numerous pro-inflammatory roles of IL-1β include increases in collagen and pro-collagenase synthesis; increase of chondrocyte protease and proteoglycan release; increase in osteoclast-activating factor release and hence bone resorption; induction of the synthesis of lipid mediators such as PGE2; and enhancement of the proliferation of fibro‐ blasts, keratinocytes, mesangial, glial cells and smooth muscle cells. IL-1β also induces che‐ motaxis of T and B cells, the synthesis of thromboxane by neutrophils and monocytes, basophil histamine release and eosinophil degranulation. Additionally, IL-1β induces syn‐ thesis of type I IFNs, endothelial plasminogen activator inhibitors, and expression of leuko‐ cyte adherence receptors on endothelial cell surfaces. Based on the above, it is not surprising that IL-1β has been implicated in numerous disorders including cardiac disease [15], rheu‐ matoid arthritis [92], and neurodegenerative diseases [71].

#### *3.3.2. Identification of IL-1 in fish*

**3.3. Interleukin-1 beta (IL-1β)**

70 New Advances and Contributions to Fish Biology

*3.3.1. Interleukin-1 cytokine family*

deal with the classical IL-1 cytokines and primarily IL-1β.

ATP receptor P2X7R, causing an efflux of K<sup>+</sup>

The interleukin-1 cytokine family is becoming increasingly diverse, with new family mem‐ bers being discovered and/or assigned to this family. In addition to the well characterized IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra) and IL-18 the new members of the family now include the IL-1F5-10, and IL-1F11 [34, 192]. Accordingly, it has been proposed that IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra) should be renamed IL-1F1, IL-1F2, IL-1F3 and IL-1F4, respectively [177]. The exact biological roles of these new IL-1 cytokines (IL-1F5-11) have not been fully elucidated, and it was reported that concentrations of 100-1000 fold greater than those of IL-1β are required to them to induce biological effects [177, 178]. These new family members will not be discussed further, instead the remainder of this section will

The IL-1α [106] and IL-1β [6] were initially identified as monocyte transcripts with only 23% amino acid identitiy but with structurally similarities. Both of these cytokines are produced as leaderless 31 kDa pro-peptides that are cleaved to generate mature 17 kDa molecules, which mediated their respective effects by binding to the IL-1RI [37, 38, 40]. The synthesis of IL-1 has been reported in several cell types, including keratinocytes, Langerhan's cells, syno‐ vial fibroblasts, mesangial cells, astrocytes, microglia, corneal cells, gingival cells, thymic ep‐ ithelial cells, in addition to myeloid and some lymphoid cell types (reviewed in references [38, 40]). Although IL-1α and IL-1β signal through the same receptor, they induce different biological functions. It has been suggested that IL-1α is produced primarily by epithelial cells and keratinocytes [125] and is involved in mediating local inflammatory processes. In contrast, IL-1β is synthesized by cells such as monocytes, macrophages, Langerhan's cells and dendritic cells [38], and mediates systemic inflammatory responses [37]. This is corrobo‐ rated by the fact that while IL-1α may act as a membrane-bound pro-IL-1α through myristo‐

lation of the protein [100, 181], IL-1β requires intracellular processing for activation.

As mentioned, IL-1α and IL-1β are both produced as pro-peptides and while IL-1α can me‐ diate biological effects as pro-IL-1α or as a mature IL-1α following enzymatic cleavage (e.g. with calpain) [96], only the proteolytically cleaved mature IL-1β, but not the pro-IL-1β can elicit immune functions. In fact, the release of IL-1β appears to be a two-step process, where‐ by the first stimulus such myeloid cell encounter of a pathogen results in increased synthesis and cytosolic accumulation of pro-IL-1β, while a second (as of yet poorly defined) stimulus induces the proteolytic processing of pro-IL-1β by caspase-1/IL-1β-converting enzyme (ICE), and the subsequent release of the biologically active mature IL-1β [39]. This processing event is further enhanced by the presence of extracellular ATP, which is recognized by the

number of mechanisms have been proposed for the release of the mature IL-1β into the ex‐ tracellular milieu. These include: exocytosis of IL-1β containing lysosomes; through micro‐ vesicular budding of the plasma membrane; release in exosomes by fusion of multivesicular bodies with the plasma membrane; export through specific membrane transporters; and cell-lysis-mediated release (reviewed in [46]). Possibly, these mechanisms are mutually ex‐

and a concomitant activation of ICE [50]. A

Fish were first suspected of possessing an IL-1 homolog when the mammalian PBL-derived IL-1 was demonstrated to enhance the proliferation of catfish T-lymphocytes in response to ConA [74], while carp epithelial cells [175], carp macrophages and granulocytes [195] and cat‐ fish monocytes [47], were shown to produce factor(s) with properties similar to those of the mammalian IL-1. Gel filtration analysis of catfish monocyte revealed two distinct bands of ap‐ proximately 70 kDa and 15 kDa, recognized by polyclonal antibodies raised against mammali‐ an IL-1α and IL-1β. Paradoxically, the catfish 70 kDa molecule activated catfish (but not mouse) T cells, while the 15 kDa molecule activated mouse (but not catfish) T cells [47].

The first fish IL-1β cDNA sequence was identified in trout and exhibited 49-56% amino acid identity to the mammalian IL-1β [170]. Notably, the trout IL-1β did not possess a putative ICE cleavage site required for the maturation-cleavage of the mammalian IL-1β, while the gene expression of this trout cytokine could be induced in tissues and head kidney leuko‐ cytes following LPS stimulation [170, 216], suggesting its pro-inflammatory nature.

Although there have been no reports of a fish IL-1α, recently a novel IL-1 family member, nIL-1F has been identified in trout and *Tetraodon* spp. and was reported to have IL-1 family sig‐ nature motif as well as a putative ICE cleavage site [198]. The expression of nIL-1F increased af‐ ter activation of macrophages with LPS or recombinant IL-1β. Furthermore, a recombinant form of the C-terminal nIL-1F abrogated the rIL-1β-induced immune gene expression in RTS-11 trout macrophage-like cells, suggesting a possible competitive inhibition through the trout IL-1RI. This mechanism would be analogous to the mammalian IL-1Ra, which also com‐ petes with IL-1β for IL-1RI binding, without inducing downstream events.

the 15 kDa protein was confirmed to be a mature carp IL-1β, with the putative carp IL-1β cleavage site situated approximately 15 amino acids downstream of the mammalian ICE site [117]. Analysis of the supernatants of the trout macrophage cell line, RTS-11, using an antitrout IL-1β polyclonal antibody detected putative native, as well as potentially cleaved trout IL-1β proteins of 29 kDa and 24 kDa, respectively [79]. When the RTS-11 cells were transfect‐

detected primarily a mature, 24 kDa IL-1β protein, confirming that the trout IL-1β under‐

In an elegant set of studies it was demonstrated that combined immune stimuli of sea bream head kidney leukocytes resulted in an accumulation of a 30 kDa, pro-IL-1β protein, which, unlike the mammalian counterpart [113], did not exhibit a maturation cleavage or secretion following stimulation of the cells with extra-cellular ATP [146]. Furthermore, sea bream per‐ itoneal acidophilic granulocytes and peripheral blood leukocytes accumulated the 30 kDa form of IL-1β following challenge with *Vibrio anguillarum* [27]. In contrast, the sea bream SAF-1 fibroblast cell line shed a mature, 22 kDa IL-1β protein through microvesicular plas‐ ma membrane budding within 30 minutes of treatment with extracellular ATP [113]. Inter‐ estingly this IL-1β maturation/shedding process could be ablated with a pharmacological inhibitor of the mammalian ICE, suggesting a presence of an orthologous sea bream enzyme

The P2X7R receptor is the primary receptor responsible for the recognition of extracellular ATP and the concomitant release of mature IL-1β [50]. Upon ATP treatment and activation of the HEK 293 mammalian cell line expressing the rat P2X7R, human ICE and sea bream IL-1β, a non-cleaved (30 kDa) sea bream IL-1β was secreted by these cells [109]. Interesting‐ ly, neither sea bream nor zebrafish P2X7R expression in ATP-stimulated HEK 293 cells re‐ sulted in sea bream or mammalian IL-1β secretion. In contrast, the expression of a chimeric P2X7R bearing the sea bream ATP-binding and rat intracellular domains led to maturation/ secretion of the mammalian IL-1β, while the outcomes of this combination on the sea bream IL-1β were not addressed [109]. The authors of this work suggest that the mechanisms in‐ volved in IL-1β secretion are conserved across vertebrates while the distinct stimuli that elic‐ it the maturation events are not. We propose that alternatively, functional specificity may stem from the P2X7R intracellular signaling, activation of species specific ICE (or alternative maturation mechanisms), and the substrate specificity of the fish Caspase1/ICE. Notably, the above group also identified the sea bream Caspase 1 and demonstrated that the recombinant form of this fish enzyme cleaved a commercially available substrate, for which the mamma‐ lian counterpart holds specificity [107]. Additional studies are needed to establish the associ‐

Majority of the IL-1 receptor signaling components have been described in fish. A fish IL-1RI was first described in Atlantic salmon, with 43-44% similarity to chicken and 31% similarity to the human IL-1RI, respectively [186]. The expression of this gene increased in fish tissues

tagged trout IL-1β, and the RTS-11 superna‐

Cytokine Regulation of Teleost Inflammatory Responses

tagged IL-1β, the authors

http://dx.doi.org/10.5772/53505

73

ed with a plasmid encoding a C-terminally His6

went maturation cleavage.

responsible for this process.

*3.3.5. IL-1 receptors of fish*

tants assayed using an Ni-NTA-column specific for the His6

ation between the sea bream Caspase I/ICE, P2X7R and IL-1β.

#### *3.3.3. Isoforms of IL-1β in fish*

Because many fish species are tetraploid and have undergone genome duplication events, it is not surprising that additional isforms of IL-1β exist in certain fish species. An identified second trout IL-1β, denoted IL-1β2, also has the 6 exon/5 intron organization, 82% amino acid identity to the trout IL-1β1 and no putative ICE cleavage site [150]. In catfish, two IL-1β genes have been described and shown to undergo distinct expression patterns following challenge with *Edwardsiella ictaluri* [201]. Additionally identified cDNA of another carp IL-1β, IL-1β2, exhibited 74% identity with the carp IL-1β1 and 95-99% identity across indi‐ vidual IL-1β2 transcripts [48]. Since several distinct IL-1β2 sequences were identified in a homozygous individual, it was suggested that there might be multiple IL-1β2 genes. The ex‐ pression of the carp IL-1β1 and IL-1β2 differed following immune stimuli where the expres‐ sion of IL-1β1 gene was on average at least ten fold greater than that of IL-1β2. It was also reported that transcripts of IL-1β2 had high substitution numbers in the coding regions, in‐ cluding key areas predicted to be involved in receptor binding. In light of the above, it was suggested that the IL-1β2 may be a pseudogene [48]. Our observations in the goldfish, a close carp relative, support this theory. Notably, the predicted goldfish IL-1β2 protein is truncated compared to the IL-1β1, and while the expression of both IL-1β isoforms is subject to change following immune stimuli, specific treatments elicit more robust changes in the expression of IL-1β1 compared to IL-1β2 (unpublished observations).

#### *3.3.4. Maturation cleavage of the fish IL-1β*

As alluded to above, despite the functional similarities of the fish IL-1βs and their mammali‐ an counterpart, all fish IL-1β proteins identified to date lack the typical ICE cleavage site necessary for the functional maturation of the mammalian IL-1β1. Despite this, evidence suggests that indeed the fish IL-1β proteins undergo cleavage. Early fish IL-1 studies using anti-mammalian IL-1α/β serum to surveys supernatants of activated fish cells demonstrated the recognition of distinct, multiple IL-1 protein species in these supernatants. While the cat‐ fish monocyte supernatants contained 70 kDa and 15 kDa molecules with IL-1-like activity [47], carp macrophages secreted 22 kDa and 15 kDa IL-1-like factors that were recognized by mammalian anti-IL-1 antibodies [195]. The immunoprecipitation experiments using the antimammalian IL-1 polyclonal antibodies recognized the 15kDa factor from the macrophage supernatants, suggesting possible maturation events of the carp IL-1 [195]. In an independ‐ ent survey of PHA-stimulated carp leukocytes using a monoclonal anti-carp IL-1β antibody,

the 15 kDa protein was confirmed to be a mature carp IL-1β, with the putative carp IL-1β cleavage site situated approximately 15 amino acids downstream of the mammalian ICE site [117]. Analysis of the supernatants of the trout macrophage cell line, RTS-11, using an antitrout IL-1β polyclonal antibody detected putative native, as well as potentially cleaved trout IL-1β proteins of 29 kDa and 24 kDa, respectively [79]. When the RTS-11 cells were transfect‐ ed with a plasmid encoding a C-terminally His6 tagged trout IL-1β, and the RTS-11 superna‐ tants assayed using an Ni-NTA-column specific for the His6 tagged IL-1β, the authors detected primarily a mature, 24 kDa IL-1β protein, confirming that the trout IL-1β under‐ went maturation cleavage.

In an elegant set of studies it was demonstrated that combined immune stimuli of sea bream head kidney leukocytes resulted in an accumulation of a 30 kDa, pro-IL-1β protein, which, unlike the mammalian counterpart [113], did not exhibit a maturation cleavage or secretion following stimulation of the cells with extra-cellular ATP [146]. Furthermore, sea bream per‐ itoneal acidophilic granulocytes and peripheral blood leukocytes accumulated the 30 kDa form of IL-1β following challenge with *Vibrio anguillarum* [27]. In contrast, the sea bream SAF-1 fibroblast cell line shed a mature, 22 kDa IL-1β protein through microvesicular plas‐ ma membrane budding within 30 minutes of treatment with extracellular ATP [113]. Inter‐ estingly this IL-1β maturation/shedding process could be ablated with a pharmacological inhibitor of the mammalian ICE, suggesting a presence of an orthologous sea bream enzyme responsible for this process.

The P2X7R receptor is the primary receptor responsible for the recognition of extracellular ATP and the concomitant release of mature IL-1β [50]. Upon ATP treatment and activation of the HEK 293 mammalian cell line expressing the rat P2X7R, human ICE and sea bream IL-1β, a non-cleaved (30 kDa) sea bream IL-1β was secreted by these cells [109]. Interesting‐ ly, neither sea bream nor zebrafish P2X7R expression in ATP-stimulated HEK 293 cells re‐ sulted in sea bream or mammalian IL-1β secretion. In contrast, the expression of a chimeric P2X7R bearing the sea bream ATP-binding and rat intracellular domains led to maturation/ secretion of the mammalian IL-1β, while the outcomes of this combination on the sea bream IL-1β were not addressed [109]. The authors of this work suggest that the mechanisms in‐ volved in IL-1β secretion are conserved across vertebrates while the distinct stimuli that elic‐ it the maturation events are not. We propose that alternatively, functional specificity may stem from the P2X7R intracellular signaling, activation of species specific ICE (or alternative maturation mechanisms), and the substrate specificity of the fish Caspase1/ICE. Notably, the above group also identified the sea bream Caspase 1 and demonstrated that the recombinant form of this fish enzyme cleaved a commercially available substrate, for which the mamma‐ lian counterpart holds specificity [107]. Additional studies are needed to establish the associ‐ ation between the sea bream Caspase I/ICE, P2X7R and IL-1β.

#### *3.3.5. IL-1 receptors of fish*

Although there have been no reports of a fish IL-1α, recently a novel IL-1 family member, nIL-1F has been identified in trout and *Tetraodon* spp. and was reported to have IL-1 family sig‐ nature motif as well as a putative ICE cleavage site [198]. The expression of nIL-1F increased af‐ ter activation of macrophages with LPS or recombinant IL-1β. Furthermore, a recombinant form of the C-terminal nIL-1F abrogated the rIL-1β-induced immune gene expression in RTS-11 trout macrophage-like cells, suggesting a possible competitive inhibition through the trout IL-1RI. This mechanism would be analogous to the mammalian IL-1Ra, which also com‐

Because many fish species are tetraploid and have undergone genome duplication events, it is not surprising that additional isforms of IL-1β exist in certain fish species. An identified second trout IL-1β, denoted IL-1β2, also has the 6 exon/5 intron organization, 82% amino acid identity to the trout IL-1β1 and no putative ICE cleavage site [150]. In catfish, two IL-1β genes have been described and shown to undergo distinct expression patterns following challenge with *Edwardsiella ictaluri* [201]. Additionally identified cDNA of another carp IL-1β, IL-1β2, exhibited 74% identity with the carp IL-1β1 and 95-99% identity across indi‐ vidual IL-1β2 transcripts [48]. Since several distinct IL-1β2 sequences were identified in a homozygous individual, it was suggested that there might be multiple IL-1β2 genes. The ex‐ pression of the carp IL-1β1 and IL-1β2 differed following immune stimuli where the expres‐ sion of IL-1β1 gene was on average at least ten fold greater than that of IL-1β2. It was also reported that transcripts of IL-1β2 had high substitution numbers in the coding regions, in‐ cluding key areas predicted to be involved in receptor binding. In light of the above, it was suggested that the IL-1β2 may be a pseudogene [48]. Our observations in the goldfish, a close carp relative, support this theory. Notably, the predicted goldfish IL-1β2 protein is truncated compared to the IL-1β1, and while the expression of both IL-1β isoforms is subject to change following immune stimuli, specific treatments elicit more robust changes in the

As alluded to above, despite the functional similarities of the fish IL-1βs and their mammali‐ an counterpart, all fish IL-1β proteins identified to date lack the typical ICE cleavage site necessary for the functional maturation of the mammalian IL-1β1. Despite this, evidence suggests that indeed the fish IL-1β proteins undergo cleavage. Early fish IL-1 studies using anti-mammalian IL-1α/β serum to surveys supernatants of activated fish cells demonstrated the recognition of distinct, multiple IL-1 protein species in these supernatants. While the cat‐ fish monocyte supernatants contained 70 kDa and 15 kDa molecules with IL-1-like activity [47], carp macrophages secreted 22 kDa and 15 kDa IL-1-like factors that were recognized by mammalian anti-IL-1 antibodies [195]. The immunoprecipitation experiments using the antimammalian IL-1 polyclonal antibodies recognized the 15kDa factor from the macrophage supernatants, suggesting possible maturation events of the carp IL-1 [195]. In an independ‐ ent survey of PHA-stimulated carp leukocytes using a monoclonal anti-carp IL-1β antibody,

petes with IL-1β for IL-1RI binding, without inducing downstream events.

expression of IL-1β1 compared to IL-1β2 (unpublished observations).

*3.3.4. Maturation cleavage of the fish IL-1β*

*3.3.3. Isoforms of IL-1β in fish*

72 New Advances and Contributions to Fish Biology

Majority of the IL-1 receptor signaling components have been described in fish. A fish IL-1RI was first described in Atlantic salmon, with 43-44% similarity to chicken and 31% similarity to the human IL-1RI, respectively [186]. The expression of this gene increased in fish tissues following LPS injection. The identification of a carp IL-1R1 was also described and the re‐ ceptor characterized in the context of acute stress conditions [121].

head kidney cells alone or in combination with the P1 peptide (at low concentrations) [145]. Intraperitoneal injections of the rtIL-1β P3 peptide induced peritoneal leukocyte migration and enhanced phagocytosis and reactive oxygen production by peritoneal cells. Additional‐ ly, the rtIL-1β P3 conferred fish resistance to viral hemorrhagic septicemia virus (VHCV) early after injection. Surprisingly, injection of rtIL-1β P1 caused an increase in *in vivo* expres‐ sion of the antiviral gene Mx and inhibition of trout TNFα expression, while having no ef‐ fects on the expression of a panel of other pro-inflammatory genes [78]. The injection of the rtIL-1β P3 peptide resulted in a more robust, widespread pro-inflammatory response with increased expression in trout rtIL-1β, IL-8 and lysozyme. This work suggests that the fish IL-1β utilizes a highly complex receptor-ligand system, possibly unique to that of mammals. The functional roles of the trout IL-1β have been corroborated in others fish species. For exam‐ ple, injection of carp with a plasmid encoding the carp IL-1β gene caused enhanced PHA-in‐ duced proliferation of carp lymphocytes, increased carp macrophage reactive oxygen production, enhanced phagocytosis and improved protection against *A. hydophila* challenge [97]. A sea bass recombinant IL-1β exhibited immuno-adjuvant properties when combined with rsIL-1β in immunization trials against the pathogen, *V. anguillarum*. The sea bass IL-1β al‐ so induced the proliferation of the murine IL-1β reporter cell line (D10.G4.1), sea bass thymo‐ cytes, enhanced kidney leukocyte phagocytosis and activated peritoneal macrophages when administered i.p. [16, 17, 29]. The orange spotted grouper rIL-1β stimulated the proliferation of grouper head kidney leukocytes and increased the gene expression of IL-1β and COX-2 through p38 MAPK and Jnk signaling pathways [112]. Together, the above findings suggest

Cytokine Regulation of Teleost Inflammatory Responses

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75

that the functions of IL-1β have been evolutionarily conserved in vertebrates.

defined by the efficacy of the individual and interdependent cytokine pathways.

A successful inflammatory response is defined by the presence and proficient coordination of cytokine networks consisting of hallmark mediators such as TNFα, IFNγ and IL-1β. The synchronized involvement of these pleiotropic yet functionally distinct agents in the recruit‐ ment, regulation and functional polarization of inflammatory cells dictates the outcome of the mounted response. Thus, it can be argued that the inflammatory processes are largely

The regulation of the vertebrate inflammatory response is complex, involving numerous mechanisms, some of which are poorly understood while others remain to be identified. This is particularly true for the teleost model systems, where lack of specific reagents for dif‐ ferent fish species hampers our ability to examine different aspects of the regulation of in‐ flammation at a mechanistic level. However, there is growing evidence that the key immune components required for effective inflammatory responses are present in teleosts. Notably, certain fish species possess additional pathways that regulate inflammatory processes (for example IFNγrel and its receptor IFNGR1-1, novel chemokines and PRRs) that are distinct from those reported in mammals. The elucidation of the coordination of inflammatory re‐ sponses by these factors may shed new light on the evolution of innate host defense mecha‐

**4. Concluding remarks**

nisms in lower vertebrates.

The trout IL-1RII was identified through a selective subtractive hybridization of genes upregulated following immune stimulation [164]. This receptor displayed low sequence identi‐ ty with the mammalian IL-1RII, but exhibited surprisingly similar overall gene organization including a very short intracellular domain. The gene expression of the identified sea bream IL-1RII increased in stimulated macrophages to levels 15 times greater than IL-1β expression [108], suggesting a conservation in the roles of the sea bream IL-1RII as a "decoy" receptor. Furthermore, sea bream IL-1RII expressed on HEK 293 cells bound the recombinant IL-1β, confirming the specificity of this receptor-ligand pair.

Enhanced understanding of the evolutionary mechanisms responsible for shaping the verte‐ brate IL-1 biological pathways will be achieved through studies that examine the IL-1 recep‐ tor-ligand relationships and biological outcomes of these interactions in teleosts.

#### *3.3.6. Inflammatory roles of the fish IL-1β*

Using the selective subtractive hybridization technique, a carp IL-1β was identified [53] and the C-terminus of the cytokine produced as a recombinant protein [206]. This recombinant carp IL-1β dimerized and enhanced carp antibody responses to *Aeromonas hydrophila*, where sera from carp co-injected with the IL-1β and *A. hydrophila* had a greater agglutinating ca‐ pacity than the respective controls.

A number of studies have since utilized recombinant technology to investigate the functions of the fish IL-1β. A recombinant form of the mature trout IL-1β (rtIL-1β) was produced by Hong and co-workers [80] and shown to enhance the expression of the MHCII β chain, IL-1β and COX-2 genes in trout head kidney leukocytes and the macrophage cell line, RTS-11. Functionally, rtIL-1β elicited the proliferation of trout head kidney cells as well as the prolif‐ eration of a murine cell line, D10.G4.1, known for its dependence on the mammalian IL-1β [80]. Also, rtIL-1β enhanced the phagocytosis of yeast particles by trout head kidney cells [80] while peritoneal admininstrations of rtIL-1β induced migration of trout leukocytes to the site of injection, enhanced phagocytosis of peritoneal cells and increased the systemic ex‐ pression of IL-1β, COX-2 and lysozyme II [77]. The injection of fish with rtIL-1β also en‐ hanced trout resistance to infection with the fish pathogen *A. salmonicida*. Additionally, the rtIL-1β and the recombinant sea bass IL-1β were demonstrated to induce Ca2+ mediated downstream signaling events, abrogated by leukocyte trypsin-treaments and indicating a re‐ quirement for receptor engagement [9]. Interestingly, these authors reported that IL-1β of trout, sea bass and humans were highly species specific, which is in contradiction of the ear‐ ly fish IL-1β carp and catfish work, where cross-reactivity was observed [9, 47, 74, 175, 195].

The distinct biological roles of individual sub-domains of the trout IL-1β were also exam‐ ined by generating appropriate peptides [78, 144, 145]. While a control scrambled peptide (P2) had no effect and the peptide corresponding to the putative trout IL-1β receptor bind‐ ing region (P1) had little effect on its own, a peptide (P3) corresponding to an alternative re‐ ceptor binding area of the mammalian IL-1β [5, 196], had chemotactic properties towards head kidney cells alone or in combination with the P1 peptide (at low concentrations) [145]. Intraperitoneal injections of the rtIL-1β P3 peptide induced peritoneal leukocyte migration and enhanced phagocytosis and reactive oxygen production by peritoneal cells. Additional‐ ly, the rtIL-1β P3 conferred fish resistance to viral hemorrhagic septicemia virus (VHCV) early after injection. Surprisingly, injection of rtIL-1β P1 caused an increase in *in vivo* expres‐ sion of the antiviral gene Mx and inhibition of trout TNFα expression, while having no ef‐ fects on the expression of a panel of other pro-inflammatory genes [78]. The injection of the rtIL-1β P3 peptide resulted in a more robust, widespread pro-inflammatory response with increased expression in trout rtIL-1β, IL-8 and lysozyme. This work suggests that the fish IL-1β utilizes a highly complex receptor-ligand system, possibly unique to that of mammals.

The functional roles of the trout IL-1β have been corroborated in others fish species. For exam‐ ple, injection of carp with a plasmid encoding the carp IL-1β gene caused enhanced PHA-in‐ duced proliferation of carp lymphocytes, increased carp macrophage reactive oxygen production, enhanced phagocytosis and improved protection against *A. hydophila* challenge [97]. A sea bass recombinant IL-1β exhibited immuno-adjuvant properties when combined with rsIL-1β in immunization trials against the pathogen, *V. anguillarum*. The sea bass IL-1β al‐ so induced the proliferation of the murine IL-1β reporter cell line (D10.G4.1), sea bass thymo‐ cytes, enhanced kidney leukocyte phagocytosis and activated peritoneal macrophages when administered i.p. [16, 17, 29]. The orange spotted grouper rIL-1β stimulated the proliferation of grouper head kidney leukocytes and increased the gene expression of IL-1β and COX-2 through p38 MAPK and Jnk signaling pathways [112]. Together, the above findings suggest that the functions of IL-1β have been evolutionarily conserved in vertebrates.
