Antimicrobial Peptides as Mediators of Innate Immunity in Teleosts

Antimicrobial peptides (AMPs) have been identified throughout the metazoa suggesting their evolutionarily conserved nature and their presence in teleosts is no exception. AMPs are short (18–46 amino acids), usually cationic, amphipathic peptides. While AMPs are diverse in amino acid sequence, with no two AMPs being identical, they collectively appear to have conserved functions in the innate immunity of animals towards the pathogens they encounter in their environment. Fish AMPs are upregulated in response to pathogens and appear to have direct broad-spectrum antimicrobial activity towards both human and fish pathogens. However, an emerging role for AMPs as immunomodulatory molecules has become apparent—the ability of AMPs to activate the innate immune system sheds light onto the multifaceted capacity of these small peptides to combat pathogens through direct and indirect means. Herein, this review focuses on the role of teleost AMPs as modulators of the innate immune system and their regulation in response to pathogens or other exogenous molecules. The capacity to regulate AMP expression by exogenous factors may prove useful in modulating AMP expression in fish to prevent disease, particularly in aquaculture settings where crowded conditions and environmental stress pre-dispose these fish to infection.


Introduction
Antimicrobial peptides (AMPs) are a diverse group of individually unique small peptides  amino acids) found in plants to animals that display broad-spectrum antimicrobial activity towards OPEN ACCESS bacteria, viruses, parasites, and fungi. Recent studies have also implicated AMPs as host defense peptides (HDPs) that function as immunomodulators adding a layer of complexity to their functional role in defense against pathogens. AMPs can be produced at a constitutive level but can also be induced in response to pathogens. The major pathway of AMP production is through the recognition of pathogen associated molecular patterns (PAMPs) via Toll-like receptors (TLRs) thereby triggering an intracellular signaling cascade involving MyD88, TRAF6, IRAK1 and IKK [1,2]. This signal transduction pathway ultimately leads to the activation and translocation of NF-B to the nucleus where it transcriptionally activates AMPs and other pro-inflammatory molecules [1,2]. The AMPs are synthesized in inactive forms as part of a prepropeptide and are enzymatically cleaved to release the functional mature peptide. The widespread nature of AMPs suggests that they are an evolutionarily ancient mechanism of host defense against pathogens and their diversity in amino acid sequence and structure points to an evolutionary pressure to cope with a specific milieu of microbes. The > 1000 AMPs identified to date has necessitated their organization in databases, such as the Antimicrobial Peptide Database [3].
Despite the reports of AMPs in amphibians and humans in the late 1970s and early 1980s, the identification and study of fish AMPs only began in the mid to late 1990s. To date, over 90 fish AMPs have been identified [3] and fall into five major families based on their structure; β-defensins, cathelicidins, hepcidins, histone-derived peptides, and the fish specific piscidins. These fish AMPs appear to retain a similar overall structure, broad-spectrum antimicrobial activity and immunomodulatory function to that of other vertebrate AMPs. The focus of this review is to explore the regulation of teleost AMPs in response to pathogen challenge and the immunomodulatory function of teleost AMPs studied thus far. A comprehensive review of the antimicrobial activity of fish AMPs towards a wide array of fish and human pathogens and the minimal inhibitory concentrations at which antimicrobial activity occurred are reviewed in [4] and will not be discussed herein. The persistence of AMPs in nature for over millions of years demonstrates the inability of microbes to develop resistance against AMPs, making AMPs an interesting target for use as antibiotic replacements in the medical field and in aquaculture as therapeutics.
The β-defensins of fish appear to be produced as a prepeptide. The prepeptide is synthesized as a precursor that is comprised of a 18-24 amino acid signal peptide and a 39-45 amino acid mature peptide, with the exception of olive flounder in which a 15 amino acid proregion is also present, and is cleaved to produce the mature peptide [5,[15][16][17]. The resulting mature β-defensin peptides are small (4-6 kDa), share the 6 conserved cysteine residues present in the vertebrate defensins, retain the CPRRYK/R motif between cysteines 4 and 5 and have an overall net cationic charge between +1 and +7 [5,6,10,11,15,16,18]. Fish β-defensins appear to fall into two main groups and have an overall three beta-strand secondary structure, although some β-defensins are predicted to possess an extra alpha-helix at the N-terminus of the three-beta strand structure [5,8].
During embryonic development β-defensin transcripts are expressed early on and at constitutive levels [8,12,17] suggesting that β-defensin may play a key role in host defense during embryogenesis. In adult fish, the expression pattern and levels of β-defensins varies greatly depending on the fish species and the defensin gene [12]. However, the primary expression of β-defensin appears to be in the immune and mucosal tissues including the intestine, gill, kidney, spleen, skin, and liver [6,13,14,[18][19][20], with expression also detected in the muscle, stomach, eye, gonad, heart, blood, monocytes and lymphocytes [5,6,11,15,16,20,21]. In some fish species, β-defensin transcripts were also detected in the pituitary, testis [7], swim bladder and peritoneum (with low levels in the skin and kidney) [8]. However, β-defensin expression is seemingly absent in the brain [16]. The broad distribution of β-defensin in fish tissues suggests that β-defensin plays a role in defense against infection in these tissues or may play broader role in fish physiology.
In fish model systems where antibodies to species-specific β-defensin have been generated, it appears that mRNA levels of β-defensins correspond to protein levels [7,11,19]. Whether this is true for all fish species or just in the fish species examined in the aforementioned study remains to be determined. However, the confirmation of mRNA transcripts to protein correlation suggests that mRNA expression is likely a good indicator of β-defensin protein production. One study noted the translocation of β-defensin peptides from the cytoplasm to the nucleus where viral replication was occurring in infected cells [18], demonstrating that β-defensins are actively mobilized to sites of pathogen replication within a cell. Recombinant or synthetic fish defensins have demonstrated antimicrobial activity towards Gram-negative bacteria [6,11,17,19,20], Gram-positive bacteria, [19,20] and viruses [8,15,18], although the antimicrobial activity was dependent on the fish species and the defensin gene. A complete list of MICs for fish β-defensins and the pathogens they have been tested against can be found in [4]. Interestingly, fish β-defensins have not been tested against fungi or parasites and represents an area of study that requires further investigation.

Regulation of Defensins by Pathogens/Exogenous Factors
The expression of β-defensins is modulated in response to a number of pathogens (Tables 1-4), similar to that observed for other teleost AMPs. Inducers of β-defensin transcript levels include pathogen components such as LPS [10], bacterial DNA [11], unmethylated CpG oligodeoxynucleotides [11] and -glucan [13], as well as pathogens including Gram-negative bacteria, Gram-positive bacteria and viruses (see Tables 1-4 and references therein). Pathogen challenge usually results in the rapid induction of β-defensin transcript levels 12 h post challenge, with transcript levels decreasing thereafter [10,18], suggesting that -defensin, like other AMPs, are important molecules in the first line of defense against pathogens. The regulation of β-defensin transcripts in tissues in response to pathogen challenge is species and gene specific, although upregulation of β-defensin is commonly observed in the head kidney, spleen, liver, skin, gills and eye [6,8,11,13,17,18]. However, in one study in which common carp were infected with carp herpes virus 3, a decrease in the β-defensin skin transcript levels was observed suggesting that this particular virus may be able to immunomodulate the host to create an immunosuppressed environment permissive to viral replication [22]. The constituents of a fish's diet can also act to regulate β-defensin expression. Gilthead seabream fed microalgae diets exhibited an increase in the expressions of defensin transcripts in the head kidney [23], while rainbow trout fed peptidoglycan spiked food showed an increase in defensin mRNA levels in the skin and gill tissues [24]. These studies suggest that pathogens, their components and entities within fish feed can modulate the expression of AMPs and may impact the susceptibility or resistance of fish to disease.
Examination of the upstream promoter region of β-defensin genes has revealed binding sites for myocyte-specific enhancer factor-2 (MEF-2), activating protein-1 (AP-1), CCAAT-binding factor (CBF), interferon regulatory factor (IRF), CCAAT/enhancer binding protein (C/EBP), IL-6 responsive element binding protein (IL-6 RE-BP), stimulating protein-1 (Sp1), lymphocyte-enhancer factor-1 (LEF-1), nuclear factor of activated T-cells (NF-AT) [17,19] and NF-B [11] transcription factors. The prediction of a multitude of transcription factor binding sites suggests that β-defensins are regulated by a number of upstream activation pathways leading to transcriptional activation of β-defensins in fish in response to varied stimuli, supported by the array of pathogen components and pathogens that can modulate the expression of β-defensins in fish.

Modulation of Immunity by Defensins
Studies in fish suggest that β-defensins may also act as HDPs and are capable of modulating the immune response. In vitro studies have demonstrated that treatment of rainbow trout RTG-2 cells with rainbow trout defensin [9] or ZF4 zebrafish embryonic fibroblast cells with zebrafish β-defensin-2 (zfBD2) resulted in the upregulation of Mx gene expression [25], suggestive of a type I interferon response. In vivo studies in fish have confirmed the increased mRNA levels of Mx and pro-inflammatory cytokines such as IL-1β and TNF-α in fish tissues in response to injection with β-defensin [18].
Furthermore, expression of zfBD2 in zebrafish ZF4 cells promoted the translocation of NF-κB from the cytoplasm to the nucleus thereby linking NF-κB pathway activation, and possibly upstream TLR signaling, as a mechanism of immune cell activation in response to β-defensins [26]. The activation of NF-κB coincides with the increased transcript levels of pro-inflammatory cytokines observed in cells and tissues upon treatment with β-defensins as NF-κB is known to transcriptionally activate AMPs and a number of pro-inflammatory molecules. Functional in vitro studies have shown defensins to be chemoattractive to gilthead seabream head kidney leukocytes [6] and to stimulate the phagocytic activity of Atlantic cod head kidney leukocytes [8], while in vivo studies with zebrafish suggest a recruitment of fish cytotoxic cells to the site of β-defensin injection [25]. It is perhaps through some or all of these immunostimulatory mechanisms that the in vivo expression of zebrafish BD2 conferred a partial enhancement of resistance towards spring viraemia of carp virus (SVCV) infection compared to wildtype zebrafish.
Amongst fish species, the majority of the cathelicidins have a four-exon/three-intron genomic structure like that of mammalian cathelicidins [24,27]. The cathelicidin transcripts encode for a 159-222 amino acid polypeptide [24,25,27,32] composed of a signal sequence, a N-terminal preproregion which contains the highly conserved cathelin domain, encoded by exons 1-3, and a highly variable C-terminal sequence, encoded by exon 4, that contains a QKIRTRR elastase cleavage site and a unique cathelicidin mature peptide [24,25,27,28,32]. The sequence of the fourth exon amongst fish is hypervariable and gives rise to the suite of distinct mature peptides that differs between and within fish species [25]. The mature peptide is 47-69 amino acids, has a charge of +8 to +17 [24,25,27,30] and studies on rainbow trout cathelicidins suggest that fish cathelicidins are mainly β-sheet and random coil structures [33]. Variations to the general structure of cathelicidins have been discovered. In Arctic charr (Salvelinus alpinus) and brook trout (Salmo trutta fario) the identified cathelicidins had a deletion of exon 3 [24]. In rainbow trout, one of the four identified cathelicidin genes was missing exons 1, 2, and 3-only exon 4 which encoded the mature peptide was present [33].
Cathelicidins are expressed early on in development and throughout fish development [34], suggesting they play a role in immunity early on. In adults, cathelicidin expression is highly variable and is dependent on the cathelicidin gene, tissue and fish species examined. For example, cathelicidin 1 is not normally expressed in rainbow trout tissues but can be induced by pathogen challenge [27,31,32], whereas S. trutta fario cathelicidin 1 is highly expressed in the head kidney, trunk kidney and spleen, followed by the skin, gill and stomach of normal animals, but not detected in the brain or the testis [25]. However, cathelicidin 2 genes appear to be ubiquitously expressed in tissues such as the gill, kidney, intestine, skin and spleen [31][32][33]. In fish species such as Arctic charr with only one cathelicidin gene, cathelicidin shows constitutive expression with highest levels present in the skin, spleen and kidney, but also detected in the gill, liver, pyloric caeca, intestine, brain, heart and muscle [24]. Antibodies directed towards rainbow trout cathelicidins found cathelicidin peptides localized in the gut mucosa and around the sinusoids of the head kidney, associated with the lymphoid cells present in these areas [33]. Thus, cathelicidins in fish appear to be broadly distributed, however their location deviates with fish species and cathelicidin class.
Fish cathelicidins have broad-spectrum activity against Gram-negative bacteria, Gram-positive bacteria [27][28][29]31,33,35], and fungi [35]. A comprehensive list of antimicrobial activity, MICs and pathogen species can be found in [4]. Similar to the observations regarding transcript distribution, the antimicrobial activity of fish cathelicidins is highly variable depending on the fish species, pathogen, and particular cathelicidin. For example, while rainbow trout cathelicidins are active against Y. ruckeri, Atlantic salmon cathelicidins are not [31]. Initial studies in rainbow trout have demonstrated cathelicidins to bind LPS in a dose-dependent manner and exert their antimicrobial activity through membrane permeabilization [33]. Deletion studies in rainbow trout also showed the N-terminus of the mature cathelicidin peptides to be important for antimicrobial activity, although the extent of loss of activity in these deletion mutants was variable depending on the cathelicidin gene [33]. The activity of cathelicidins likely occurs as a result of the hypervariable nature of the mature cathelicidin peptide, suggesting their structural differences at the C-terminus are important in determining activity and selectivity against pathogens.

Regulation of Cathelicidins by Pathogens/Exogenous Factors
The expression of cathelicidin transcripts can be induced by pathogens and pathogen components in a time dependent manner and is species and gene copy dependent [36]. The promoter region of cathelicidin contains predicted sites for NF-IL-6, C/EBPβ, α-IRE, γ-IRE, NF-1 and NF-κB transcription factor binding sites [27,37], further supporting the role of cathelicidin as an immune responsive gene in fish. Indeed, cathelicidins transcript levels are induced in vivo in response to an array of Gram-negative and Gram-positive bacteria (Tables 1-4) primarily in the spleen, kidney, gill, liver and intestine in a time dependent manner, usually within 24 h post challenge [24,27,31,32,38]. Treatment of rainbow trout cell lines with Saprolegnia parasitica revealed the differential regulation of cathelicidin 1 versus cathelicidin 2; cathelicidin 1 transcripts were upregulated in the rainbow trout cell lines RTL and RTS11 while cathelicidin 2 increased in RTG-2, RTGill, RTL and RTS11 rainbow trout cell lines [39]. Similar in vitro studies have demonstrated a time-, dose-, and cathelicidin class dependent increase in cathelicidin transcripts in Chinook salmon embryo cell lines (CHSE-214) in response to live bacteria, bacterial DNA and flagellin, occurring within 12 to 24 h post challenge [30,36]. Increasing amounts of bacteria led to a concentration-dependent increase in cathelicidin transcripts and this increase was time dependent, peaking at 24 h post challenge and decreasing sharply thereafter [36]. Furthermore, the Golgi apparatus, microtubules and PI3K signaling pathway appear to be important for inducing cathelicidin transcription in CHSE-214 fish cells in response to flagellin [36], suggesting a role for TLR5 and the downstream signaling pathway in the regulation of cathelicidin transcription. Cathelicidin transcript levels are also increased in response to pro-inflammatory cytokines such as IL-1β1 and IL-6 in rainbow trout macrophages and RTL cells [39,40]. Exogenous molecules can also influence cathelicidin transcript levels. For example, zymosan treatment of the rainbow trout intestinal epithelial RTgutGC cell line with zymosan in vitro or feeding rainbow trout a zymosan supplemented diet induced cathelicidin and IL-1β expression which were co-localized in the intestinal epithelial cells in vivo [41]. In addition, treatment of Atlantic salmon with 17β-estradiol or testosterone decreased the overall parasite load of the salmon louse ectoparasite and these fish were found to have higher mRNA levels of cathelicidin suggesting that cathelicidin may be an important factor in decreasing parasite load [42]. The modulation of cathelicidin in response to pathogens, pathogen components, inflammatory cytokines, and hormones suggest a role for cathelicidins in the immune response of fish towards pathogens.

Modulation of Immunity by Cathelicidins
Limited studies have been performed on teleost cathelicidins regarding their immunomodulatory role in fish. Both rainbow trout [33] and Atlantic salmon [31] cathelicidins induce the expression of IL-8 in fish peripheral blood leukocytes (PBLs) at early, but not later, time points following treatment of cells with cathelicidins [31]. However, Atlantic salmon cathelicidins did not induce IL-1 or IL-18 transcript levels, suggestive of selective induction of pro-inflammatory cytokines involved in chemotaxis [31]. Lastly, rainbow trout cathelicidin 2 treatment of rainbow trout RTgutGC cells was shown to induce IL-1β transcript levels [41]. Taken together, it appears that the regulation of pro-inflammatory transcripts by cathelicidins in fish occurs rapidly and is species, cathelicidin and cell type specific. Future studies on the immunomodulatory properties of teleost cathelicidins are needed in order to elucidate the role of cathelicidins in fish immunity.
Piscidins are produced as a 64-89 amino acid prepropeptide that undergoes proteolytic cleavage at the N-terminus to remove the signal peptide and at the C-terminus to cleave away the prodomain, releasing a 18-26 amino acid mature peptide that is ~2.5 kDa [43,55,56,[60][61][62]. However, the piscidin 4 genes result in a 44 amino acid mature peptide that is ~5.5 kDa [56,57]. The mature piscidin peptides have an amphipathic -helical conformation with the hydrophilic side being cationic and capable of interacting with pathogen membranes in a parallel fashion [48,[62][63][64]. Collectively, piscidin family members seem to be undergoing positive selection [53,65], suggesting they may be undergoing diversification as a means to adapt to rapidly changing pathogens and would support the low sequence identity amongst the mature peptides of piscidin family members. Differential tissue expression of piscidin genes within a species and also between fish species exists [58,59]. Piscidins are expressed early during fish development and generally increase in expression with fish development [54,58,59]. In adult tissues, piscidin transcripts and peptides are usually found at high levels in the skin, skin mucus, gill, intestine/gastrointestinal tract, head kidney, and spleen [43,45,47,49,50,54,[57][58][59]66], while some piscidins are expressed more broadly and can also be detected in the liver, blood, gall bladder, pyloric caeca, stomach, rectum, muscle, heart, and brain [50,54,58,60]. Within these tissues, expression of piscidins have been detected in blood leukocytes, head kidney and spleen phagocytes, kidney hematopoietic cells [67], epithelial and basal cells of the dorsal skin, epithelial cells in the gill, multi-granular cells in the muscle, chondrocytes from cartilaginous tissues, epithelial columnar cells and multi-granular cells in the intestine, liver hepatocytes, and neuronal cells in the swim bladder [67]. However, piscidins are most commonly found in mast cells from the gills [46,47,51,68] at concentrations that are found to be inhibitory to fish pathogens in vitro [68,69], in mast cells and rodlet cells of the skin, gill and intestine [70], in eosinophilic granular cells [71], epithelial mucous cells [55,61,62], intestinal goblet cells [61,62], and in phagocytic granulocytes of the intestinal mucosa [72]. Furthermore, upon phagocytosis of bacteria by intestinal phagocytic granulocytes, the piscidin-containing granules fused with the bacteria-filled phagosome [72]. Together, these results support the involvement of piscidins in mucosal innate immunity [61], that piscidins are produced at sufficient concentrations in vivo at mucosal locations to have direct antimicrobial activity, and that they play an important inflammatory role in the elimination of pathogens. Furthermore, the lowered levels of piscidin in diseased and stressed fish [69] suggests the regulation of piscidin in vivo may be used as a marker for the health status of fish.
Piscidin family members demonstrate antimicrobial or growth inhibition activity towards a wide array of Gram-negative and Gram-positive bacteria [48,52,[54][55][56]58,60,62,73] with differing bacterial selectivity in a time-dependent fashion [62] and over a considerable temperature and salt range [55]. Piscidins also possess anti-viral activity under a broad range of physiological conditions and temperatures [74], anti-fungal activity [75,76], anti-parasite activity at physiological concentrations of piscidins found in fish species [49,54,[77][78][79] and anti-tumor activity towards mammalian cells [80][81][82][83]. Piscidins can also act in synergy with other AMPs to enhance their activity [61,84,85]. Studies on the mechanism of action surrounding the anti-bacterial activity exhibited by piscidins show they interact with acidic phospholipids and form toroidal pores in the membrane [86][87][88]. While the extent of anti-fungal and anti-mammalian tumor activity varies with the piscidin gene expressed, the piscidin anti-fungi and anti-mammalian tumor activity appears to occur through membrane permeabilization and pore formation as well as the induction of reactive oxygen species and apoptotic pathways [75,76,[80][81][82][83]89]. The in vitro antimicrobial studies performed support piscidins as direct defense molecules against a wide array of pathogens in vivo and their key role in the fish immune system.

Regulation of Piscidins by Pathogens/Exogenous Factors
The upstream promoter regions of members of the piscidin family contain putative binding sites for NF-IL-6, alpha-IRE, gamma-IRE [58,61], AP-1 [58], C/EBPβ [55] and hepatocyte nuclear factor (HNF-1) [44] transcription factors, with HNF-1 shown to be required for the transcriptional activation of epinecidin in response to LPS [44]. Although the receptors, intracellular signaling pathway and transcription factors involved in the regulation of piscidin family members in response to pathogens have yet to be explicitly identified and characterized, it appears that LPS [43], Gram-negative and Gram-positive bacteria [45,47,48,54,60,66,90], poly I:C [47,66], viruses [47,48] and parasites [46,51,[91][92][93][94] enhance the expression of piscidin family members in fish spleen, kidney, skin, and intestinal tissues as well as in mast cells (Tables 1-4 and references therein). However, it is important to note that the regulation of piscidin transcript and protein levels is pathogen, fish, tissue and piscidin dependent, thus requiring detailed studies of each fish species, pathogen, tissue, and piscidin family member. Despite the general induction of piscidin family members in fish challenged with pathogens, some systems exist in which piscidin family members are not induced in response to pathogens such as the case with moronecidin transcripts which remained unchanged in hybrid sea bass challenged with S. iniae [55]. That fish piscidin family members are largely inducible in response to a broad array of pathogen or pathogen components provides support for their essential role in the immune response for defense against pathogens.

Modulation of Immunity by Piscidins
Members of the piscidin family possess immunomodulatory activity. In vitro studies have shown treatment of the rainbow trout RTS11 macrophage cell line with pleurocidin to induce the expression of IL-1 and COX1 transcripts in a dose and time dependent manner, but did not impact Mx, antigen presentation or JAK/STAT gene expressions [73]. However, unlike that of other AMPs, treatment of RTS11 fish cells with LPS and pleurocidin did not affect the LPS inflammatory induced response [73]. Meanwhile, studies in the cured barramundi brain (cBB) fish cell line showed grouper epinecidin-1 to induce Mx transcripts [84]. Thus, it appears that induced gene expression is dependent on the piscidin family member and the cell line employed.
Collectively, in vivo studies have shown piscidins to aid in inhibiting pathogen replication, lowering pathogen load and increasing host survival in a number of fish species when challenged with bacteria such as Vibrio vulnificus [95][96][97][98] or viruses such as nervous necrosis virus (NNV) [84,99,100]. While these studies share the overall finding of piscidin protection of fish against pathogens, the mechanism of how this is achieved appears to vary depending on the piscidin gene, route of piscidin administration, fish species and pathogen used for challenge. For example, in grouper (Epinephelus coioides) Mx2 and Mx3 transcript levels were down regulated in response to piscidin [84]. Meanwhile, transgenic zebrafish expressing epinecidin-1 showed induced MyD88 expression [98], which differed from zebrafish injected with epinecidin-1 that induced IL-10, IL-15, IL-17c, and MAPK gene expressions but failed to induce any changes in MyD88, TLRs, IFN, IL-1, IL-12, IL-4/IL-13A, leptin, NF-B, SP1, TNF- or REL-A [95]. Interestingly, in a study examining the differential expression of proteins in zebrafish injected with epinecidin-1, researchers failed to identify any differences in immune proteins and instead found changes in the levels of proteins involved in cell assembly, organization, function, and maintenance [101]. Finally, when fish were fed E. coli expressing epinecidin-1, enhanced expressions of TNF, TLR4, IL-1, nitric oxide synthase 2, and NF-B were observed [96]. However, in this case it is difficult to discern whether the gene expressions observed were solely the result of epinecidin-1 or whether the E. coli in conjunction with epinecidin-1 were responsible for the transcriptional activation of key players in the pro-inflammatory response. Despite the studies performed on fish piscidins and their immunomodulatory role in fish, the immunomodulatory roles of fish piscidins remains somewhat clouded and further studies are needed to elucidate the mechanism of action in which piscidins appear to confer resistance towards a number of pathogens in vivo.
Fish hepcidins are encoded as prepropeptides having three domains; a 22-24 amino acid leader domain, a 40-47 amino acid proregion domain, followed by a 20-26 amino acid mature peptide domain that contains the 6-8 cysteine residues that are important in the formation of the 3-4 disulphide bonds [105,117,[123][124][125][126], yielding a 2-3 kDa mature peptide. However, it should be noted that some fish hepcidins only possess four cysteine residues in their mature peptides [105], such as the red-spotted grouper (Epinephelus akaara) [127], the orange-spotted grouper (Epinephelus coioides) [118,119] and rainbow trout (Oncorhynchus mykiss) [113]. Sequences of the mature hepcidin peptides from fish to mammals show high conservation of the 8 cysteine residues found within the mature peptide, with one of the four disulphide bridges being a vicinal disulphide bridge, are rich in beta sheets and cationic with a tendency to primarily form amphipathic molecules [105,113,114,117,121,125]. Conservation of the cysteine residues required for disulphide bridges points to their functional importance in conferring antimicrobial activity towards pathogens and their amphipathic nature is likely important for the interaction with and insertion of hepcidins into microbial membranes or interaction with intracellular components.
Expression of hepcidins occurs early during development and in some fish species appears to increase in expression with development [106,124,127]. In adult fish, hepcidin is primarily expressed in the liver, as is the case in the red-spotted grouper (Epinephelus akaara) [127]. However, depending on the fish species and the isoform, hepcidin can also be found expressed in the skin, gill, head kidney, trunk kidney, intestine, muscle, ovary, gonad, heart, spleen, brain, stomach, blood and peritoneal leukocytes [106][107][108]121,123,128]. Furthermore, studies in gilthead seabream demonstrated that hepcidin is most highly expressed in acidophilic granulocytes compared to monocytes/macrophages and lymphocytes [128].
In those fish species that have multiple hepcidin isoforms, expression is tissue specific, displaying differing patterns of expression between fish for similar isoforms, tissue specific for multiple isoforms within the same fish species and their regulation in response to pathogen challenge is also isoform specific [116,119,120,[129][130][131]. It is proposed that the multiple copies of hepcidin have arisen due to the whole genome duplications that have occurred in fish and that the dual role of mammalian hepcidin has been sub-functionalized between the hepcidin copies observed in fish, i.e. one hepcidin has iron regulatory roles while the second hepcidin copy has retained the antimicrobial role [114,132,133]. This is supported by the inducible expression of one hepcidin copy while the other lacks inducible expression in response to pathogen challenge and rather appears to be iron responsive [114,117,119,133,134]. In those fish with only a single hepcidin gene, the dual function in iron homeostasis and as an AMP appears to be conserved [135]. However, for fish species that have greater than two hepcidin isoforms, the sub-functionalization or neo-functionalization of these isoforms have yet to be clearly elucidated. It appears that positive selection is occurring in the hepcidins of fish [136,137].
Collectively, the hepcidins of fish possess antimicrobial activity towards a broad range of pathogens in vitro, including Gram-negative bacteria, Gram-positive bacteria, viruses, and parasites and demonstrate varying minimal inhibitory concentrations depending on the particular pathogen, the fish species the hepcidin is derived from, and the hepcidin isoform itself. The antimicrobial activity of recombinant AMPs and synthesized AMPs from a number of fish species, in a dose-dependent manner, has been demonstrated [111,126,138]. A comprehensive list of the MICs associated with each fish hepcidin and the pathogen they demonstrate antimicrobial activity against can be found in [4].
Studies examining the structure-function relationship of fish hepcidins have shown a number of key features to be necessary for antimicrobial function. In zebrafish, the cysteine residues in a recombinant hepcidin-2 peptide were found to be essential for the binding of recombinant zebrafish hepcidin-2 to LPS, LTA and PGN and direct antimicrobial effects observed, suggesting that zebrafish hepcidin-2 acts as a pattern recognition molecule [126]. Studies on trout hepcidin revealed an amine terminal Cu 2+ and Ni 2+ binding (ATCUN) motif that is important for metal binding and was essential for DNA hydrolysis [139]. However, the truncation of this ATCUN motif in trout hepcidin did not result in loss of antimicrobial activity towards E. coli and P. salmonis [139]. Membrane permeabilization studies and confocal microscopy suggested that the mechanism of bacterial growth inhibition observed in response to trout hepcidin was not through bacterial membrane permeabilization or destruction, but rather appeared to be through the interaction with the bacterial DNA, regardless of the presence or absence of the ATCUN motif [139]. However, another study on turbot hepcidins found turbot hepcidin to be capable of binding to E. tarda and this interaction caused the bacteria to look transparent as a result of alteration of the bacterial surface, suggesting that turbot hepcidins were disrupting the outer membrane of the bacteria [111]. Furthermore, pre-treatement of E. tarda with turbot hepcidins prior to infection of flounder FG fish cells greatly reduced the number of viable intracellular bacteria present in the cell cultures [111]. Collectively, these studies demonstrate key structural properties of fish hepcidins and the multiple mechanisms in which they can exert their microbial action.
A few studies have demonstrated the antimicrobial activity of fish hepcidin in vivo. Prior in vivo administration of synthetic tubort hepcidins greatly reduced the E. tarda bacterial load or megalocytivirus load recovered from the kidney, spleen and liver of infected fish compared to that of non-treated infected control fish [111]. Studies have also employed the use of transgenic zebrafish for the over expression of tilapia hepcidin 1-5 (TH1-5) in live animals. Overexpression of TH1-5 in transgenic zebrafish conferred resistance to challenge with V. vulnificus or S. agalactiae compared to wildtype zebrafish [140,141]. Lastly, in vitro studies in hybrid striped bass demonstrate synergy between antimicrobial peptides [142], suggesting that the current manner in which in vitro and in vivo studies are performed (i.e., measuring MICs for single AMPs at a time) may not be reflective of the actual physiological conditions found within an organism that often expresses more than a single AMP. Although these studies do demonstrate the antimicrobial capacity of fish AMPs in vivo, the levels at which the AMPs were artificially simulated may be outside of the physiological range of AMP expression in wildtype fish, or may require "partner" AMPs in order to attain optimal activity. Further studies are required in order to ascertain whether AMPs have a direct antimicrobial action on fish pathogens under physiological conditions.

Regulation of Hepcidin by Pathogens/Exogenous Factors
In accord with the role of hepcidin in iron homeostasis in mammals, fish hepcidin is upregulated in response to iron or anemia and can be modulated by transferrin [104,121,135,143,144], suggesting that hepcidin isoforms also participates in iron homeostasis in fish. Furthermore, hepcidin expression in fish can be rapidly upregulated in response to IL-6 and bone morphogenic protein (BMP) [40,[145][146][147], bacterial components such as LPS, Gram-negative bacteria, Gram-positive bacteria, viruses or viral mimics and parasites (see Tables 1-4 and references therein), suggesting that hepcidin is an acute phase protein and involved in an inflammatory response akin to that of hepcidin in mammals [104,148,149]. The tissues in which hepcidin expression can be induced in response to pathogen challenge are broad, often depending on the fish species and hepcidin isoform studied. For example, bass hepcidin is upregulated to the greatest extent in the liver and to a lesser extent in the skin, gill, intestine, spleen, kidney and blood in Streptococcus iniae challenged fish [108]. However in some fish species such as catfish, hepcidin levels in the liver did not increase upon bacterial challenge, yet increased instead in kidney and spleen tissues [106]. The transcriptional activation of fish hepcidins in response to iron levels, IL-6, and pathogens is in accord with the presence of putative binding sites for the HNF, STAT, NF-B, and C/EBP transcription factors found in the fish hepcidin promoter regions [106,108,109,120], as IL-6 signals primarily though the JAK/STAT pathway and detection of pathogens and pathogen components can occur via TLRs and downstream NF-B activation. It is interesting to note that fish hepcidins can also be modulated in response to environmental contaminants [150,151] and high temperatures [152], implicating environment impacts on the potential health of fish. Together, these studies support the role of hepcidin as an APP required for the sequestering of iron during an immune response to limit pathogen growth and as an AMP with direct antimicrobial activity towards pathogens, both of which act together to limit the spread of infection.

Modulation of Immunity by Hepcidin
Fish hepcidins, in addition to having iron-regulating and antimicrobial properties, have demonstrated immune-modulating properties, albeit limited studies have been performed demonstrating this attribute. Simple in vitro studies in which Chinook salmon embryo (CHSE-214) cells were treated with tilapia hepcidin 1-5 (TH1-5) lead to an immediate (2 h post treatment) induction of annexin and metallothionein mRNA levels [153], but returned to basal levels 16 h post treatment. In vivo studies in which transgeneic zebrafish overexpressing TH1-5 were challenged with V. vulnificus and S. agalactiae exhibited enhanced resistance to infection with these pathogens [140,141]. Examination of immune gene regulation between the transgenic and wildtype zebrafish revealed that transgenic TH1-5 zebrafish exhibited higher mRNA levels of IL-10, IL-21, IL-22, lysozyme, NF-B, TNF- and TLR-1 compared to that of wildtype zebrafish. These data suggest that TH1-5 acts to upregulate these immune molecules in fish, leading to immunoregulation in the host that confers resistance to V. vulnificus and S. agalactiae in vivo. [140,141]. In some cases, fish hepcidins are used to treat mammalian cells in attempts to elucidate their immunomodulatory properties and the pathways through which they work. Treatment of mammalian RAW264.7 macrophage cells with tilapia hepcidin 2-3 (TH2-3) induced a elongated shape in mammalian RAW264.7 cells, found to be triggered through PKC (inhibits PKCµ-744 phosphorylation but promotes PKCµ-916 phosphorylation) and MEK pathways and resulted in a decrease in the mRNA levels of markers associated with dendritic cells [153,154]. However, the action of TH2-3 also appeared to abrogate the dendritic like shape induced by LPS in the mammalian RAW264.7 cells and was found to suppress TNF-, IL-1, IL-1, IL-6, and cyclooxygenase-2 (COX-2) expression in the presence of LPS [153,154]. Despite the latter study using a fish hepcidin in a mammalian model system, these data seem to suggest that fish hepcidin may act as an immunostimulatory peptide in the absence of pathogen components, but may act to immunomodulate and dampen a potentially exaggerated an harmful pro-inflammatory response in the presence of bacterial components. Thus, it appears that hepcidin may also act as an important HDP to regulate inflammation.

Conclusions
The past 20 years of research on teleost AMPs have identified over 90 peptides from five classes of AMPs and reported on their genomic organization, copy number, conserved motifs and structures, transcript distribution over fish development and within tissues, antimicrobial activity towards an array of fish and human pathogens, and their expression in response to pathogens. Studies on teleost AMPs have only just begun to investigate the potential synergy between AMPs, their transcriptional regulation in response to host pro-inflammatory molecules, the immunomodulatory activities AMPs possess in the absence or presence of infection and the receptors, intracellular signaling molecules and transcription factors that regulate them. Of particular interest is the receptor(s) to which AMPs bind in fish to elicit an immunostimulatory response in the absence of pathogens and how this pathway converges with pathogen-activated pathways to then immunomodulate and dampen pro-inflammatory pathways in the presence of pathogens. It is intriguing to ponder if the binding of AMPs to pathogens at sub-MIC levels may act to opsonize the pathogen and enhance immune receptor signaling leading to immune activation and destruction of the AMP-bound pathogen in fish, particularly in light of their chemoattractive activity. It may be possible that there is a dose-dependency to the type of response elicited from teleost immune cells in response to AMPs. With over 28,000 species of teleosts, the large diversity of teleost AMPs, each with their own unique species-specific tissue/cell distribution, antimicrobial activity and immunomodulatory activity, in conjunction with the positive selection occurring on AMPs speaks to their adaptation to the fish-specific pathogens they defend against. In light of the expansive number of teleost species, it is likely that many fish AMPs will be identified in future studies.
The immunomodulatory activity of teleost AMPs has broad application to fish health, particularly in aquaculture settings where high density rearing that are permissive for rapid pathogen transmission and stress-induced immunosuppression can occur. The addition of compounds, such as TLR ligands, to fish feed that induce mucosal AMP expression may provide fish with a means of protection against mucosal fish pathogens, either as a preventative measure or during active infection. However, further studies are required in order to optimize levels of AMP-inducers in fish feed to prevent disregulation in AMP expression in fish, perhaps leading to a prolonged inflammatory response and tissue destruction in fish if applied in too high of a dose or for too long. Furthermore, monitoring the expression of AMPs has the potential to be an indicator of fish health, allowing for appropriate and timely strategies to be employed to maintain fish health [175].
Teleost AMPs may also have applications in the medical field. Antibiotic resistant bacteria are increasing at an alarming rate, necessitating the development of other antimicrobial agents for the therapeutic treatment of patients. Of particular consideration is the use of AMPs as a replacement strategy for the failing antibiotics due to the demonstrated antimicrobial activity of teleost AMPs both in vitro and in vivo, broad specificity and ability to modulate the immune response in mammals [176][177][178]. However, while this is an emerging area, much research is still needed in order to translate the use of AMPs as antibiotic replacements into the medical field.