K. Tsukamoto, T. Kawamura, T. Takeuchi, T. D. Beard, Jr. and M. J. Kaiser, eds.Fisheries for Global Welfare and Environment, 5th World Fisheries Congress 2008, pp. 263–276.© by TERRAPUB 2008.

Molecular Innate Immunity in Teleost Fish:Review and Future Perspectives

Takashi Aoki,* Tomokazu Takano,Mudjekeewis D. Santos, Hidehiro Kondo

and Ikuo Hirono

Laboratory of Genome ScienceTokyo University of Marine Science and Technology

Konan 4-5-7 Minato-ku, Tokyo 108-8477, Japan

*E-mail: aoki@kaiyodai.ac.jp

Innate immune response is a primitive form of defense mechanism existent in plantsand animals. It is a complex system which, in vertebrates, is composed of cellular andhumoral responses. The vertebrate teleost fish, which diverged from the tetrapod lin-eage about 450 million years ago, has an innate immune component that showsconsiderable conservation with higher vertebrates particularly in mammals highlightedby the presence of orthologous pattern recognition receptors (PRRs) and stimulatedcytokines. However, there is also increasing evidence of teleost fish components andfunctions that are not observed in mammals suggesting complexity and diversity inteleost fish innate immune function. Knowledge about the network and function ofthese innate immune-related molecules in model and/or economically important fishis therefore important and new molecular biological tools such as large scale geneexpression profiling including DNA microarrays and protein-based techniques willhelp elucidate such information. This review focuses on the molecular innate immunemechanisms in teleost fish in reference to the known mammalian system. It also in-cludes future perspectives on how this basic knowledge coupled with existing mo-lecular biological techniques can contribute to the control of fish disease and therebyimprove aquaculture.

KEYWORDS molecular immunity; teleost fish; pathogen recognition receptors; in-flammatory cytokines

1. Introduction

Innate immunity is the first line of defenseagainst infection and is regarded as the pri-

meval and hence the universal form of hostdefense (Janeway and Medzitov 2002). Itexists in animals (vertebrates and inverte-brates) and plants, and recent data suggeststhat it is a product of convergent rather than

264 T. AOKI et al.

divergent evolution (Ausubel 2005). Com-mon features of the system across speciesinclude conserved receptors for microbe-rec-ognition, antimicrobial peptides and mi-togen-associated protein kinase signalingcascades.

Innate immunity is generally subdividedinto two parts, the cellular and humoraldefense responses. Cellular responses in-clude the physical barrier such as mucus andepithelial tissues lining the skin, gills andstomach, which keeps infectious microor-ganisms from entering the body, and special-ized cells (like monocytes/macrophages,granulocytes and nonspecific cytotoxic cells)capable of killing and digesting pathogensif the latter breaches the physical barriers.These cells are recruited in the infection siteprimarily by inflammatory cytokines. Hu-moral responses, on the other hand, employa variety of proteins and glycoproteins ca-pable of destroying or inhibiting growth ofinfectious microorganisms, which includeamong others anti-bacterial peptides,proteases, complement, transferrins and theantiviral myxovirus resistance-1 protein(Mx1).

In mammals, innate immunity is trig-gered by the recognition of conserved mi-crobial products by receptors generallycalled pathogen recognition receptors or pat-tern recognition receptors (PRRs) (Fig. 1).These molecules can distinguish ‘infectiousnonself’ from the ‘noninfectious’ self. PRRs,which include both extracellular andcytosolic recognition, mediate several path-ways that give rise to the production of in-flammatory cytokines and interferons (Leeand Kim 2007). In teleost fish, such mecha-nisms are believed to be conserved mainlybecause of the presence of PRR and cytokineorthologs (Plouffe et al. 2005; Stein et al.2007). Furthermore, microarray analysis hasrevealed transcriptional modulation of vari-ous cloned fish PRRs and cytokines follow-ing bacterial agents (Kurobe et al. 2005;Gerwick et al. 2007; Peatman et al. 2007;

Peatman et al. 2008). On the other hand, in-creasing evidence also shows that there aremolecules and mechanisms that are specificto teleost fish (Plouffe et al. 2005; Stein etal. 2007).

In this paper, we review the innate im-mune system network in fish gathered fromavailable information and studies includingpathogen recognition and the ensuingcytokine cascade output as well as innateimmune-related cellular functions. We drawcomparisons of these with the known mol-ecules and mechanisms in mammals, in theabsence/limited functional information inteleost fish, as well as highlight recent andgrowing innate immune functions that areteleost fish-specific. We also present somefuture perspectives on the basic study of fishimmunology and the techniques that wouldhelp produce this knowledge, as well as thepossible applications that could be generatedfor the improvement of aquacultural produc-tivity.

2. Mammalian Innate ImmuneResponses

2.1. Pathogen recognition

In mammals, innate immune response is ini-tiated through the recognition of pathogensby the PRRs. PRR families that have beenidentified thus far include the extracellularToll-like Receptors (TLRs), C-type lectinsand complement receptors CRs, and thecytosolic nucleotide-binding domain andLRR proteins (NLRs) (Table 1). It is knownthat these PRRs are able to trigger variousinnate immune responses including comple-ment pathway, apoptosis, leukocyte activa-tion and migration, and cytokine production(Lee and Kim 2007).

TLRs, now one of the most studied bio-logical molecules discovered because of theToll receptor in Drosophila, are consideredto be vital to microbial pathogen recognition.Its extracellular leucine-rich repeat (LRR)

Molecular innate immunity in teleost fish 265

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Molecular innate immunity in teleost fish 267

domains are responsible for specifically rec-ognizing molecular signatures of variouspathogens while the cytoplasmic Toll/IL-1R(TIR) domain directs signals that inducespro-inflammatory cytokine, Type I interferonand chemokines production (Kawai andAkira 2005). To date, 10 members of TLRshave been identified in human, and 13 inmouse (Krishnan et al. 2007).

On the other hand, C-type lectins, ofwhich the mannose-binding lectin (MBL) thebest characterized, recognize enveloped vi-rus, bacteria and fungi structure via cell tocell interactions following Ca2+-dependentmanner (Epstein et al. 1996; Vasta et al.1999). Through the binding of extracellularMBL to the pathogen then to MBP-associ-ated serine proteases (MASPs), the lectincomplement pathway is induced and pro-duces pro-inflammatory cytokines (Guptaand Surolia 2007). Recently, another C-typelectin, dectin1, was found to recognize fun-gus and β-glucan (Robinson et al. 2006).Upon binding of dectin1 to pathogen, theimmunoreceptor tyrosine-based activationmotif (ITAM) also induces immune re-sponses (Ariizumi et al. 2000; Brown 2006).Another lectin, the mammalian intelectin(IntL), is a Ca2+-dependent enteric moleculethat has specific affinities to D-pentoses andD-galactofuranosyl residues as well as rec-ognizes Nocardia rubra arabinogalactan(Tsuji et al. 2001). Pentraxins recognize awide range of pathogenic substances and al-tered self molecules and acute phase proteins(Mantovani et al. 2007). Examples of whichare the C-reactive protein (CRP), serumamyloid P component (SAP) and Pentraxin3 (PTX3) (Mantovani et al. 2007).

The complement receptors such as CR3and CR4 are able to mediate phagocytosisof opsonized pathogens and/or apoptoticcells. Further, C5aR is known as immuno-regulatory signal generator. C5a is a potentinflammatory agent that has diverse func-tions including chemotaxis, phagocytosisenhancement, reduction of neutrophil

apoptosis, and IL-12 production throughC5aR. The PI-3K-Akt and ERK signalingpathways are known C5aR cascade that leadto the expression of the anti-apoptotic pro-tein Bcl2 so far (Lee and Kim 2007).

The NLR family, are recently discoveredPRRs involved in the cytoplasmic recogni-tion of whole bacteria and their products suchas flagellin and peptidoglycan (Kaparakis etal. 2007). It is composed of 2 major sub-families, the NODs and the NACHT-LRR-PYD-containing proteins, or NALPs. NODsand NALPs induce the production of pro-inflammatory cytokines and even antimicro-bial peptides upon pathogen recognition viathe NLR signaling pathway and the“inflammasome” pathway, respectively(Kaparakis et al. 2007). The other cytoplas-mic PPRs of retinoic acid-inducible gene I(RIG-I) and melanoma-differentiation-asso-ciated gene 5 (MDA5) possesses RNAhelicase activity to mediate the antiviral re-sponses (Kaparakis et al. 2007).

2.2. Cytokine cascade

Cytokines, specifically pro-inflamma-tory cytokines and Type I interferons, areknown to be induced following the activa-tion by signal pathways as result of the stimu-lation of PRRs (Table 2). Cytokines havepleiotropic (multiple), overlapping andsometimes contradictory functions such thattheir classification is oftentimes difficult.These molecules have been grouped basedon general names (e.g. lymphokines,monokines, chemokines, interleukins,interferons, tumor necrosis factors, colonystimulating factors etc); on functions (e.g.pro- and anti-inflammatory or innate andadaptive immunity-related); on structure(e.g. short and long chain cytokines); and onreceptors used (immunoglobulin superfam-ily, hematopoetic growth factor (type 1-fam-ily) , interferon family (type 2-family), tumornecrosis factor (type 3-family) and chemo-kine receptors (7 transmembrane helix

268 T. AOKI et al.

family). Cytokines here were grouped basedon available recent reviews for each of thestated group; Cytokine Class 1, CytokineClass 2, Chemokines, TNF Super-family andIL1 Family (Table 1).

Class 1 cytokines are often involved inthe proliferation, recruitment to inflamma-tion/infection sites, survival and maturationof cells (Huising et al. 2006). Because oftheir pleiotropic nature, they can also be in-volved in other physiological processes likereproduction, food intake and metabolism.They share similar 4α-helical-2β sheet 3Dstructure and use the class 1 cytokinereceptors. Although they share little primarysequence identity, these cytokines are con-sidered to have expanded from a single an-cestor (Bazan 1990).

Class II cytokines, in contrast to ClassI, generally acts on minimizing damage tohost after noninfectious and infectious insult,and include the interferons which mediatesantiviral responses and IL10 known to beanti-inflammatory in nature (Krause andPestka 2005). Their structure is likewise dif-ferent with Class 1 cytokines in that their βsheets have been replaced by α-helices(Langer et al. 2004).

Chemotactic cytokines or chemokines,are mostly involved in leukocyte migrationand are known as “second-order” cytokines,being induced by pro-inflammatory “first-order” cytokines like IL-1s, TNFs, IFNs.These are a family of small proteins thatregulate cell migration under both inflam-matory conditions or homeostasis (Peatmanand Liu 2007). Chemokines are subdividedbased on the arrangement of the 1st 2 of the4 cysteine residues; CC, CXC, C and CX3C(Bacon et al. 2003).

TNF ligand superfamily (TNSF)cytokines, which includes the tumor necro-sis factor-α (TNF-α), lymphotoxin-α andlymphotixin-β, are structurally related pro-teins, either soluble or membrane-bound, thatare produced during lymphoid organ devel-opment and inflammation. (Gruss 1996)

These molecules are involved mainly in cel-lular regulation such as tumor cell killingduring immune responses and inflammatoryreactions.

Interleukin 1 family is one of the criti-cal early pro-inflammatory cytokines thatdown- or up-regulate other cytokines duringinfection or inflammation (Huising et al.2004). These molecules are produced bymonocytes, activated macrophages, neutro-phils, endothelial cells, fibroblasts, Langer-hans cells of the skin and other types of cells.IL18, which is a recently discovered mem-ber, was observed to stimulate (IFN)-γ pro-duction, T-helper1 (Th1) cell differentiationand enhances cell cytotoxity of natural killercells (Akira 2000).

3. Teleost Innate Immune Responses

3.1. Pathogen recognition

Many components of the innate immunesignaling are conserved between teleost fishand mammals, with very clear orthologousrelationships. Examples of the latter are thepresence of teleost fish PRRs such as TLRs,Lectins, complemenet recptors, NLRs (Ta-ble 1).

Teleost fish have been shown to func-tionally react to a number of TLR agonistsand that this recognition induces cytokineexpression similar to those observed in mam-mals (Purcell et al. 2006). In particular, TLRsignaling molecules have been establishedto be evolutionary conserved in teleost fishincluding myeloid differentiation factor 88(MYD88), TIR domain-containing adapterprotein (TIRAP), TIR-domain containingadaptor including INF-β(TRIF), TRIF-re-lated adaptor protein (TAM), sterile α andHEAT-Armadillo motifs (SARM), interferonregulatory factor 3 (IRF3) and interferonregulatory factor 7 (IRF7) (Purcell et al.2006). As such, it is now believed that thebasic program of gene regulation by TLR-signaling pathway is conserved across

Molecular innate immunity in teleost fish 269

vertebrates. A total of 11 types of teleost fishTLRs (TLR 1, 2, 3, 5, 7, 8, 9, 14, 21, 22 and23) have been including isotypes for TLR 1,4, 8 and 21 (Jault et al. 2004; Roach et al.2005). Their orthology suggests that theyalso possess the same type of pathogen rec-ognition mechanism as with their mamma-lian counterparts.

The complement system of teleost fish,which is composed of soluble plasma pro-teins involved in immune responses, hasbeen reviewed recently (Boshra et al. 2006).It has been shown to be involved inopsonization, phagocytosis and inflamma-tion. Fish complement components includethe C1q/MBL family, C1r/C1s/MASP fam-ily, C2/Bf and factor D molecules,C3/C4/C5family, membrane attack complex (MAC)assembly, complement regulatory proteinsand complement receptors.

Lectin is well studied in various teleostfish, although the detail of correlation to thefish immunity is unclear (Russell andLumsden 2005). Teleost fish MBL is able torecognize mannose, N-acetyl-D-glucosamine(GlcNAc) and glucose (Nakao et al. 2006).This has also been demonstrated with lam-prey MBL suggesting that lectin complementpathway exist as early as in agnathans(Takahashi et al. 2006). IntL homologs havelikewise been identified in teleost fishthrough gene expression profiling (Gerwicket al. 2007; Peatman et al. 2007; Peatman etal. 2008).

3.2. Cytokine cascade

Similar to PRRs, there are also clearorthologous cytokines in teleost fish, particu-larly of the Class I, Class II, chemokines,TNF superfamily and IL1 families (Table 2).Coupled with the PRR orthologues, this in-formation suggests that the innate immunesystem in teleost fish also exhibits the samegeneral mechanism of recognition and cas-cade seen in mammals. Recent reviews alsodiscussed the similarities of innate immune

mechanisms between teleost fish and mam-mals where the presence of fish orthologuesfor antimicrobial peptides, pro-inflammatorycytokines, e.g. TNF-α, IL-1β, IL-18,interferons, chemo-kines and IL-8, and com-plement have been established (Plouffe et al.2005).

Gene or protein orthology and conser-vation allows speculation for similarity offunction but functional genomics and pro-tein assays are desirable to ascertain func-tion. With the whole genome sequences infish, recent publications have started to re-port functionalities of immune-related genesand their proteins. An example of gene struc-ture and function conservation between fishand humans is the action of erythropoietin(EPO) and erythropoietin receptor (EPOR)in red blood cell formation and hemato-poiesis and under hypoxic conditions(Paffett-Lugassy et al. 2007). Another is themacrophage colony-stimulating factor(CSF-1), which like in mammals, was ob-served to induce proliferation of monocytesto macrophages (Hanington et al. 2007).

4. Only in Teleost Fish

The Whole Genome Duplication (WGD) thathappened early in the life of ray-finned fishesis now increasingly believed to have hap-pened about 350 to 450 million year ago andis the main reason for the explosion of thefish species diversity at >23, 500 spp. (Volff2005). The gene duplication that happenedresulted in the creation of numerous novelor semi-novel genes and functions in fish,known as “more genes in fish than mam-mals” concept (Ohno 1970). It is thereforenot surprising that there are already 5 ge-nomic databases sequenced thus far for thistaxon; the zebrafish, medaka, stickleback,tiger pufferfish and the green spottedpufferfish (www.ensembl.org). Large scaleteleost fish genome analysis has revealednumerous gene duplications that are thoughtto originate from the WGD (Stein 2007). This

270 T. AOKI et al.

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toki

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Molecular innate immunity in teleost fish 271

was clear with genes involved in signal trans-duction downstream of the transmembranereceptors but not with molecules involvedin interactions with pathogen components.Genetic diversity translates to protein diver-sity and as such it is therefore very possiblethat in teleost fish, there will be a lot ofunique and differing functionalities amidstthe background of conserved functions. Infact, many of these fish-specific features arenow starting to be unraveled.

4.1. Pathogen recognition

Teleost fish TLR14, 21, 22 and 23 have notbeen found from mammals so far. TLR 14 isreported form lamprey, teleosts and amphib-ians, TLR 21 in teleosts, amphibians andbirds, TLR 22 in teleosts and amphibians,TLR 23 in Fugu (Roach et al. 2005). Thesoluble form TLR5 (sTLR5) is also uniquein teleost fish (Tsujita et al. 2004). Its pro-duction is facilitated by the NF-κB activatedby membrane TLR5 (mTLR5) upon recog-nition of Gram-negative bacteria flagellin(Tsujita et al. 2004). Conversely, there areno reports yet of mammalian TLR6orthologues in fish.

Fish complement/anaphylatoxin PUEF-8, which is composed of C3a, C4a and C5a,greatly enhance particle uptake in teleost fishleukocytes, increased the number of phago-cytic cells by 3- to 4-fold and acted as strongchemoattractant to peripheral blood and kid-ney leukocytes. These observations were notobserved in mammalian anaphylatoxins andare therefore novel functions only found infish (Boshra et al. 2006).

NLRs have been identified in teleost fishincluding a conserved NACHT-domain pro-tein which is unique in fish and species-spe-cific subgroup of NLRs with a novel N-ter-minal domain were found from zebrafish andpufferfish (Stein et al. 2007). Taken together,the divergence of the fish PRRs may indi-cate the species-specific pathogen recogni-tion pathways and immune responses.

4.2. Cytokine cascade

The differing functional attributes betweenteleost fish and mammals is now also beingobserved not only at the genetic but at theprotein level as well. Th2 cytokines (IL4,IL5, IL13) as well as the neighbor IL3 andgranulocyte-macrophage colony stimulatingfactor (GM-CSF) are apparently missing inteleost fish (Huising et al. 2006). Recently,there have been reports of members of theIL6-ctokine subfamily that appear to only befound in teleosts. A novel class-1 helicalcytokine M17 (named after the original clonenumber of the carp leukocyte cDNA library),cloned in 2003, was shown to be induced byalginate by (Fujiki et al. 2003) and latershown to be induced by nitric oxide and canstimulate proliferation of monocytes tomacrophages (Hanington and Belosevic2007). Its apparent paralogue named M17homologue (MSH) have been cloned re-cently and was shown to be induced by bac-terial agents, lipopolysaccharide, peptidog-lycan and the dsDNA, viral mimic polyI:C(Hwang et al. 2007). Another recently pub-lished fish-specific report showed that gold-fish possesses a soluble macrophage colonystimulating factor receptor (CSF1R), Interms of cytokine which can inhibit CSF1-induced proliferation of monocytes intomacrophages and monocyte-like cells(Hanington et al. 2007).

In terms of cytokine receptors, therehave also been reports of such genes that donot possess clear orthology to knownreceptors and appears to be more of ances-tral in structure. The Leukemia InhibitoryFactor Receptor (LIFR)-like molecule iden-tified in goldfish was suggested to be theancestral molecule for mammalian LIFR andOncostatin M Receptor (OSMR) (Haningtonand Belosevic 2005) although it still not clearwhether the LIFR-like genes predicted in thechromosome 12 of the green spottedpufferfish is orthologous to the goldfish

272 T. AOKI et al.

LIFR-like genes or to teleost OSMR as well(Jaillon et al. 2004). Another class 1-cytokinereceptor in fish which appears to be ances-tral in structure is the Japanese flounderglycoprotein 130 homologue or JfGPH dis-covered very recently (Santos et al. 2007).This receptor, which contains the classicWSXWS motif, the cytokine binding do-mains, and the Jak/STAT signaling boxes 1to 3 in its cytoplasmic region, does not pos-sess a detectable Ig-like N-terminal domain.It appears to be an ancestral gene for class 1cytokine receptors that is ubiquitously ex-pressed in tissues and down-regulated dur-ing viral-induced pathways.

Novel immune-type receptor (NITRs) isanother group of molecules belonging to theimmunoglobulin superfamily that have noknown homolog in mammals. These are ex-pressed mainly in spleen, kidney and intes-tine and were suggested to play importantroles in the regulation of cellular immuneresponses (Hawke et al. 2001). Recently, wehave identified an NITR in Japanese floun-der that is localized in both the T and B cellspointing to its specific importance in adap-tive immunity (Piyaviriyakul et al. 2007).

4.3. Cellular mechanisms

Fish rely heavily on innate immune re-sponses to deal with pathogens. With this isthe use of cells to combat pathogens. Noveltypes of cells that have been found in teleostfish but not mammals include non-specificcytotoxic cells (NCCs) (Plouffe et al. 2005).

Because of cellular and morphologicaldifferences, blood cells between mammalsand teleost fish, has always been suggestedto be functionally different (Rowley et al.1988). Recently, direct studies have identi-fied immune-related cellular functions in fishthat defies present paradigms in mammals.For example, unlike mammalian B cells,rainbow trout B cell populations were shownto have potent phagocytic capability i.e. fol-lowing uptake of particles, these cells could

induce “downstream” degradation pathwaysleading to formation of phagolysosome andintracellular killing of microbes (Li et al.2006). Zebrafish primitive macrophage lin-eage was shown to give rise to a number ofneutrophilic granulocytes in contrast tomammalian primitive macrophage which isconsidered to give rise to no other cell types.Only a small fraction of these zebrafish lar-val neutrophils phagocytose microbes ascompared to the macrophages and are moreattracted to stressed and malformed tissuessuggesting that these cells are involved in awider role than biodefense (Guyader et al.2007). The same functional adaptation hasbeen seen in seabream professional phago-cytic granulocytes, where these cells, otherthan immune surveillance, have a role in thereorganization of the testis during post-spawning (Chaves-Pozo et al. 2005).

5. Future Perspective

5.1. Basic knowledge in fish innateimmunity

There are undoubtedly numerous innate im-mune mechanisms in teleosts that resembleor are conserved with mammalian processes.This is not unexpected since it is regardedthat innate immunity is the universal andancient form of host defense against infec-tion (Janeway and Medzitov 2002). Presentstudies on orthologous teleost fish genes,albeit many are only up to expression stud-ies, points to general similarity in functionto higher vertebrates. Going forward, how-ever, it is the fish-specific molecules andprocesses that would usher a new era in ba-sic teleost fish immunological studies. Theapparent duplication of many of the teleostfish genes adds to the complexity of fishimmunology and could potentially revealnew mechanisms unheard of in the study ofimmunology. The availability of 5 fish ge-nomic databases (for zebrafish, fugu, greenspotted pufferfish, stickleback and medaka)

Molecular innate immunity in teleost fish 273

will certainly fast track the study of thesefish-specific phenomenon. This will allowfor a better understanding of fish immunityas well as how this relate to the evolution ofinnate immunity in vertebrates.

5.2. Technologies for fish innateimmunity

The advent of various molecular biologicaltechniques have paved the way in increas-ing our knowledge of fish innate immunityand these same technologies will still be veryuseful in uncovering this new information.For example, we have successfully used theExpressed Sequence Tag (EST) platform toidentify orthologous, paralogous and novelgenes in Japanese flounder (Aoki andHirono, 2005). We likewise have success-fully employed cDNA microarray platformto evaluate efficacy of vaccination using dif-ferent vaccine agents like formalin-killedcells, DNA and recombinant protein vaccines(Byon et al. 2006; Byon et al. 2005) as wellas comparing between vaccine agents likeviral G and N protein DNA vaccines(Yasuike et al. 2007). Technologies that areimportant for the future include PCR-basedtechniques including Quantitive Real-TimePCR, immunological-bases assays,recombinant protein analysis and microarraystudies. In mammals, these assays have beenvery effective in elucidating innate immu-nity and is expected that it will do the samefor fish.

5.3. Application to aquaculture

The detailed analysis of the innate immune-related molecules fish including their func-tion and network will certainly generate newtechnologies that can be applied to improveaquaculture.

5.3.1. Biomarkers

The production of healthy quality fish isimportant to consumers in terms of foodsafety and nutrient content, and to aquacul-

ture in terms of profitability. Biomarkerscould be utilized to monitor such quality. Ininnate immune responses, several importantmolecules are involved and the up- or down-regulation of these molecules provides somemeasure of the biological state of the fish,whether it is in normal condition or stresseddue to infection. For instance, C-reactiveprotein is an indicator for the internal inflam-matory responses in mammals. From ourstudies of the innate immune responses ofJapanese flounder at the molecular level, wehave discovered a number of potentialbiomarker genes such as antimicrobialagents, acute stress response factors, andvarious cytokines during the infectiouspathogen invasion (Aoki and Hirono 2005).The antiviral Mx1 gene is an interesting ex-ample of a biomarker to measure vaccineefficiency. We observed that it was only ex-pressed by rhabdovirus glycoprotein genevaccinated but not in rhabdovirus nucleocap-sid gene vaccinated fish (Yasuike et al.2007).

5.3.2. Vaccine/adjuvant

Our immunostimulation studies with the aidof extensive gene expression profiling likeESTs, cDNA microarray and Serial Analy-sis of Gene Expression (SAGE) generatedinformation on various pro-inflammatorycytokines that are potential for vaccine/adjuvant development. For example, we havepreviously shown that Japanese flounderIL-1β can induce various cytokines, cell sur-face antigens, signal transduction genes, sug-gesting that IL-1β is a potential vaccineadjuvant (Emmadi et al. 2005).

5.3.3. Transgenic

Fish transgenesis to produce transgenic fishwith commercially important traits such asgrowth enhancement and disease resistanceis still being pursued and many moleculesinvolved in innate immunity are target mol-ecules for such. We have, for example, suc-cessfully shown that the TNF promoter can

274 T. AOKI et al.

be a tissue-specific, inducible promoter thatcan be utilized to express foreign genes inJapanese flounder (Yazawa et al. 2005a–c).The transgenic technique itself could be usedto augment or complement the fish innateimmune response. For instance, the re-combinant Japanese flounder lysozyme hasbeen shown to be infective against Japaneseflounder pathogens like Edwardsiella tarda(Hikima et al. 2001). However, when chicken

lysozyme was expressed in transgeniczebrafish, the fish acquired resisatnce againstthe bacteria (Yazawa et al. 2006).

Acknowledgements

This study was supported in part by a research grantfrom the Grants-in-Aid for Scientific Research (S) fromthe Ministry of Education, Culture, Sports, Science andTechnology of Japan.

References

Akira S. The role of IL-18 in innate immunity. Curr. Opin. Immunol. 2000; 12: 59–63.Aoki T, Hirono I. Molecular immunity in fish-pathogen interactions. In: Leung, KY (ed). Current Trends in the

Study of Bacterial and Viral Fish and Shrimp Diseases. World Scientific Publishing, Singapore. 2004;256–291.

Ariizumi K, Shen GL, Shikano S, Xu S, Ritter R III, Kumamoto T, Edelbaum D, Morita A, Bergstresser PR,Takashima A. Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractivecDNA cloning. J. Biol. Chem. 2000; 275: 20157–20167.

Ausubel FM. Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 2005;10: 973–979.

Bacon K, Baggiolini M, Broxmeyer H, Horuk R, Lindley I, Mantovani A, Matsushima K, Murphy P, NomiyamaH, Oppenheim J. Chemokine/chemokine receptor nomenclature. Cytokine 2003; 21: 48–49.

Bazan JF, Haemopoietic receptors and helical cytokines. Immunol. Today 1990; 11: 350–354.Boshra H, Li J, Peters R, Hansen J, Matlapudi A, Sunyer JO. Cloning, expression, cellular distribution, and

role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in anonmammalian species. J. Immunol. 2004; 172: 4381–4390.

Brown GD. Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat. Rev. Immunol. 2006; 6: 33–43.Byon JY, Ohira T, Hirono I, Aoki T. Use of a cDNA microarray to study immunity against viral hemorrhagic

septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination. Fish Shell-fish Immunol. 2005; 135–147.

Byon JY, Ohira T, Hirono I, Aoki T. Comparative immune responses in Japanese flounder, Paralichthys olivaceusafter vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNAvaccine using a microarray analysis. Vaccine 2006; 24: 921–930.

Chaves-Pozo E, Mulero V, Meseguer J, Ayala AG. Professional phagocytic granulocytes of the bony fishgilthead seabream display functional adaptation to testicular microenvironment. J. Leukoc. Biol. 2005;75: 345–351.

Emmadi D, Iwahori A, Hirono I, Aoki T. cDNA microarray analysis of interleukin-1β induced Japanese floun-der Paralichthys olivaceus kidney cells. Fish. Sci. 2005; 71: 519–530.

Epstein J, Eichbaum Q, Sheriff S, Ezekowitz RA. The collectins in innate immunity. Curr. Opin. Immunol.1996; 8: 29–35.

Fujiki K, Nakao M, Dixon B. Molecular cloning and characterisation of a carp (Cyprinus carpio) cytokine-likecDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF. Dev. Comp.Immunol. 2003; 27: 127–136.

Gerwick L, Corley-Smith G, Bayne CJ. Gene transcript changes in individual rainbow trout livers followingan inflammatory stimulus. Fish Shellfish Immunol. 2007; 22: 157–171.

Gruss HJ. Molecular, structural and biological characteristics of the tumor necrosis factor ligand superfamily.Int. J. Clin. Lab. Res. 1996; 26:143–159.

Gupta G, Surolia A. Collectins: sentinels of innate immunity. Bioessays 2007; 29: 452–464.Guyader DL, Redd MJ, Colucci-Guyon E, Murayama E, Kissa K, Briolat V, Mordelet E, Zapata A, Shinomiya

H, Herbomel P. Origins and unconventional behavior of neutrophils in developing zebrafish. Blood2008; 111: 134–141.

Molecular innate immunity in teleost fish 275

Hanington PC, Belosevic M. Characterization of the leukemia inhibitory factor receptor in the goldfish(Carassius auratus). Fish Shellfish Immunol. 2005; 18: 359–369.

Hanington PC, Wang T, Secombes CJ, Belosevic M. Growth factors of lower vertebrates: characterization ofgoldfish (Carassius auratus) macrophage colony-stmulating factor—1. J. Biol. Chem. 2007; 282: 31865–31872.

Hawke NA, Yoder JA, Haire RN, Mueller MG, Litman RT, Miracle AL, Stuge T, Shen L, Miller N, Litman GW.Extraordinary variation in a diversified family of immune-type receptor genes. Proc. Natl. Acad. Sci.USA 2001; 98:13832–13837.

Hikima J, Minagawa S, Hirono I, Aoki T. Molecular cloning, expression and evolution of the Japanese floun-der goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochim. Biophys.Acta 2001; 1520: 35–44.

Huising MO, Stet RJM, Savelkoul HFJ, Lidy Verberg-van Kemenede BM. The molecular evolution of theinterleukin-family of cytokines; IL 18 in teleost fish. Dev. Comp. Immunol. 2004; 28: 395–413.

Huising MO, Kruiswijk CP, Flik G. Phylogeny and evolution of class-I helical cytokines. J. Endocrinol. 2006;189: 1–25.

Hwang JY, Santos MD, Kondo H, Hirono I, Aoki T. Identification, characterization and expression of novelcytokine M17 homologue (MSH) in fish. Fish Shellfish Immunol. 2007; 23: 1256–1265.

Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C,Bernot A et al. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early verte-brate proto-karyotype. Nature 2004; 431: 946–957.

Janeway CA, Medzhitov R. Innate immune recognition. Ann. Rev. Immunol. 2002; 20: 197–216.Jault C, Pichon L, Chluba J. Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Mol.

Immunol. 2004; 40: 759–771.Kaparakis M, Philpott DJ, Ferrero RL. Mammalian NLR proteins; discriminating foe from friend. Immunol.

Cell. Biol. 2007; 85: 495–502.Kawai T, Akira S. Toll-like receptor downstream signaling. Arthritis. Res. Ther. 2005; 7: 12–19.Kim HJ, Yasuike M, Kondo H, Hirono I, Aoki T. Molecular characterization and gene expression of a CXC

chemokine gene from Japanese flounder, Paralichthys olivaceus. Fish Shellfish Immunol. 2007; 23:1275–1284.

Kono T, Zou J, Bird S, Savan R, Sakai M, Secombes CJ. Identification and expression analysis of lymphotoxin-beta like homologues in rainbow trout Oncorhynchus mykiss. Mol. Immunol. 2006; 43: 1390–1401.

Krause CD, Pestka S. Evolution of the class 2 cytokines and receptors, and discovery of new friends andrelatives. Pharmacol. Ther. 2005; 106: 299–346.

Krishnan J, Selvarajo K, Tsuchiya M, Lee G, Choi S. Toll-like receptor signal transduction. Exp. Mol. Med.2007; 39: 421–438.

Kurobe T, Yasuike M, Kimura T, Hirono I, Aoki T. Expression profiling of immune-related genes from Japa-nese flounder Paralichthys olivaceus kidney cells using cDNA microarrays. Dev. Comp. Immunol. 2005;29: 515–523.

Langer JA, Cutrone EC, Kotenko S. The class II cytokine receptor (CRF2) family: overview and patterns ofreceptor-ligand interactions. Cytokine Growth. Factor. Rev. 2004; 15: 33–48.

Lee MS, Kim YJ. Signaling pathways downstream of pattern-recognition receptors and their cross talk. Ann.Rev. Biochem. 2007; 76: 447–480.

Li J, Barreda DR, Zhang YA, Boshra H, Gelman AE, LaPatra S, Tort L, and Sunyer JO. B lymphocytes fromearly vertebrales have potent phagocytic and microbicidal abilities. Nature Immunol. 2006; 7: 1116–1124.

Mantovani A, Garlanda C, Doni A, Bottazzi B. Pentraxins in Innate Immunity: From C-reactive protein to thelong Pentraxin PTX3. J. Clin. Immunol. 2008; 23: 1–13.

Nakao M, Kajiya T, Sato Y, Somamoto T, Kato-Unoki Y, Matsushita M, Nakata M, Fujita T, Yano T. Lectinpathway of bony fish complement: identification of two homologs of the mannose-binding lectinassociated with MASP2 in the common carp (Cyprinus carpio). J. Immunol. 2006; 177: 5471–5479.

Ohno S. Evolution by Gene Duplication. Springer, New York. 1970.Paffet-Lufassy N, Hsia N, Fraenkel PG, Paw B, Leshinsky I, Barut B, Bahary N, Caro J, Handin R, Zon LI.

Functional conservation of erythropoietin signaling in zebrafish. Blood 2007; 110: 2718–2726.Peatman E, Liu Z. Evolution of CC chemokines in teleost fish: a case study in gene duplication and implica-

tions for immune diversity. Immunogenetics 2007; 59: 613–623.

276 T. AOKI et al.

Peatman E, Baoprasertkul P, Terhune J, Xu P, Nandi S, Kucuktas H, Li P, Wang S, Somridhivej B, Dunham R,Liu Z. Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) afterinfection with a Gram-negative bacterium. Dev. Comp. Immunol. 2007; 31: 1183–1196.

Peatman E, Terhune J, Baoprasertkul P, Xu P, Nandi S, Wang S, Somridhivej B, Kucuktas H, Li P, Dunham R,Liu Z. Microarray analysis of gene expression in the blue catfish liver reveals early activation of theMHC class I pathway after infection with Edwardsiella ictaluri. Mol. Immunol. 2008; 45: 553–566.

Piraviriyakul P, Kondo H, Hirono I, Aoki T. A novel immune type receptor of Japanese flounder (Paralichthysolivaceus) is expressed in both T and B lymphocytes. Fish Shellfish Immunol. 2007; 22: 467–476.

Plouffe DA, Hanington PC, Walsh JG, Wilson EC, Belosevic M. Comparison of select innate immune mecha-nisms of fish and mammals. Xenotransplantation 2005; 12: 266–77.

Purcell MK, Smith KD, Hood L, Winton JR, Roach JC. Conservation of Toll-Like Receptor Signaling Pathwaysin Teleost Fish. Comp. Biochem. Physiol. Part D. Genomics. Proteomics. 2006; 1: 77–88.

Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A. The evolution ofvertebrate Toll-like receptors. Proc. Natl. Acad. Sci. USA 2005; 102: 9577–9582.

Robertsen B. The interferon system of teleost fish. Fish Shellfish Immunol. 2006; 20: 172–191.Robinson MJ, Sancho D, Slack EC, LeibundGut-Landmann S, Reis e Sousa C. Myeloid C-type lectins in

innate immunity. Nat. Immunol. 2006; 7: 1258–1265.Rowley AF, Hunt T, Page M, Mainwaring G. Fish. In: Rowley AF, Ratcliffe NA (eds). Vertebrate Blood Cells.

Cambridge University Press, Cambridge, UK. 1988; 19–128.Russell S, Lumsden JS. Function and heterogeneity of fish lectins. Vet. Immunol. Immunopathol. 2005; 108:

111–120.Santos MD, Yasuike M, Kondo H, Hirono I, Aoki T. A novel type-1 cytokine receptor from fish involved in

the Janus kinase/signal transducers and activators of transcription (Jak/STAT) signal pathway. Mol.Immunol. 2007; 44: 3355–3363.

Stein C, Caccamo M, Laird G, Leptin M. Conservation and divergence of gene families encoding compo-nents of innate immune response systems in the zebrafish. Genome Biol. 2007; 8: R251.

Takahashi M, Iwaki D, Matsushita A, Nakata M, Matsushita M, Endo Y, Fujita T. Cloning and characterizationof mannose-binding lectin from lamprey (Agnathans). J. Immunol. 2006; 176: 4861–4868.

Tsuji S, Uehori J, Matsumoto M, Suzuki Y, Matsuhisa A, Toyoshima K, Seya T. Human intelectin is a novelsoluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J. Biol.Chem. 2001; 276: 23456–23463.

Tsujita T, Tsukada H, Nakao M, Oshiumi H, Matsumoto M, Seya T. Sensing bacterial flagellin by membraneand soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mykiss). J. Biol. Chem.2004; 279: 48588–48597.

Vasta GR, Quesenberry M, Ahmed H, O’Leary N. C-type lectins and galectins mediate innate and adaptiveimmune functions: their roles in the complement activation pathway. Dev. Comp. Immunol. 1999; 23:401–420.

Volff JN. Genome evolution and biodiversity in teleost fish. Heredity 2005; 94: 280–294.Yasuike M, Kondo H, Hirono I, Aoki T. Difference in Japanese flounder, Paralichthys olivaceus gene expres-

sion profile following hirame rhabdovirus (HIRRV) G and N protein DNA vaccination. Fish ShellfishImmunol. 2007; 23: 531–541.

Yazawa R, Hiono I, Ohira T, Aoki T. Functional analysis of tumor necrosis factor gene promoter from Japa-nese flounder, Paralichthys olivaceus, using fish cell lines. Dev. Comp. Immunol. 2005a; 29: 73–81.

Yazawa R, Hiono I, Ohira T, Aoki T. Induction of Japanese flounder TNF promoter activity by lipopolysac-charide in zebrafish embryo. Mar. Biotechnol. 2005b; 7: 625–633.

Yazawa R, Hiono I, Aoki T.Characterization of promoter activities of four different Japanese flounder pro-moters in transgenic zebrafish. Mar. Biotechnol. 2005c; 7: 231–235.

Yazawa R, Hiono I, Aoki T. Transgenic zebrafish expressing chicken lysozyme show resistance against bac-terial disease. Transgenic Res. 2006; 15: 385–391.