Dietary methionine improves the european seabass (dicentrarchus labrax) immune status, inflammatory response, and disease resistance

ORIGINAL RESEARCH published: 20 No embe 2018 doi: 10.3389/ immu.2018.02672 F on ie s in Immunology | www. on ie sin.o g 1No embe 2018 | Volume 9 | A icle 2672 Edi ed by: Alexande G. Haslbe ge , Uni e si ä Wien, Aus ia Re iewed by: Zhonghai Yan, Columbia Uni e si y, Uni ed S a es Zhuo an Yin, Ha a d Medical School, Uni ed S a es *Co espondence: Ma ina Machado mcasimi o@ciima .up.p Benjamín Cos as bcos as@ciima .up.p Special y sec ion: This a icle was submi ed o Nu i ional Immunology, a sec ion o he jou nal F on ie s in Immunology Recei ed: 04 Sep embe 2018 Accep ed: 29 Oc obe 2018 Published: 20 No embe 2018 Ci a ion: Machado M, Aze edo R, Fon inha F, Fe nández-Boo S, Conceição LEC, Dias J and Cos as B (2018) Die a y Me hionine Imp o es he Eu opean Seabass (Dicen a chus lab ax) Immune S a us, In lamma o y Response, and Disease Resis ance. F on . Immunol. 9:2672. doi: 10.3389/ immu.2018.02672 Die a y Me hionine Imp o es he Eu opean Seabass (Dicen a chus lab ax) Immune S a us, In lamma o y Response, and Disease Resis ance Ma ina Machado1,2,3,4*, Ri a Aze edo1,3, Filipa Fon inha1,3, Se gio Fe nández-Boo1, Luis E. C. Conceição5, Jo ge Dias5and Benjamín Cos as1,3* 1Cen o In e disciplina de In es igação Ma inha e Ambien al, Ma osinhos, Po ugal, 2Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 3Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o, Po o, Po ugal, 4Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal, 5Spa os Lda, Olhão, Po ugal Me hionine p esen s a pi o al ole in he egula ion o many cellula e en s wi h c ucial impac on he immune sys em, such as in p ocesses in ol ed in he con ol o in lamma ion and polyamines syn hesis. Acco dingly, he p esen s udy aimed o assess he modula o y e ec s o die a y me hionine on he Eu opean seabass (Dicen a chus lab ax) immune s a us, in lamma o y esponse and disease esis ance o Pho obac e ium damselae subsp. piscicida (Phdp). Fo his pu pose, ish we e andomly dis ibu ed in h ee independen g oups ( h ee eplica es pe g oup) and each was ed he co esponding die : a con ol die (CTRL) o mula ed o mee he es ablished amino acid equi emen s o he species; a die supplemen ed wi h me hionine a 0.5% o eed weigh ela i e o he CTRL die (8.2% o me hionine concen a ion abo e CTRL); and one supplemen ed wi h me hionine a 1% o eed weigh o he CTRL die (11.8% o me hionine concen a ion abo e CTRL). To e alua e he immune s a us o ish ed wi h each o he die s be o e being submi ed o bac e ial in ec ion ish we e sampled om each g oup a 2 and 4 weeks a e he beginning o eeding. Non-sampled ish we e injec ed in ape i oneally wi h Phdp (5 ×103c u/ ish) a 4 weeks a e ini ia ion o eeding and he in lamma o y esponse (a 4, 24, and 48 h pos -in ec ion) and su i al (las ing 21 days pos -in ec ion) e alua ed. Fish hema ological p o ile, pe iphe al cell dynamics, plasma humo al immune pa ame e s, leucocy e mig a ion o he in lamma o y ocus and head-kidney gene exp ession we e e alua ed. Resul s show ha me hionine die a y supplemen a ion imp o es seabass cellula immune s a us wi hou e idence o ac i a ion o p o-in lamma o y mechanisms. Addi ionally, he obse ed enhanced immune s a us p o ided by me hionine supplemen a ion ansla ed in o an imp o ed immune esponse o in ec ion, as highe cellula di e en ia ion/p oli e a ion and ec ui men o he in lamma o y ocus, imp o ed plasma humo al immune pa ame e s and modula ion o key immune- ela ed genes was obse ed. Las ly, a e a bac e ial challenge, highe su i al was obse ed in ish ed supplemen ed die s, ul ima ely co obo a ing he posi i e e ec o me hionine adminis a ion o 4 weeks in he cellula immune s a us. Keywo ds: amino acids, cell p oli e a ion, in lamma ion, immunos imula ion, ish, nu aceu ics Machado e al. Die a y Me hionine Immunomodula ion INTRODUCTION A dependency o he immune sys em upon he a ailabili y o amino acids (AA) has been associa ed o hei ole as signaling molecules essen ial o cellula unc ion as e iewed in (1–4), bu also as me hyl g oup dono s and p ecu so s o physiological impo an molecules, such as ho mones, bioac i e amines, enzymes, neu o ansmi e s and ni ic oxide. Se e al s udies ha e epo ed ha AA de iciency educes hei plasma concen a ion, ul ima ely comp omising he immune sys em epe oi e (5,6). In ac , AA equi emen s may inc ease as a di ec consequence o me abolic changes associa ed wi h in lamma ion and in ec ion (7). Me hionine is an example o an indispensable AA wi h a ecognized ole in he immune sys em and i s die a y supplemen a ion p o ed o enhance mammalian hos immuni y (8). By gene a ing S-adenosylme hionine (SAM), me hionine is a me hyl g oup dono ha pa icipa es in he me hyla ion o DNA, ul ima ely in luencing gene exp ession (8). Addi ionally, me hionine akes pa on he polyamine (i.e., spe midine and spe mine) biosyn hesis h ough he aminop opyla ion pa hway, whe e deca boxyla ed SAM successi ely adds aminop opane o he o ming polyamines, equi ed o cell p oli e a ion (9). Du ing he anssul u a ion pa hway, me hionine is also p ecu so o cys eine, one o he h ee glu a hione (GSH) elemen s, a molecule in ol ed in sca enging ee adicals, hence p o ec ing cells om oxida i e s ess du ing in lamma ion (1). Me hionine also plays a pi o al ole in p ocesses esponsible o he con ol o in lamma ion and apop osis, such as p o ein ubiqui ina ion and au ophagy (10). By inducing SAM- media ed me hyla ion, me hionine has been shown o inhibi au ophagy and p omo e g ow h in yeas (11). In ac , me hionine and i s downs eam me aboli e SAM a e esponsible o au ophagy modula ion (12). As a esul , and knowing ha he ideal in lamma o y esponse is apid, ye speci ic and sel -limi ing (13), me hionine p esen s an impo an po en ial as immunomodula o du ing in ec ion. S ill, u he in-dep h s udies a e needed o unde s and he immune mechanisms ha his pa icula AA is ac i a ing be o e and a e in ec ion episodes. Me hionine die a y immunomodula ion also adds a p ac ical pe spec i e o mode n animal p oduc ion. Fo ins ance, he impo ance o me hionine as a nu aceu ical supplemen o con ol en e ic p ocesses and oxida i e s ess in mammals has been ecen ly e iewed (14). Mo eo e , Bunchasak (15) and Jankowski e al. (16) e iewed he many bene icial e ec s o die a y me hionine (and o he sul u -con ainig AA) on poul y immune mechanisms and i s use on poul y indus y. Howe e , he ole o me hionine as an immunomodula o y addi i e in aqua eeds s ill needs o be explo ed so o imp o e sus ainabili y and ish wel a e in ish a ming. Recen s udies showed ha me hionine supplemen a ion inc ease Eu opean seabass (Dicen a chus lab ax) cellula immune s a us as well as immune esponse o an in lamma o y insul wi h UV- inac i a ed Pho obac e ium damselae subsp. piscicida (Phdp) (17). An inc eased pe iphe al leucocy es concen a ion was also obse ed in ju enile Jian ca p (Cyp inus ca pio a . Jian) a e being ed g aded le els o me hionine hyd oxy analog, a syn he ic me hionine sou ce, esul ing in inc eased su i al a e and s onge humo al and cellula esponse a e injec ion wi h Ae omonas hyd ophila (18). Likewise, Tang and co-wo ke s (19) obse ed an inc ease in plasma lysozyme ac i i y, complemen ac o s and IgM o Jian ca p gi en die a y me hionine supplemen a ion du ing 8 weeks. The e o e, die a y me hionine also seems o be an impo an nu i ional addi i e o ish heal h managemen . The main goal o he p esen s udy was o ga he e idence on he speci ic ole o me hionine o ches a ing he Eu opean seabass immune esponse be o e and a e a Phdp in ec ion. MATERIALS AND METHODS Expe imen al Design Eu opean seabass ju eniles we e acqui ed o a ce i ica ed ha che y (MARESA, Spain) and main ained in qua an ine o 2 weeks a he Ins i u o de In es igação e Ino ação em Saúde (i3S; Uni e si y o Po o, Po ugal) ish holding acili ies unde he cul u e condi ions desc ibed below. A e his pe iod, ish we e weighed (Table 4) and andomly dis ibu ed in o 9 anks (200 l; 3 g oups wi h 3 eplica es o 50 ish each) o a eci cula ion seawa e sys em in which O2sa u a ion (7.38 ±0.01 mg/l), salini y (35 pp ) and pho ope iod (10 h da k: 14 h ligh ) we e kep unchanged h oughou he expe imen (Figu e 1). The empe a u e was main ained a 20 ±0.5◦C un il he ime he bac e ial in ec ion was ca ied ou (4 weeks a e eeding wi h he es die s), whe e i was inc eased o 24 ±0.5◦C un il de end o he expe imen so as o mimic he empe a u e inc ease which ypically igge s piscine ou b eaks. Ammonium and ni i e le els we e kep below 0.025 and 0.3 mg l−1, espec i ely. A e 1 week, du ing which ish we e all ed wi h he comme cial die wi h which hey we e being ed p e iously, he expe imen was s a ed by eeding o each g oup wi h he espec i e eed 3 imes a day a an a e age a ion o 2.5% biomass pe day (daily adjus ed ±0.5% based on he assessmen o he non-consumed eed): (i) one g oup was ed a con ol die (CTRL); (ii) ano he g oup was ed a die supplemen ed wi h 0.5% me hionine o eed weigh o he CTRL die (MET0.5); and inally, (iii) ano he g oup was ed a die supplemen ed wi h 1% me hionine o eed weigh o he CTRL die (MET1). A 2 and 4 weeks a e eeding he es die s, 36 ish om each g oup (12 pe eplica e) we e eu hanized by an o e dose o anes he ic (2-phenoxye hanol; Me ck, e . 807291, Ge many), weighed, and collec ed blood and head kidney samples. Also a 4 weeks, ish ha we e no sampled (78 pe g oup, 26 pe eplica e) we e in ec ed in ape i oneally (i.p.) wi h 100 µl o a Phdp suspension (5 ×104c u ml−1). O hese, 60 ish pe g oup (20 pe eplica e) we e placed back in hei anks, eed eplenished acco ding o he p e ious egimen and mo ali y eco ded o 3 weeks and he ela i e pe cen age su i al (RPS) calcula ed. A e eu hanasia o he mo ibund ish, he animals we e weighed and he p esence o Phdp in he head-kidney checked by g owing on TSA-2 pla es. The emaining in ec ed ish (6 pe g oup, 3 pe eplica e) we e e-alloca ed in a simila eci cula ion sys em (Tempe a u e: 24 ±0.5◦C; Salini y: 35 pp ; Pho ope iod: 10 h da k: 14 h ligh ) acco ding o die a y ea men and 6 ish pe F on ie s in Immunology | www. on ie sin.o g 2No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion FIGURE 1 | Expe imen al design. g oup we e eu hanized a 4, 24, and 48 h pos -in ec ion ( ime- cou se) and blood, head-kidney and pe i oneal exuda es sampled om each ish, o in es iga e he immunomodula o y e ec o he die s du ing he ini ial in lamma o y esponse o Phdp. The expe imen s we e app o ed by he i3S Animal Wel a e Commi ee and ca ied ou in a egis e ed ins alla ion (license numbe 0421/000/000/2018). Expe imen s we e pe o med by ained scien is s in ull compliance wi h na ional ules and ollowing he Eu opean Di ec i e 2010/63/EU o he Eu opean Pa liamen and he Eu opean Union Council on he p o ec ion o animals used o scien i ic pu poses. Expe imen al Die s The 3 die s (Table 1) we e o mula ed and manu ac u ed by Spa os Lda (Olhão, Po ugal). The CTRL die was o mula ed o include an indispensable AA p o ile mee ing he ideal pa e n es ima ed o Eu opean seabass (20). Acco ding o esul s om p e isous wo ks (17,21) wo o he die s we e o mula ed (MET 0.5 and MET 1, espec i ely) o be iden ical o he CTRL bu supplemen ed wi h DL-Me hionine a 0.5 o 1% o eed weigh , a he expenses o whea glu en and whea meal. A e AA analysis he pe cen age o me hionine in ela ion o he o al AA amoun was o 2.6% o CTRL and 2.8 and 3.2% o MET 0.5 and MET 1, espec i ely, p esen ing hese die s 8.2 and 11.8% mo e me hionine han CTRL. Main ing edien s we e g ound (below 250 µm) in a mic opul e ize hamme mill (SH1; Hosokawa Mic on, B.V., Doe inchem, The Ne he lands). Powde ing edien s and oils we e hen mixed acco ding o he a ge o mula ion in a paddle mixe (RM90; Mainca, S.L., G anolle s, Spain). All die s we e manu ac u ed by empe a u e-con olled ex usion (pelle sizes: 1.5 mm) by means o a low-shea ex ude (P55; I alplas , S. .l., Pa ma, I aly). Upon ex usion, all eed ba ches we e d ied in a con ec ion o en (OP 750-UF; LTE Scien i ics, Oldham, UK) o 4 h a 45◦C. Fo mula ion o expe imen al die s is p esen ed in Table 1. P oxima e composi ion analysis was conduc ed by he ollowing me hods: d y ma e , by d ying a 105◦C o 24 h; ash, by combus ion a 550◦C o 12 h; c ude p o ein (N×6.25), by a lash combus ion echnique ollowed by gas ch oma og aphic sepa a ion and he mal conduc i i y de ec ion (LECO FP428); a , a e pe oleum e he ex ac ion, by he Soxhle me hod; o al phospho us, acco ding o he ISO/DIS 6491 me hod, using he anado-molybda e eagen ; g oss ene gy, in an adiaba ic bomb calo ime e (IKA). Die s we e analyzed o o al AA con en . Die samples we e hyd olysed in 6 M HCl a 116◦C o 2 h in ni ogen- lushed glass ials. Samples we e hen p e-column de i a ised wi h Wa e s AccQ Fluo Reagen (6-aminoquinolyl-N-hyd oxysuccinimidyl ca bama e) using he AccQ Tag me hod (Wa e s, USA). Analyses we e done by ul a-high pe o mance liquid ch oma og aphy (UPLC) in a Wa e s e e sed-phase AA analysis sys em, using no aline as an in e nal s anda d. Du ing acid hyd olysis aspa agine is con e ed o aspa a e and glu amine o glu ama e, so he epo ed alues o hese AA ep esen he sum o he espec i e amine and acid. Since i is pa ially des oyed by acid hyd olysis, yp ophan was no de e mined. The esul an peaks we e analyzed wi h EMPOWER so wa e (Wa e s, USA). The AA p o ile o he expe imen al die s and he ela i e pe cen age o me hionine supplemen a ion is p esen ed in Table 2. Collec ion o Blood, Head Kidney, and Pe i oneal Exuda es Blood Collec ion Blood was collec ed om he caudal ein using hepa inized sy inges one pa being used o hema ological analysis and he emainde cen i uged a 10,000 ×g 10 min a 4◦C and he F on ie s in Immunology | www. on ie sin.o g 3No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion TABLE 1 | Ing edien and chemical composi ion o he expe imen al die s. Ing edien s CTRL MET 0.5 MET 1 % Fishmeal LT70 (Sou h Ame ican)1 11.00 11.00 11.00 Fishmeal 60217.00 17.00 17.00 Soy p o ein concen a e312.00 12.00 12.00 Whea glu en48.00 7.70 7.40 Co n glú en54.00 4.00 4.00 Soybean meal 48614.00 14.00 14.00 Rapeseed meal76.00 6.00 6.00 Whea meal810.00 9.80 9.60 Fish oil98.50 8.50 8.50 Rapeseed oil10 5.00 5.00 5.00 Vi amin and mine al p emix11 1.00 1.00 1.00 B ewe ’s yeas 12 3.00 3.00 3.00 Soy leci hin13 0.50 0.50 0.50 DL-Me hionine14 −0.50 1.00 To al 100 100 100 Pelle size, mm 1.50 1.50 1.50 PROXIMATE ANALYSES (% DRY WEIGHT) D y ma e (g/100 g) 5.20 5.54 5.09 P o ein (g/100 g) 45.83 45.62 46.25 Fa (g/100 g) 18.80 19.00 18.10 Ash (g/100 g) 7.74 7.58 7.81 Ene gy (kJ/g) 22.48 22.70 22.55 1LT70 s eam d ied, 70.7% c ude p o ein (CP), 8.1% c ude a (CF), Pesque a Diaman e, Pe u. 2COFACO 60: 62.3% CP, 8.4% CF, COFACO, Po ugal. 3Soycomil P: 63% CP, 0.8% CF, ADM, The Ne he lands. 4VITAL: 83.7% CP, 1.6% CF, ROQUETTE F è es, F ance. 5Co n glu en meal: 61% CP, 6% CF, COPAM, Po ugal. 6Dehulled sol en ex ac ed soybean meal: 47% CP, 2.6% CF, CARGILL, Spain. 7De a ed apeseed meal: 34% CP, 2% CF, P emix Lda, Po ugal. 8Whea meal: 10.2% CP; 1.2% CF, Casa Lanchinha, Po ugal. 9SAVINOR UTS, Po ugal. 10Hen y Lamo e Oils GmbH, Ge many. 1120 PREMIX Lda, Po ugal: Vi amins (IU o mg/kg die ): DL-alpha ocophe ol ace a e, 100 mg; sodium menadione bisulpha e, 25 mg; e inyl ace a e, 20,000 IU; DL-cholecalci e ol, 2,000 IU; hiamin, 30 mg; ibo la in, 30 mg; py idoxine, 20 mg; cyanocobalamin, 0.1 mg; nico inic acid, 200 mg; olic acid, 15 mg; asco bic acid, 500 mg; inosi ol, 500 mg; bio in, 3 mg; calcium pan ho ena e, 100 mg; choline chlo ide, 1,000 mg, be aine, 500 mg. Mine als (g o mg/kg die ): coppe sul a e, 9 mg; e ic sul a e, 6 mg; po assium iodide, 0.5 mg; manganese oxide, 9.6 mg; sodium seleni e, 0.01 mg; zinc sul a e,7.5 mg; sodium chlo ide, 400 mg; excipien whea middlings. 12 PREMIX Lda, Po ugal. 13 Lecico P700IPM, LECICO GmbH, Ge many. 14 DL-Me hionine o Aquacul u e: 99% Me hionine, E onik Nu i ion & Ca e GmbH, Ge many. plasma collec ed, ozen on d y ice and s o ed a −80◦C o e alua ing inna e humo al immune esponse pa ame e s. O he ish sampled a 2 (36 ish pe g oup; 12 pe eplica e) and a 4 (36 ish pe g oup; 12 pe eplica e) weeks, 9 ish om each g oup (3 pe eplica e) we e used pe ime poin o he hema ological analysis. Fo he assessmen o inna e humo al immune esponse, TABLE 2 | Amino acid composi ion o expe imen al die s. Amino acids CTRL MET 0.5 MET 1 mg AA/g DW die Me hionine 10.8 11.8 13.2 A ginine 39.5 39.6 39.3 His idine 11.9 11.9 11.7 Lysine 27.9 27.8 28.4 Th eonine 17.4 16.8 17.5 Isoleucine 15.9 16.1 15.8 Leucine 32.3 32.5 32.3 Valine 20.1 21.0 20.3 Phenylalanine 22.4 22.7 22.4 Cys eine 3.1 3.1 3.0 Ty osine 16.0 16.0 15.9 Aspa ic acid +Aspa agine 32.2 32.6 32.5 Glu amic acid +Glu amine 70.6 71.0 70.3 Alanine 22.0 21.3 22.0 Glycine 23.0 22.4 23.4 P oline 26.7 27.4 26.4 Se ine 17.8 17.3 16.9 Tau ine 1.2 1.2 1.2 T yp ophan was no analyzed. plasma om all sampled ish we e used, al hough he plasma was pooled om e e y 3 indi iduals (12 pools pe ea men ). O he ish sampled a 4, 24, and 48 h a e bac e ial in ec ion (6 ish pe g oup; 3 pe eplica e) he hema ological analysis and he e alua ion o he inna e humo al immune esponse pa ame e s we e pe o med o each indi idual. Head-Kidney Collec ion Head-kidneys we e also ha es ed om he 9 ish sampled a 2 and 4 weeks and used o blood collec ion and hema ological analysis. Likewise, he head-kidneys o all ish sampled a 4, 24, and 48 h a e in ec ion we e collec ed. A e ha es ing, he kidneys we e immedia ely ozen on d y ice and s o ed a −80◦C un il p ocessed o gene exp ession analysis. Pe i oneal Exuda es Collec ion Pe i oneal cells we e only collec ed om ish sampled a 4, 24, and 48 h pos -in ec ion ( ime-cou se), acco ding o he p ocedu e desc ibed by Cos as e al. (22). B ie ly, ollowing ish anes hesia and bleeding by he caudal essel, 5 ml o cold Hank’s balanced sal solu ion (HBSS) supplemen ed wi h 30 uni s hepa in ml−1 was injec ed in o he pe i oneal ca i y. The pe i oneal a ea was hen sligh ly massaged in o de o dispe se pe i oneal cells in he injec ed HBSS. The i.p. injec ed HBSS con aining suspended cells we e collec ed and o al pe i oneal leucocy es coun s we e pe o med wi h a hemocy ome e . Analysis o Hema ological Pa ame e s The hema ological p o ile was conduc ed acco ding o Machado e al. (17) and comp ised he o al whi e (WBC) and ed (RBC) blood cells coun s, as well as haema oc i (H ) F on ie s in Immunology | www. on ie sin.o g 4No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion and hemoglobin (Hb; SPINREACT ki , e . 1001230, Spain) assessmen s. A e wa ds, he mean co puscula olume (MCV), mean co puscula hemoglobin (MCH) and mean co puscula hemoglobin concen a ion (MCHC) we e also calcula ed (17). H was no assessed in ish sampled a 4, 24, and 48 h pos -in ec ion. Immedia ely a e blood collec ion, blood smea s we e pe o med om homogenized blood and ai d ied. A e ixa ion wi h o mol-e hanol (10 o 37% o maldehyde in absolu e e hanol) de ec ion o pe oxidase was ca ied ou as desc ibed by A onso e al. (23) in o de o acili a e iden i ica ion o neu ophils. Blood smea s we e hen s ained wi h W igh ’s s ain (Haemacolo ; Me ck) Slides we e examined (1,000×), and a leas 200 leucocy es we e coun ed and classi ied as h ombocy es, lymphocy es, monocy es and neu ophils. Absolu e alue (×104 ml−1) o each cell ype was calcula ed acco ding o he o al blood WBC coun . Analysis o Inna e Immune Response Pa ame e s Lysozyme Ac i i y Lysozyme ac i i y was measu ed using a u bidime ic assay as desc ibed by Cos as e al. (22). A solu ion o Mic ococcus lysodeik icus (0.5 mg ml−1, 0.05 M sodium phospha e bu e , pH 6.2) was p epa ed. In iplica es, 15 µl o plasma was added o a mic opla e and 250 µl o he abo e suspension we e pipe ed o gi e a inal olume o 265 µl. The eac ion was ca ied ou a 25◦C and he abso bance (450 nm) was measu ed a e 0.5 and 4.5 min in a Syne gy HT mic opla e eade . Se ial dilu ed, lyophilized hen egg whi e lysozyme (Sigma) in sodium phospha e bu e (0.05 M, pH 6.2), was used o de elop a s anda d cu e. The amoun o lysozyme in he sample was calcula ed using he o mula o he s anda d cu e. Pe oxidase Ac i i y To al pe oxidase ac i i y in plasma was measu ed ollowing he p ocedu e desc ibed by Quade and Ro h (24). In iplica es, 15 µl o plasma was dilu ed wi h 135 µl o HBSS wi hou Ca+2 and Mg+2in la -bo omed 96-well pla es. Then, 50 µl o 20 mM 3,3′,5,5′- e ame hylbenzidine hyd ochlo ide (TMB; Sigma) and 50 µl o 5 mM H2O2we e added. A e 2 min, he colo -change eac ion was s opped by adding 50 µl o 2 M sulphu ic acid and he op ical densi y was ead a 450 nm in a Syne gy HT mic opla e eade . Wells wi hou plasma we e used as blanks. The pe oxidase ac i i y (uni s ml−1plasma) was de e mined by de ining one uni o pe oxidase as ha which p oduces an abso bance change o 1 OD. Bac e icidal Ac i i y The bac e icidal ac i i y assay was pe o med using Phdp s ain PP3. Bac e ia we e cul u ed in yp ic soy b o h (TSB) (Di co Labo a o ies) supplemen ed wi h NaCl o a inal concen a ion o 2% (w/ ) (TSB-2) and exponen ially g owing bac e ia we e esuspended in s e ile HBSS and adjus ed o 1 ×106c u ml−1. Pla ing se ial dilu ions o he suspensions on o TSA-2 pla es and coun ing he numbe o c u ollowing incuba ion a 22◦C con i med bac e ial concen a ion o he inoculum. Plasma bac e icidal ac i i y was hen de e mined ollowing he me hod desc ibed by G aham and Secombes (25) wi h modi ica ions (17). B ie ly, 20 µl o plasma we e added o duplica e wells o a U-shaped 96-well pla e. HBSS was added o some wells ins ead o plasma and se ed as posi i e con ol. To each well, 20 µl o Phdp (1 ×106c u ml−1) we e added and he pla e was incuba ed o 2.5 h a 25◦C. 25 µl o 3-(4, 5 dime hyl-2-yl)-2,5-diphenyl e azolium b omide (MTT, 1 mg ml−1; Sigma) we e hen added o each well and incuba ed o 10 min a 25◦C o allow he o ma ion o o mazan. Pla es we e hen cen i uged a 2,000 ×g o 10 min and he p ecipi a e was dissol ed in 200 µl o dime hyl sul oxide (Sigma). The abso bance o he dissol ed o mazan esul ing om he educ ion o MTT in di ec p opo ion o he numbe o iable bac e ia p esen , was measu ed a 560 nm. Viable bac e ia was exp essed as pe cen age, calcula ed om he di e ence be ween he dissol ed o mazan in samples and he one o med in he posi i e con ols (100%). The bac e icidal ac i i y was calcula ed as he pe cen age o non- iable bac e ia. Al e na i e Complemen Pa hway Ac i i y Al e na i e complemen pa hway ac i i y (ACH50) was e alua ed as desc ibed by Sunye and To (26). Th ee bu e s we e p e iously p epa ed: GVB (Iso onic e onal bu e ed saline), pH 7.3, con aining 0.1% gela in; EDTA-GVB, as p e ious one bu con aining 20 mM EDTA; and Mg-EGTA-GVB, which is GVB wi h 10 mM Mg2+and 10 mM EGTA. Rabbi ed blood cells (RaRBC; P obiologica Lda., Po ugal) we e washed ou imes in GVB and esuspended in he same bu e o a concen a ion o 2.5 ×108cells ml−1. Then, 10 µl o RaRBC suspension we e added o 40 µl o se ially dilu ed plasma in Mg-EGTA-GVB bu e in iplica es. Following an incuba ion ime o 100 min a oom empe a u e wi h con inuous shaking, he eac ion was s opped by adding 150 µl o cold EDTA-GVB. Samples we e hen cen i uged o 2.5 min a 120 ×g and he ex en o haemolysis was es ima ed by measu ing he op ical densi y o he supe na an a 414 nm. The ACH50 uni s we e de ined as he concen a ion o plasma inducing 50% haemolysis o RaRBC. Gene Exp ession Analysis To al RNA isola ion was conduc ed wi h NZY To al RNA Isola ion ki (NZYTech, Lisbon, Po ugal) ollowing manu ac u e ’s speci ica ions. Fi s -s and cDNA was syn hesized wi h NZY Fi s -S and cDNA Syn hesis Ki (NZYTech, Lisbon, Po ugal). Quan i a i e PCR assays we e pe o med wi h an Eppendo Mas e cycle ep ealplex, using 1 µl o dilu ed cDNA (1:5 dilu ion) mixed wi h 10 µl o NZYSpeedy qPCR Mas e Mix and 0.4 µl (10 µM) o each speci ic p ime in a inal olume o 20 µl. cDNA ampli ica ion was ca ied ou wi h speci ic p ime s (Table S1) o genes ha ha e been selec ed o hei in ol emen in immune esponses and me hionine me abolism (Table 3). P ime s we e designed wi h NCBI P ime Blas Tool acco ding o known qPCR es ic ions (amplicon size, Tm di e ence be ween p ime s, GC con en and sel -dime o c oss-dime o ma ion). Sequences encoding Eu opean seabass l 2, s a 3, m o , c3ze a, cc 3, mcs 1 1, and cd8βwe e iden i ied a e ca ying ou a sea ch in he da abases 1.0c seabass genome (27) and designed F on ie s in Immunology | www. on ie sin.o g 5No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion TABLE 3 | Immune- ela ed genes analyzed by eal- ime PCR. Gene Ac onym Gene Ac onym 40s Ribossomal p o ein (House-Keeping) 40s Clus e o di e en ia ion 8 be a cd8β In e leukin 1 βil1βToll-like ecep o 9 l 9 In e leukin 8 il8 Toll-like ecep o 2 l 2 In e leukin 6 il6 Mac ophage colony-s imula ing ac o 1 ecep o 1 mcs 1 1 T ans o ming g ow h ac o -be a g βMa ix- me allop o einase 9 mmp9 Tumo nec osis ac o -alpha n αComplemen ac o 3 c3 Cyclo-oxygenase 2 cox 2 Mechanis ic a ge o apamycin m o In e leukin 10 il10 Caspase 3 casp 3 C-C chemokine ecep o ype 3 cc 3 Caspase 1 casp 1 Chemokine CXC ecep o 4 cxc 4 Signal ansduce s and ac i a o s o ansc ip ion s a 3 Supe oxide dismu ase sod Melanoco in 2 ecep o mc2 Gu a hione pe oxidase gpx Hea shock p o ein 70 hsp70 Hepcidin hep Hea shock p o ein 90 hsp90 Ni ic oxide-inducible gene p o ein noxin Spe mine/spe midine N (1)-ace yl ans e ase sa 1 Majo his ocompa ibili y complex II an igen be a chain mhc II Adenosylme hionine deca boxylase 1 amd 1 Clus e o di e en ia ion 3 ze a chain c3ze a as p e iously desc ibed. S was used o analyse he e iciency o he p ime pai s by calcula ing he slope o he eg ession line o he cycle h esholds (C ) s. he ela i e concen a ion o cDNA. Accession numbe , e iciency alues, annealing empe a u e, p oduc leng h,and p ime s sequences a e p esen ed in Table S1. Mel ing cu e analysis was also pe o med o e i y ha no p ime dime s we e ampli ied. The s anda d cycling condi ions we e 94◦C ini ial dena u a ion o 2 min, ollowed by 40 cycles o 94◦C dena u a ion o 30 s, p ime annealing empe a u e (Table S1) o 30 s and 72◦C ex ension o 30 s. All eac ions we e ca ied ou as echnical duplica es. The exp ession o he a ge genes was no malized using he exp ession o Eu opean seabass ibosome 40s subuni (40s). Analysis o he Pe i oneal Leukocy e Popula ions Pe i oneal cells we e collec ed in ish om he ime-cou se ial, acco ding o he p ocedu e desc ibed in he Pe i oneal Exuda es Collec ion sec ion. The i.p. injec ed HBSS con aining suspended cells was collec ed and o al pe i oneal leucocy es coun s we e pe o med wi h a haemocy ome e . Cy ospin p epa a ions we e hen made wi h a THARMAC Cellspin appa a us and s ained as indica ed abo e o blood smea s. Lymphocy es, mac ophages and neu ophils in he pe i oneal exuda es we e di e en ially coun ed, and he pe cen age o each cell ype was es ablished a e coun ing a minimum o 200 cells pe slide. Concen a ion (×104 ml−1) o each leucocy e ype was also calcula ed. Bac e ial Challenge Fo he bac e ial challenge, Phdp, s ain PP3, isola ed om yellow ail (Se iola quinque adia a; Japan) by D And ew C. Ba nes (Ma ine Labo a o y, Abe deen, UK), was used. Bac e ia we e ou inely cul u ed a 22◦C in yp ic soy b o h (TSB) o yp ic soy aga (TSA) (bo h om Di co Labo a o ies) supplemen ed wi h NaCl o a inal concen a ion o 2% (w/ ) (TSB-2 and TSA-2, espec i ely) and s o ed a −80◦C in TSB-2 supplemen ed wi h 15% ( / ) glyce ol. To p epa e he inoculum o injec ion in o he ish pe i oneal ca i ies, 100 µL o s ocked bac e ia we e cul u ed o e nigh a 22◦C on TSA-2. Exponen ially g owing bac e ia we e collec ed and e-suspended in s e ile TSB-2 and adjus ed o a inal concen a ion o 5 ×104 colony o ming uni s (c u) ml−1, as con i med by pla ing he esul ing cul u es on TSA-2 pla es and coun ing o c u, and each ish inocula ed in ape i oneally wi h 100 µl (5 ×103c u pe ish) o he bac e ial suspension. Da a Analysis All esul s a e exp essed as mean ±s anda d de ia ion (mean ±SD). Da a was analyzed o no mali y and homogenei y o a iance and, when necessa y, ans o med be o e being ea ed s a is ically. All da a exp essed as pe cen age we e a csine ans o med (28). Da a was analyzed by wo-way ANOVA, wi h ime and die as ac o s and ollowed by Tukey pos -hoc es o iden i y di e ences in he expe imen al ea men s. All s a is ical analyses we e pe o med using he compu e package STATISTICA 12 o WINDOWS. The le el o signi icance used was P≤0.05 o all s a is ical es s. Sampling poin 4 weeks was used as ime 0 h du ing ime-cou se da a analysis, as hey ep esen uns imula ed animal p io o in ec ion. The Chi-squa e es was pe o med o iden i y di e ences on he cumula i e mo ali y among die a y ea men . RESULTS Immune S a us Fish G ow h Pe o mance Thi y six ish pe g oup (12/ eplica e) we e sampled and weigh ed a 2 and 4 weeks a e eeding wi h he expe imen al die s in o de o e alua e he e ec o he die s on he g ow h pe o mance (Table 4). Wi hin each g oup, no di e ences we e ound be ween eplica e a any sampling poin and be ween expe imen al die s in any o he g ow h pa ame e s e alua ed. Hema ology and Pe iphe al Leucocy e Responses The blood o 9 ish om each g oup (3 pe eplica e), sampled a 2 and 4 weeks, was used o e alua ion o hema ological pa ame e s. The hema ological p o ile showed ew changes h oughou he 2–4 weeks pe iod, wi h no al e a ion in he haema oc i . An inc ease o ed blood cells (RBC) numbe s om 2 o 4 weeks was obse ed wi hin each die a y ea men , F on ie s in Immunology | www. on ie sin.o g 6No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion TABLE 4 | Da a on he ini ial weigh and g ow h pe o mance o Eu opean seabass sampled a 2 and 4 weeks a e being ed h ee di e en die s. Die a y ea men s Pa ame e s CTRL MET 0.5 MET 1 2 weeks 4 weeks 2 weeks 4 weeks 2 weeks 4 weeks Ini ial weigh (g) 8.75 ±1.02 8.37 ±0.46 8.35 ±0.48 Final weigh (g) 9.74 ±0.58* 11.43 ±0.33 9.48 ±0.05* 11.37 ±0.85 9.74 ±0.29* 11.57 ±0.42 Weigh gain1(%) 14.74 ±11.32* 34.74 ±11.68 15.67 ±12.77* 38.42 ±12.75 13.16 ±5.92* 42.30 ±10.52 RGR2(% day−1) 0.95 ±0.73 1.06 ±0.32 0.90 ±0.38 1.09 ±0.43 1.11 ±0.26 1.17 ±0.11 Values a e p esen ed as means ±SD (n =36). P- alues om wo-way ANOVA (p ≤0.05). I in e ac ion was signi ican , Tukey pos -hoc es was used o iden i y di e ences in he expe imen al ea men s. 1Weigh gain =( inal weigh ×100)/ini ial weigh . 2Rela i e G ow h Ra e =(e ((ln ( inal weigh ) – ln (ini ial weigh ))/days−1) – 1) ×100. As e isk s ands o signi ican di e ences be ween imes o he same die . TABLE 5 | Haema oc i , hemoglobin, mean co puscula olume (MCV), mean co puscula hemoglobin (MCH), mean co puscula hemoglobin concen a ion (MCHC), ed blood cells (RBC), and whi e blood cells (WBC) in Eu opean seabass ed die a y ea men s du ing 2 and 4 weeks. Pa ame e s Die a y ea men s CTRL MET 0.5 MET 1 2 weeks 4 weeks 2 weeks 4 weeks 2 weeks 4 weeks Haema oc i (%) 21.50 ±2.60 22.63 ±4.55 20.63 ±3.12 22.50 ±4.56 22.56 ±2.17 20.43 ±4.81 Hemoglobin (g dl) 1.09 ±0.20 1.40 ±0.68 1.11 ±0.18 1.23 ±0.40 1.16 ±0.16 1.18 ±0.49 MCV (µm3) 190.57 ±67.55 122.44 ±7.64 159.77 ±16.44 126.48 ±49.00 156.15 ±19.13 106.09 ±8.71 MCH (pg cell−1) 9.37 ±2.34 8.64 ±2.47 8.50 ±1.36 6.72 ±2.73 8.02 ±0.98 6.21 ±2.53 MCHC (g 100 ml−1) 5.33 ±0.85 5.56 ±1.94 5.49 ±0.48 5.66 ±1.74 5.15 ±0.40 6.59 ±2.44 RBC (×106µl−1) 1.21 ±0.29 1.83 ±0.34 1.32 ±0.19 1.92 ±0.42 1.46 ±0.14 1.95 ±0.39 WBC (×104µl−1) 7.83 ±1.71 4.67 ±0.80 9.09 ±3.28 5.73 ±0.65 9.66 ±1.59 6.40 ±1.45 Two-way ANOVA Pa ame e s Die Time Die Time ×Die CTRL MET 0.5 MET 1 Haema oc i Ns ns ns – – – Hemoglobin ns ns ns – – – MCV <0.001 ns ns – – – MCH 0.032 ns ns – – – MCHC ns ns ns – – – RBC <0.001 ns ns – – – WBC <0.001 0.032 ns B AB A Values a e p esen ed as means ±SD (n =9). P- alues om wo-way ANOVA (p ≤0.05). I in e ac ion was signi ican , Tukey pos -hoc es was used o iden i y di e ences in he expe imen al ea men s. Di e en capi al le e s indica e di e ences among die s ega dless ime. al hough he hemoglobin (Hb) le els ha e emained una ec ed. Wi h he excep ion o he mean co puscula hemoglobin concen a ion (MCHC), which emain unchanged be ween 2 and 4 weeks, all o he pa ame e s analyzed (mean co puscula olume, MCV; mean co puscula hemoglobin, MCH; whi e blood cells, WBC) dec eased om 2 o 4 weeks in each die . Among he di e en die s, and despi e dec easing om 2 o 4 weeks, he WBC numbe was inc eased in he die supplemen ed wi h 1% me hionine when compa ed o he alues obse ed a equi alen imes o he CTRL die (Table 5), being his inc ease due o a g ea e numbe o neu ophils (Table 6). In ac , wi h espec o he concen a ion o each ype o leukocy e analyzed in he blood, he only di e ence de ec ed be ween he die s was a highe numbe o neu ophils in he blood o he ish ed wi h he MET 1 die compa ed o hose ed wi h he die CTRL and MET 0.5, bu no di e ences we e obse ed be ween 2 and 4 weeks wi hin each g oup. Howe e , o h ombocy es, lymphocy es and monocy es, al hough hey did no a y among he ish ed he di e en die s, he e was a dec ease in hei numbe om 2 o 4 weeks wi hin each ea men , co ela ing wi h he dec ease in WBC om 2 o 4 weeks in each die . Thus, while he dec ease in he numbe o WBC om 2 o 4 weeks obse ed in he ish ed wi h each o he die s was due F on ie s in Immunology | www. on ie sin.o g 7No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion TABLE 6 | Absolu e alues o pe iphe al blood leucocy es ( h ombocy es, lymphocy es, monocy es, and neu ophils) o Eu opean seabass ed die a y ea men s du ing 2 and 4 weeks. Pa ame e s Die a y ea men s CTRL MET 0.5 MET 1 2 weeks 4 weeks 2 weeks 4 weeks 2 weeks 4 weeks Th ombocy es (×104µl−1) 4.16 ±1.05 2.93 ±0.50 4.99 ±2.19 2.71 ±0.88 4.68 ±1.03 2.96 ±0.81 Lymphocy es (×104µl−1) 3.63 ±0.89 1.58 ±0.40 4.88 ±2.20 2.07 ±0.52 4.40 ±1.21 2.52 ±0.91 Monocy es (×104µl−1) 0.23 ±0.08 0.12 ±0.05 0.23 ±0.14 0.15 ±0.09 0.25 ±0.10 0.20 ±0.13 Neu ophils (×104µl−1) 0.06 ±0.07 0.03 ±0.02 0.02 ±0.02 0.08 ±0.11 0.12 ±0.04 0.17 ±0.12 Two-way ANOVA Pa ame e s Die Time Die Time ×Die CTRL MET 0.5 MET 1 Th ombocy es <0.001 ns ns – – – Lymphocy es <0.001 ns ns – – – Monocy es 0.018 ns ns – – – Neu ophils ns 0.004 ns B B A Values a e p esen ed as means ±SD (n =9). P- alues om wo-way ANOVA (p ≤0.05) (n =9). I in e ac ion was signi ican , Tukey pos -hoc es was used o iden i y di e ences in he expe imen al ea men s. Di e en capi al le e s indica e di e ences among die s ega dless ime. TABLE 7 | Plasma lysozyme, pe oxidase, ACH50, and bac e icidal ac i i ies o Eu opean seabass ed die a y ea men s du ing 2 and 4 weeks. Pa ame e s Die a y ea men s CTRL MET 0.5 MET 1 2 weeks 4 weeks 2 weeks 4 weeks 2 weeks 4 weeks Lysozyme (µg mg ml−1) 3.51 ±0.92a* 1.08 ±0.91 1.62 ±0.95b0.89 ±0.61 1.39 ±0.84b0.97 ±0.41 Pe oxidase (uni s ml−1) 124.45 ±32.04 89.80 ±17.36 126.56 ±201.13 132.42 ±38.96 129.04 ±48.06 118.52 ±40.32 Bac e icidal ac i i y (%) 30.39 ±7.16 25.82 ±10.81 30.13 ±6.57 22.04 ±11.65 41.15 ±7.52 25.86 ±4.23 ACH50 (uni s ml−1) 74.71 ±24.46 197.32 ±60.91 78.19 ±21.27 120.47 ±42.80 96.85 ±28.88 119.00 ±37.06 Two-way ANOVA Pa ame e s Die Time Die Time ×Die CTRL MET 0.5 MET 1 Lysozyme <0.001 <0.001 <0.001 A A B Pe oxidase ns ns ns – – – Bac e icidal ac i i y <0.001 0.002 ns AB B A ACH50 0.007 ns ns – – – Values a e p esen ed as means ±SD (n =12). P- alues om wo-way ANOVA (p ≤0.05). I in e ac ion was signi ican , Tukey pos -hoc es was used o iden i y di e ences in he expe imen al ea men s. Di e en lowe case le e s s and o signi ican di e ences among die a y ea men s o he same ime, while as e isk s ands o signi ican di e ences be ween imes o he same die . Di e en capi al le e s indica e di e ences among die s ega dless ime. o he dec ease in he numbe o h ombocy es, monocy es and lymphocy es, he highes numbe o WBC obse ed in ish ed wi h he die supplemen ed wi h 1% me hionine was exclusi ely due o a highe numbe o neu ophils, sugges ing he s imula ion o an in lamma o y esponse by me hionine supplemen a ion. Humo al Inna e Immune Response Fo he e alua ion o he inna e humo al esponse, 36 ish we e collec ed om each expe imen al g oup (12 pe eplica e) and, o easons o quan i y limi a ion, he plasma om each 3 ish was pooled. Humo al inna e immune pa ame e s assessed in plasma a e p esen ed in Table 7. Two weeks a e he beginning o eeding o he expe imen al die s, plasma o ish ed die s supplemen ed wi h me hionine (MET 0.5 and MET 1) p esen ed lowe lysozyme concen a ion han ha ound in he plasma o ish ed wi h he con ol die . Fu he mo e, a dec ease om 2 o 4 weeks was obse ed in he lysozyme concen a ion o all die s, al hough only F on ie s in Immunology | www. on ie sin.o g 8No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion s a is ically signi ican o ish ed CTRL. Such dec ease o lysozyme concen a ion could explain he educ ion o he o al bac e icidal ac i i y wi h ime o all die s. Plasma bac e icidal ac i i y was ound o be highe in ish ed MET 1 ela i e o hose ed MET 0.5 Rega ding he al e na i e complemen pa hway, he e we e no di e ences in ac i i y be ween he di e en ea men s, al hough i s ac i i y inc eased om 2 o 4 weeks in ish ed any o he die s. Head-Kidney Gene Exp ession To e alua e he exp ession o genes ela ed o immune esponse and me hionine me abolism ole in immune esponse (Table 3), cDNA was isola ed om head-kidneys collec ed om 9 ish om each g oup (3 pe eplica e). High a iabili y in he exp ession o many o he analyzed genes was obse ed, wi h s a is ically signi ican di e ences in he exp ession o he genes coding o IL-1b, Noxin, CD8β, Caspase-3, Melanoco in 2 ecep o , and Spe mine/spe midine N (1)-ace yl ans e ase. The no malized sa 1 exp ession le el showed a dec ease be ween bo h sampling imes (Table S2). Mo eo e , il1β (Figu e 2A), noxin (Figu e 2B), casp3 (Figu e 2C), and sa 1 (Figu e 2D) mRNA exp ession le el was lowe in ish ed MET 1 han in ish ed CTRL, while ish ed MET 0.5 and MET 1 p esen ed lowe cd8β(Figu e 2E) exp ession le els han ish ed CTRL die a y ea men . Fish ed MET 0.5 and Me 1 p esen ed dec eased mc2 (Figu e 2F) ansc ip s compa ed o ish ed CTRL a e 2 weeks o eeding. The da a ega ding gene exp ession du ing he eeding ial is p esen ed in Table S2 as Supplemen a y Da a. Bac e ial Challenge To e alua e a possible p o ec i e e ec o a die supplemen ed wi h me hionine du ing a bac e ial in ec ion, 60 ish om each g oup (20/ eplica e) we e inocula ed wi h Phdp and hei mo ali y ollowed o 3 weeks (Figu e 3). Fish ed any o he die s supplemen ed wi h me hionine, MET0.5 and MET1, showed lowe mo ali y han ish ed he CTRL die , wi h a ela i e pe cen age su i al (RPS) o ish ed he CTRL die o 32 and 43%, espec i ely. Al hough no s a is ically signi ican di e ences we e de ec ed be ween he RPS obse ed be ween he expe imen al g oups, his is mos p obably due o he ac ha he numbe o ish pe g oup calcula ed (powe analysis) assuming ha he supplemen ed die s would p omo e an RPS o o ish ed he CTRL die highe han 60%. In ec ion Response To examine he in luence ha me hionine supplemen a ion may ha e on he ini ial in lamma o y esponse ollowing Phdp in ec ion, samples o blood, head kidney, and pe i oneal exuda es we e collec ed a 4, 24, and 48 h pos -in ec ion om ish o each expe imen al g oup (6 ish om each expe imen al die by ime- poin ). Sampling poin 4 weeks was used as ime 0 h du ing ime- cou se da a analysis, as hey ep esen uns imula ed animal p io o in ec ion. Thus, he collec ed samples we e used o analyze whe he he die s supplemen ed wi h me hionine, compa ed o he con ol die , caused hema ological al e a ions, in luenced he inna e immune esponse and he exp ession o genes in he head kidney as well as induced changes in he in ape i oneal leukocy e popula ions. Hema ology and Pe iphe al Leucocy e Responses The concen a ion o RBC in he ish blood was highe a 4 h a e in ec ion wi h Phdp ega dless o he die a y ea men , wi h no e ec o me hionine supplemen a ion obse ed, since no di e ences we e obse ed be ween he expe imen al g oups (Table 8). Rega ding he hemoglobin concen a ion and he MCH index, no changes we e obse ed o e ime wi hin each expe imen al g oup no be ween ish ed he di e en die s. Simila ly o he RBCs, an inc ease in he numbe o WBCs in ish blood was also obse ed 48 h a e in ec ion when compa ed o he numbe o WBCs a ea lie imes. Howe e , in a manne di e en om ha obse ed o he concen a ion o RBC, i appea s ha me hionine supplemen a ion had an in luence on he numbe o WBC in he blood o he ish ed wi h he supplemen ed die s, since hei numbe is inc eased compa ed o he numbe o WBC in he blood o ish ed he con ol die (Table 9). As al eady no ed be o e in ec ion, his inc ease in he numbe o WBC in he blood o ish ed die s supplemen ed wi h me hionine seems o be due o he inc ease in ci cula ing neu ophils since he numbe o hese cells no only inc eases o e ime wi hin any expe imen al g oup, as i is inc eased ela i e o ha obse ed in he blood o he ish ha we e ed he con ol die , while no di e ences be ween ea men s we e de ec ed ela i e o he numbe o o he leukocy es. Howe e , wi hin each ea men , and as obse ed o neu ophils, he numbe o h ombocy es, lymphocy es and monocy es we e inc eased in ish blood a e 48 h a e in ec ion compa ed o he ime immedia ely be o e in ec ion (0 h), bu : (i) in he case o h ombocy es, he inc ease was obse ed a 4 h a e in ec ion, emaining high and wi hou a ia ion un il 48 h: (ii) in he case o lymphocy es, he e was a dec ease in he ini ial imes pos - in ec ion (4 and 24 h) inc easing hei numbe a 48 h; and (iii) in he case o monocy es, hei numbe emained cons an un il 24 h, inc easing a 48 h. Analysis o he Pe i oneal Leucocy es Responses To al and di e en ial pe i oneal leucocy es coun s we e only pe o med in in ec ed ish wi h he aim o assess cell mig a ion dynamics o he in lamma ion si e ollowing bac e ial injec ion, and a e p esen ed in Table 10. Fish ed MET 1 displayed a highe leucocy e popula ion in he pe i oneal ca i y a 48 h han ish ed CTRL and MET 0.5, ma ching wi h he la ge numbe o lymphocy es, mac ophages and neu ophils a he same ime compa ed o hose ed wi h he o he die s, al hough, due o he high a iabili y obse ed in he mac ophage coun , no s a is ically signi ican di e ence was de ec ed in he numbe o his ype o cells. In ac , in gene al, an inc ease o all leukocy e popula ions o e ime was obse ed in he pe i oneal ca i y o ish ed he die wi h highe me hionine supplemen a ion, which suppo s he occu ence o a s onge local in lamma o y esponse a e he in ape i oneal in ec ion wi h Phdp in ish ed wi h his die . F on ie s in Immunology | www. on ie sin.o g 9No embe 2018 | Volume 9 | A icle 2672 Machado e al. Die a y Me hionine Immunomodula ion as i imp o ed Eu opean seabass cellula immune s a us wi hou igge ing p o-in lamma o y indica o s. Fu he mo e, i was shown ha his enhanced immune s a us ansla es in o an imp o ed in lamma o y esponse agains Phdp, as highe cellula di e en ia ion/p oli e a ion and ec ui men o he in lamma o y ocus was obse ed, as well as imp o ed plasma humo al immune pa ame e s oge he wi h a modula ion o key immune- ela ed genes. Las ly, his wo k s ongly sugges s ha die a y me hionine supplemen a ion o 4 weeks imp o es disease esis ance agains Phdp in a dose-dependen manne . AUTHOR CONTRIBUTIONS MM, LC, and BC concei ed he expe imen s. MM and FF conduc ed he expe imen al ial. RA and SF-B assis ed wi h analy ical p ocedu es. MM di ec ed mos labo a o y echniques and w o e he manusc ip unde he supe ision o RA, LC, and BC. JD o mula ed and p oduced he expe imen al die s. All au ho s con ibu ed o and app o ed he manusc ip . ACKNOWLEDGMENTS This wo k was pa ially suppo ed by P ojec s ALISSA ( e e ence ALG-01-0247-FEDER-3520) and F/00197/2015, inanced by Po ugal and he Eu opean Union h ough FEDER, COMPETE 2020 and CRESC Alga e 2020, in he amewo k o Po ugal 2020, and h ough he COMPETE and Ope a ional Human Po en ial P og ammes and na ional unds h ough Fundação pa a a Ciência e a Tecnologia (FCT, Po ugal). MM and BC we e suppo ed by FCT, Po ugal (SFRH/BD/108243/2015 and IF/00197/2015, espec i ely). The au ho s also acknowledge D . Nuno San os and D . Ana do Vale (i3S/IBMC) suppo du ing he s udy and o c i ically e iewing he manusc ip . SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ immu. 2018.02672/ ull#supplemen a y-ma e ial REFERENCES 1. G imble RF, G imble GK. Immunonu i ion: ole o sul u amino acids, ela ed amino acids, and polyamines. 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(2013) 203:563–74. doi: 10.1083/jcb.201306041 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2018 Machado, Aze edo, Fon inha, Fe nández-Boo, Conceição, Dias and Cos as. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Immunology | www. on ie sin.o g 17 No embe 2018 | Volume 9 | A icle 2672