Teixeira-Carvalhod, Olindo Assis Martins-Filhod, Amanda Fortes Franciscoa, Jamille Mirelle Cardosoa, Fernando Augusto Siqueira Mathiasa, Rodrigo Correa-Oliveirae,f, Mariângela Carneirog, Wendel Coura-Vitalb,f,g, Alexandre Barbosa Reisa,b,f, ∗

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l h o me p a g e:w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Immunological

profile

of

resistance

and

susceptibility

in

naturally

infected

dogs

by

Leishmania

infantum

Gleisiane

Gomes

de

Almeida

Leal

_,

_Bruno

_Mendes

_Roatt

,

Rodrigo

Dian

de

Oliveira

Aguiar-Soares

,

Cláudia

Martins

Carneiro

,

Rodolfo

Cordeiro

Giunchetti

_,

_Andréa

_{Teixeira-Carvalho}

_,

Olindo

Assis

Martins-Filho

_,

_Amanda

_Fortes

_Francisco

_,

Jamille

Mirelle

Cardoso

_,

_Fernando

_Augusto

_Siqueira

_Mathias

_,

Rodrigo

Correa-Oliveira

,

Mariângela

Carneiro

,

Wendel

Coura-Vital

,

Alexandre

Barbosa

Reis

a_{LaboratóriodeImunopatologia,NúcleodePesquisasemCiênciasBiológicas/NUPEB,UniversidadeFederaldeOuroPreto,OuroPreto,}

MinasGerais,Brazil

b_{LaboratóriodePesquisasClínicas,DepartamentodeAnálisesClínicas,EscoladeFarmácia,UniversidadeFederaldeOuroPreto,Ouro}

Preto,MinasGerais,Brazil

c_{LaboratóriodeBiologiadasInterac¸õesCelulares,DepartamentodeMorfologia,UniversidadeFederaldeMinasGerais,BeloHorizonte,}

MinasGerais,Brazil

d_{LaboratóriodeBiomarcadoresdeDiagnósticoeMonitorac¸ão,CentrodePesquisasRenéRachou,Fundac¸ãoOswaldoCruz-FIOCRUZ,}

BeloHorizonte,MinasGerais,Brazil

e_{LaboratóriodeImunologiaCelulareMolecular,CentroRenéRachou,Fundac¸ãoOswaldoCruz,BeloHorizonte,MinasGerais,Brazil} f_{InstitutoNacionaldeCiênciaeTecnologiaemDoenc¸asTropicais,INCT-DT,Brazil}

g_{P´os-Graduac¸˜aoemInfectologiaeMedicinaTropical,FaculdadedeMedicina,UniversidadeFederaldeMinasGerais,BeloHorizonte,}

MinasGerais,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23January2014

Receivedinrevisedform21August2014

Accepted25August2014

Keywords:

Caninevisceralleishmaniasis

Leishmaniainfantum

Clinicalforms

Immunophenotyping Cytokines

a

b

s

t

r

a

c

t

Visceralleishmaniasishasagreatimpactonpublichealth,anddogsareconsideredthemain domesticreservoirofLeishmaniainfantum,thecausalparasite.Inthisstudy,159animals naturallyinfectedbyL.infantumfromanendemicareaofBrazilwereevaluatedthroughan analysisofcellularresponses,usingflowcytometry,andofthehematologicalparameters. Theresultsconfirmedthatdiseaseprogressionisassociatedwithanemiaandreductions ineosinophils,monocytesandlymphocytes.Theinvestigationoftheimmuneresponse, basedontheimmunophenotypicprofileofperipheralblood,showeddeclinesinthe abso-lutenumbersofTlymphocytesCD5+_{andtheirsubsets(CD4}+_andCD8+_)andadropofB lymphocytesinasymptomaticseropositive(AD-II)andsymptomaticseropositive(SD)dogs. Neutrophils,whenstimulatedwithsolubleantigenofL.infantum,showedhighersynthesis ofinterferon(IFN)-␥+_{inAD-IIandSDgroups,withdecreasedproductionofinterleukin}

∗ _{Correspondingauthorat:LaboratóriodeImunopatologia,NúcleodePesquisasemCiênciasBiológicas,ICEBII,MorrodoCruzeiro,UniversidadeFederal}

deOuroPreto,OuroPreto,MGCEP35400-000,Brazil.Tel.:+55213135591694;fax:+55213135591680.

E-mailaddresses:alexreisufop@gmail.com,alexreis@nupeb.ufop.br(A.B.Reis).

http://dx.doi.org/10.1016/j.vetpar.2014.08.022

(IL)-4+_{inasymptomaticseronegativedogspositiveforL.infantuminfectionbasedon} poly-merasechainreactiontesting(AD-Igroup).IntheAD-IIandSDgroups,subpopulationsof stimulatedlymphocytes(CD4+_andCD8+_{)alsoexhibitedgreatersynthesisofIFN-␥}+_and IL-4+_{inculture.TheseresultssuggestthattheanimalsoftheAD-IIandSDgroupsexhibited} amixedimmuneresponse(Type1and2)andtheAD-Igrouppresentinganimmuneprofile verysimilartonormalcontrolanimals.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Visceral leishmaniasis(VL) caused by theprotozoan

Leishmania(Leishmania)infantum,isoneofthemost

impor-tantzoonoticdiseasesaffectingdogsandhumansinSouth andCentral America,theMediterraneanbasin andparts of Asia (World Health Organization, 2010).Dogs (Canis

familiaris)are the mostimportant reservoir of the

par-asite in urban areas, especially those that have a high parasite burden in the skin and a high prevalence in this environment (Giunchetti et al., 2006; Coura-Vital etal.,2011b).Ithasoftenbeenobservedthatanincrease in caninevisceral leishmaniasis (CVL) cases precedes a rise inhuman cases(Fragaet al., 2012;Grimaldi et al., 2012).

CVL may evolve from a nonapparent infection to a severeandsystemicdisease,whichusuallyculminatesin death. Asymptomaticdogscanrecoveror develop clini-calsymptomaticdisease,ortheymayremaininfectedfor years, evenlifelong,withoutclinicalmanifestation (Reis etal.,2009).Recentlyithasbeenshownthatahigh per-centage ofasymptomaticinfecteddogsarePCR positive butseronegative(Coura-Vitaletal.,2011b).Theseanimals, althoughtheirinfectionstatusisnotdetectedby conven-tionalserology,aremorelikelytoseroconvert(Coura-Vital etal.,2013).Theyalsoapparentlyhaveadifferenttypeof immuneresponsethatseemstoberelatedtoresistance andischaracterizedbyhighproportionsofCD4+_T lym-phocytesandCD21+_{BcellsandhighexpressionofIFN-␥} (Reisetal.,2006b;Coura-Vitaletal.,2011a;Menezes-Souza etal.,2011).

The components of innate and adaptive immunity engageinarangeofinteractionsthatisremarkablydiverse andcomplex(Reisetal.,2009,2010).ThecourseofCVL isinterconnectedwiththehostimmuneresponseandthe persistenceandproliferationoftheparasitesthroughout theskinandvisceralorgans.Theinnateimmuneresponse hasarelevantroleinprotectingagainsttheparasitein addi-tiontoswitchingontheadaptiveresponsethatcancontrol

theLeishmaniainfectionwithoutthedevelopmentofa

spe-cificadaptiveimmunity(MorenoandAlvar,2002).Studies indicatethatthesuccessfulresolutionofLeishmania infec-tions depends on the ability of the host to mount a specificT-cellresponse,withtheactivationofmacrophages mediatedbycytokinesderivedfromTcells(Carrilloand Moreno,2009).Symptomaticdogsthatdevelopsevere dis-easealreadyexhibitclearsuppressionofspecifictypesof cell-mediatedimmunity,particularlyCD8+_Tlymphocytes (Pinellietal.,1994).Resistancetoinfectionisassociated witha Type 1response, witha predominance ofIL-12,

IFN-␥,IL-2andtumornecrosisfactor(TNF)-␣,whichwill increase theefficiencyof phagocyticcells and cytotoxic lymphocytes, triggeringa protective immune response. On theother hand, susceptibilityto infectionis associ-atedwithaType2response,withpredominanceofIL-4, IL-5,IL-10, IL-13and TGF-␤(Pinelliet al.,1995,1999a; Correaetal.,2007;Lageetal.,2007;Menezes-Souzaetal., 2011).

In a previously reported study, our group showed that asymptomatic dogs (seronegative/PCR+ [AD-I] and seropositive[AD-II])appeartohaveadichotomous infec-tion spectrum that influencesthe humoral and cellular immunologicalstatusinCVL(Coura-Vitaletal.,2011a).The aimofthepresentstudywastoinvestigateimmunological eventsinnaturallyinfecteddogsbasedonthedichotomy betweenasymptomatic groups (AD-I and AD-II) and to identifybiomarkersassociatedwithresistanceand suscep-tibilitytoinfectionbyL.infantum.

2. Materialsandmethods

2.1. Experimentaldesign

Thepresentstudyincluded159mongreldogsofboth sexes(81maleand78 female)fromanendemicareaof Brazil.Themeanagewas49.8months(SD37.8),andthe medianwas42months(IQR24;66).Thesampleswere col-lectedfromdomesticdogsattheZoonosesControlCentre ofBeloHorizonte.Serologicaltests(immunofluorescence antibodytest[IFAT]andELISA)wereperformedfollowing themanufacturer’sinstructions.Dogs withanIFAT titer <1/40wereconsideredseronegative,anddogswithIFAT titer≥1/40wereconsideredseropositiveandinfectedwith

Leishmaniaspp. Theserologicaltestswereperformedin

theLaboratoryof ZoonosisattheBeloHorizonteHealth Department.Becauseofthehighprevalenceof seroneg-ative/PCR+ dogsintheendemic area(Coura-Vital et al., 2011b),theseronegativedogsweresubjectedto molec-ular testing. Molecular testing (PCR) was performed in buffycoatsampleswithprimersfromaconservedregion

of the Leishmania kDNA minicircle (P150–152) (Passos

2.2. Serologicalassays

TheELISAwasperformedusingtheEIE-LVC®_kit

(Bio-Manguinhos/Fiocruz,Rio deJaneiro,Brazil) accordingto themanufacturer’sinstructions.Thereactionswere per-formedandsampleswithopticaldensityabovethecut-off were considered positive. The cut-off was defined on each platebyconsidering themeanof theoptical den-sityofthenegativecontrolsmultipliedbytwo.Theassays were read on an automatic EL 800G ELISA microplate reader.

TheIFAT wasconductedusingtheIFI-LVC® _kit

(Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil). The tests wereexecutedaccordingtothemanufacturer’sinstruction. The slides were prepared and examined using a fluo-rescentmicroscopewith40×objective(OlympusBX40).

Theresultswereconsideredpositivewhenthefluorescent parasiteswereobservedatserumtiterof1:40ormore.

2.3. Clinicalgroups

Dogs wereclinically classified accordingto serologi-cal(ELISAandIFAT)andmoleculartests(PCR-RFLP)and by clinical features, as described by Coura-Vital et al. (2011a), and were divided into fourgroups. Dogs with noclinicalsigns andnegativeserologicaland molecular resultscomposedthecontrolgroup(CD;n=44). Seroneg-ativedogs withoutclinicalsigns but positive molecular resultsfor L.infantum were classified as asymptomatic dogsI(AD-I;n=53).Dogswithpositiveserologicalresults

for Leishmaniaspp. but noclinical signswere classified

asasymptomaticdogsII(AD-II;n=20).Dogswith clini-calsigns and positiveserologicalresultswere classified as symptomatic dogs (SD; n=38). With regard to gen-der,meanage(inmonths)andstandarddeviationthese groupshave thefollowing characteristics: CD (27♂and 17♀;[52.4;SD38.2]);AD-I(24♂and29♀;[51.0;SD38.4]); AD-II(9♂and11♀;[45.4;SD17.7])andSD(20♂and18♀; [45.5;SD22.8]).

For immunophenotyping and intracytoplasmic cytokine assays, 124 of the 159 dogs were grouped as follows: CD (n=28), AD-I (n=34), AD-II (n=20) and SD (n=42). Alreadythesegroups thegender ageof the animalsandstandarddeviationwere:CD(18♂and10♀; [50.6;SD39.5]);AD-I(17♂and17♀;[50.9;SD35.8]);AD-II (9♂and11♀;[45.4;SD17.7])andSD(21♂and21♀;[46.8; SD22.6]).

2.4. Bloodsamplesandhematologicalevaluation

Peripheral blood (5mL) from the brachiocephalic or jugularveinwascollectedintotubescontainingethylene diaminetetraceticacid(EDTA)atafinalconcentrationof 1mg/mLforthehemogram,bloodfilmand immunologi-calevaluation byimmunophenotyping(exvivo)byflow cytometry.Erythrocytesand leukocyteswere quantified usinganautomaticcellcounter(Model2800Vet,Mindray). Morphologicalcharacteristicsoftheblood cellsand dif-ferentialleukocytecountswereobtainedbybloodsmear analysisafterpriorstainingbyroutinemethods.Toconduct thetestsforevaluatingtheimmuneresponseafterinvitro

stimulation,5mLofaheparinizedperipheralbloodsample fromeachdogwastransferredtosterileheparinizedtubes (BDPharmingen,SanDiego,CA).

2.5. Caninebloodleukocyteimmunophenotyping

Immunophenotyping of peripheral blood by flow cytometry wascarried outusing simple markupof five primary 5-mL polystyrene tubes (Falcon® _{2054, Becton}

Dickinson, San Diego, USA) with the following mAbs: dilutedphycoerythrin(PE)-labeledanti-canineCD5(1:400, mouseIgG2a,cloneYKIX322.3),anti-canineCD4(1:1000, mouse IgG2a,cloneYKIX302. 9)and dilutedfluorescein isothiocyanate(FITC)anti-canineCD8(1:80,mouseIgG1, cloneYCATE55.9)(Serotec,USA).ThemAbswereusedinan indirectimmunofluorescenceprocedureinwhichpooled normal ratserum(diluted1:6000)wasusedasthe iso-typiccontrol.Amouseanti-humanCD21PE(1:100,mouse IgG1,cloneIOBla;ImmunotechCo.Marseille,France)and a diluted PE/Cy-5 conjugated mouse anti-human CD14 (1:300mL,1:200,IgG2a,cloneTÜK4;Serotec,USA)were alsoused.FiftymicrolitersofbloodcollectedinEDTAwas added to tubes containing 50␮L of antibody and incu-batedfor30minatroomtemperature(RT).Afterwards,the erythrocyteswerelysedbyadding2mLoflysissolution (FACSbrandlysingsolution;BectonDickinson,SanDiego, CA,USA),whichwasfollowedbyincubationfor10minat RT.Theleukocyteswerethenwashedtwicewith2mLof PBS(phosphatebufferedsaline0.15M,pH7.2)and cen-trifugedat400×gfor10minatRT.Then,thelabeledcells

werefixedfor30minatRTwith200␮LofFACSFIX solu-tion(10g/Lparaformaldehyde;10.2g/Lsodiumcacodylate and 6.65g/L sodiumchloride, pH 7.2). Flow cytometric measurementswereperformedonaFACScaliburTM instru-ment (Becton Dickinson, Mountain View, CA), and the datawereanalyzedusingFlowJo®_{software(20,000events}

acquiredpersample).Theresultswereexpressedin abso-lute counts (cellnumber/mm3_{) through the product of} the percentage of positive cells (CD5+_{, CD4}+_{, CD8}+ _and CD21+_{)withingatedlymphocytesbyabsolutelymphocyte} counts.Theabsolutecountsformonocyteswereobtained throughtheproductsofCD14+_{cellswithinungated} leuko-cytes by the selection of the region of interest, based on morphometric and immunophenotypic graphics of distribution CD14/FL3 versus SSC, to identify the pop-ulation of CD14+High _{and SSC}Intermediate_{, minimizingthe} contaminationof thispopulationwithlymphocytesand neutrophils.

2.6. Antigenproductionforinvitroassays

Soluble L. infantum (MHOM/BR/1070/BH46) antigen (SLAi)waspreparedasdescribedbyReisetal.(2006c)from promastigotes harvested from stationary-phase in liver infusiontryptosecultures.Theconcentrationofproteinin theSLAisolutionwasdeterminedaspreviouslydescribed Lowryetal.(1951)andadjustedto1mg/mL.DilutedSLAi wasdividedintosmallportionsandstoredat−80◦Cuntil

Table1

HematologicalparametersofdogsnaturallyinfectedwithLeishmaniainfantumanduninfecteddogs.

Hematologicalparameters Clinicalgroups

CD AD-I AD-II SD

Erythrocytes(106_/mm3_{) 7.3(6.8–8.2) 7.41(6.6–7.9) 6.2(5.3–6.5)}a,b _{4.6(3.9–5.5)}a,b

Hemoglobin(g%) 15.0(13.6–16.2) 15.6(13.8–17.1) 13.0(11.5–13.9)a,b _{9.4(7.5–11.5)}a,b,c

Hematocrit(%) 40.8(37.0–45.0) 41.4(39.0–46.3) 40.9(34.5–44.9) 29.8(24.6–34.1)a,b,c

Platelets(103_/mm3_{) 222.0(159.0–321.0) 258.0(171.0–342.0) 199(148.0–240.0) 202.5(114.8–250.0)}b

Leucocytes(103_/mm3_{) 11.2(9.0–13.4) 11.1(9.6–14.1) 8.4(7.6–11.3)}b _{10.8(7.2–12.8)}

Totalneutrophils 5.2(4.3–7.0) 5.8(4.5–7.3) 5.4(4.3–7.9) 7.0(4.2–9.6)

Eosinophils 0.5(0.4–0.7) 0.4(0.3–0.7) 0.3(0.2–0.5)a,b _{0.3(0.1–0.4)}a,b

Monocytes 0.5(0.4–0.7) 0.5(0.4–0.6) 0.3(0.2–0.4)a,b _{0.3(0.2–0.4)}a,b

Lymphocytes 4.3(3.6–5.5) 4.4(3.6–5.5) 2.7(2.4–3.7)a,b _{2.5(2.0–3.4)}a,b

Resultsareshownasmedianvaluesandthebracketsfirst(Q1)andthird(Q3)quartile.

CD,controldogs;AD-I,asymptomaticdogsI;AD-II,asymptomaticdogsII;SD,symptomaticdogs.

a_{StatisticallysignificantdifferencescomparedwithCD.}

b_{StatisticallysignificantdifferencescomparedwithAD-I.}

c_{StatisticallysignificantdifferencescomparedwithAD-II.}

2.7. Immunostainingforcellsurfacemarkersand

intracellularcytokines

Bloodsampleswerecollectedinsteriletubes contain-ingsodiumheparinatafinalvolumeof5mLofperipheral blood.Monoclonalantibodiesusedtodetectcellsurface markersincludedanti-canineFITC-CD4 antibody(1:200, mouseIgG2a,cloneYKIX322.3)andanti-canineFITC-CD8 antibody (1:100,mouse IgG2a,clone YKIX302 9). Addi-tionally,mAbscross-reactivewithcaninecytokineswere usedforintracytoplasmicstaining,includinganti-bovine PE-IFN-␥antibody(cloneCC302) andanti-bovine PE-IL-4 antibody (clone CC303), all purchased from Serotec (Oxford,UK).

Two14-mLpolypropylenetubeswerepreparedforeach animalstudied;oneservedasacontroltube(1mLRPMI plus1mLofwholebloodinheparin),andinthestimulated

tubeL.infantumantigenwasaddedatfinalconcentration

of25␮g/mL.Thetubeswereincubatedfor12handkeptat 37◦_{Cinanincubatorwith5%CO}

2.BrefeldinA–BFA(Sigma, StLouis,MO,USA)wasaddedtoeachtubeatafinal con-centrationof10␮g/mL,andcultureswerethensubmitted toanadditional 4hofincubationin 5%CO2 humidified incubatorat37◦_{C.AtubecontainingPMAatafinal} con-centrationof25ng/mL wasusedasapositivecontrolat final4hofincubationasBFA.Firststainingwasperformed formonoclonalanti-surfacemolecules(CD4+ _andCD8+_). Afterresuspensionoftheselabeledcells,weproceededto stainintracytoplasmiccytokines(anti-IFN-␥andanti-IL-4) inU-bottom96-wellplates.Themicrotubeswerekeptat 4◦_{Cuntiltheacquisitionofcountsontheflowcytometer} (FACScalibur–BectonDickinson,SanJose,CA,USA),which evaluatedatleast30,000eventspertube.

Distinct gating strategies were used to select the leukocyte subpopulations. The canine neutrophils were identifiedandselectedbasedontheiruniqueexpression ofCD4cellsurfacemarker,usingsidescatter(SSC)versus FL1/anti-CD4FITCdotplotdistributions,thusminimizing contaminationof theselectedregionby monocytesand eosinophils.Theeosinophilswereidentifiedandselected based ontheir autofluorescence, using nonrelated FL-3 channel versus forward scatter (FSC) dot plot distribu-tions.TheanalysisofthecytokineprofileofCD4+_andCD8+

T-cellsubsetswasperformedbyfirstestablishinga scat-teringgateonthelymphocytepopulation,usinglaserSSC versusFL1dotplotdistributions.Afterselectingtheregion ofinterest(R1)dotplotswereconstructedforFSCversus IFN-␥/FL2orIL-4/FL2todeterminethepercentageof

IFN-␥+orIL-4+_{cellswithinthepopulationofneutrophilsand} eosinophilspreviouslyselectedinR1.ToevaluatetheCD4+ andCD8+_{Tsubsets, dotplotswereusedforCD4/FL1or} CD8/FL1versus IFN-␥/FL2 or IL-4/FL2. Theresults were expressedinpercentageofcells(IFN-␥+neutrophils,IL-4+ neutrophils,IFN-␥+eosinophils,IL-4+_{eosinophils,IFN-␥}+ CD4+_,IL-4+_CD4+_,IFN-␥+_CD8+_andIL-4+_CD8+_).

2.8. Statisticalanalysis

Statistical analysis was performed using GraphPad Prism5.0.Thenormalityofthedatawasassessedusing theKolmogorov–Smirnofftest.Consideringthe nonpara-metricnatureofalldatasets,Kruskal–Wallistestswere usedtoinvestigatedifferencesbetweenthegroups, fol-lowedbyDunn’stestforpairwisecomparisons.Spearman’s rank correlation was computed to investigate associa-tions between cell immunophenotyping and cytokine-producingcellsandtheclinicalgroups.TheChi-squaretest andKruskal–Wallistestwereusedtoevaluateifthe clin-icalgroupswerehomogeneousinrelationtogenderand agerespectively.Significantdifferenceswereconsideredat

p<0.05.

2.9. Ethicalstatement

3. Results

Tocheckfortheinfluenceofgenderandageofthe ani-malsontheresultsweevaluatedthehomogeneityofthe clinicalgroups.Itwasobservedthatthereisnosignificant differencebetweenthegroupsregardinggenderandageof animals(datanotshow).

3.1. Hematologicalprofile

Evaluationofhematologicalparametersshowedsevere anemia in the SD group, with significant decreases (p<0.0001)inthenumberoferythrocytes,hemoglobinand hematocrit.Decreasederythrocytecountswereobserved intheAD-IIandSDgroupscomparedwiththeCDand AD-Igroups. Furthermore,hemoglobinconcentrations were reducedintheAD-IIgroupcomparedwiththeCDandAD-I groups.IntheSDgroup,hemoglobinandhematocritwere reducedcomparedwithallothergroups.Thewhiteblood cellsanalysisrevealedreductionsin leukocytecountsin theAD-IIgroupcomparedwiththeAD-Igroup(p=0.0052). Absolutevalues of eosinophils,monocytesand lympho-cytesweredecreasedintheAD-IIandSDgroupscompared withtheCDandAD-Igroups(p<0.0001).Plateletcounts werereduced(p=0.0098)intheSDgroupcomparedwith theAD-Idogs(Table1).

3.2. ImmunophenotypingofcirculatingTlymphocytes

andtheirsubsets,Blymphocytesandmonocytes

AnimalsfromtheAD-IIandSDgroupsshowed signifi-cantlydecreasedabsolutevaluesofTlymphocytes(CD5+₎ andtheirsubpopulations(CD4+_andCD8+_{)comparedwith} theCD andAD-Igroups(p<0.0001). Anegative correla-tionwasobservedwiththeclinicalstatus.TheCD4+_/CD8+ TlymphocyteratiowassignificantlyhigherintheAD-Iand SDgroupscomparedwiththeCDandAD-IIgroups(Fig.1). Inaddition,theAD-IIandSDdogshadlowerB-lymphocyte counts(CD21+_{)thantheCDandAD-Igroups(p<0.0001).} Nodifferencewasobservedinmonocytescomparedwith allgroups.Anegativecorrelationwasalsofoundbetween the number of B lymphocytes, monocytes and clinical groups(Fig.2).

3.3. IntracytoplasmicsynthesisofIFN-+andIL-4+by

eosinophilsandneutrophils,afterinvitroantigen-specific

stimulation

After stimulation with SLAi, neutrophils had higher frequencyofIFN-␥+ _{intheAD-IIandSD groupsin} com-parisonwiththeCD andAD-Igroups.SynthesisofIL-4+ wasreduced intheAD-Igroupcomparedwithallother groups (p<0.0001). In addition, a positive correlation between clinical ongoing CVL and IFN-␥+ or IL4+ neu-trophils was observed, and the IFN-␥+/IL-4+ _{ratio was} increasedintheAD-IIandSDgroupscomparedwiththeCD group(p=0.0002).However,nosignificantchangeswere observedincytokine-producingeosinophilsafterinvitro SLAistimulation(Fig.3).

T

CD

5

+Ly

mphoc

yt

es

T

CD

4

+Ly

mphoc

yt

es

T

CD

8

+Ly

mphoc

yt

es

T

CD

4

+/CD

8

+

Ly

mphoc

yt

e

s

ratio

0 5000 10000

0 2000 4000

0 2000 4000

0 1 2

Cel

l

number/mm

3

Clinical groups

CD AD-I AD-II SD

r= - 0.5759

p< 0.0001

r= - 0.4043

p< 0.0001

r= - 0.2966

p= 0.0002

Fig.1.Immunophenotypicprofileoflymphocytesinperipheralbloodof

dogsnaturallyinfectedbyL.infantumcategorizedbytheirclinicalstatus

andlaboratoryresultsasasymptomatic-I(AD-I),asymptomatic-II

(AD-II)andsymptomatic(SD).Noninfecteddogswereusedascontrols(CD).

Resultsareexpressedasabsolutecellcountsinscatterplots,andbars

rep-resentmedianandinterquartilerange.Significantdifferences(p<0.05)

areindicatedbyconnectinglinesbetweenthegroups.ThePearson

cor-relation(r)andpvalueshowninthegraphicsdemonstratecorrelations

betweenTCD5+_{lymphocytesandtheTsubsets(CD4}+_andCD8+_)with

CD21

_{B Lymphoc}

_ytes

_CD1

₄

_Monocytes

CD AD-I AD-II SD

0 1250 2500

CD AD-I AD-II SD

Cel

l

number/mm

3

Clinical groups

P< 0.0001

r= - 0.2126 P= 0.0108

Fig.2.ImmunophenotypicprofileofBlymphocytesandmonocytesinperipheralbloodofdogsnaturallyinfectedbyL.infantumcategorizedbytheir

clinicalstatusandlaboratoryresultsasasymptomatic-I(AD-I),asymptomatic-II(AD-II)andsymptomatic(SD).Noninfecteddogswereusedascontrols

(CD).Resultsareexpressedasabsolutecellcountsinscatterplots,andbarsrepresentmedianandinterquartilerange.Significantdifferences(p<0.05)are

indicatedbyconnectinglinesbetweenthegroups.ThePearsoncorrelation(r)atp<0.05showedcorrelationbetweenproductionofCD21+_Blymphocytes

andCD14+_{monocyteswithclinicalprogression.}

3.4. IntracytoplasmicsynthesisofIFN-+andIL-4+by

lymphocytesubsets(CD4+_andCD8+_{)afterinvitro}

antigen-specificstimulation

IncreasedpercentagesofIFN-␥producingCD4+_orCD8+ lymphocyte subsets wereobservedin theAD-II andSD groupscomparedwiththeCDandAD-Igroups(p<0.0001). Moreover,weobservedanincreasedpercentageofIL-4+ CD4+_{lymphocytesintheSDgroupascomparedwiththe} CDgroup(p=0.0005).ThepercentageofIL-4+_CD8+ lym-phocyteswashigherintheAD-IIandSDgroupscompared withtheCDandAD-Igroups,andapositivecorrelationwas foundbetweenthesynthesisofIFN-␥+ orIL-4+_bythese cellsandtheclinicalstatus(p<0.0001).Inananalysisof theIFN-␥+_/IL-4+_CD4+_orCD8+_{cellratio,nodifferencewas} observedintheexperimentalgroups(Fig.4).

4. Discussion

Studiesofthehematologicalandimmunological pro-files of dogs naturally infected by L. infantum and presenting different clinical profiles are important in assessingbiomarkersrelatedtothepathogenesisand prog-nosisofCVL(Reisetal.,2009).Thesearchforbiomarkers maybeimportanttobetterpredicttheevolutionofcanine disease,sincetheprogressionofinfectiondependsonthe efficiencyoftheimmuneresponseofthehost.Thus,the identificationandcharacterization ofbiomarkerscanbe importantinthedevelopmentofdiagnosis/prognosistests, vaccinesandtherapiesevaluationsappliedtoCVL.

Inthepresentstudy,diseaseprogressionwasassociated withchanges in varioushematological parameterssuch assignificantreductionoferythrocytes,hemoglobinand hematocrit,whichcharacterizedsevereanemia,especially in symptomaticdogs.Our findingscorroborateprevious

studiesthatidentifiedanemiaasacommon hematologi-caleventinactiveandsevereCVL(Reisetal.,2006a;da Costa-Valetal.,2007;deFreitasetal.,2012).DeLunaetal. (2000)suggested that anemiawould result in impaired erythrocytemembranefluidityinCVL,whichwouldfavor mechanicalsequestrationoferythrocytesintothespleen and/or alter receptor–ligand erythrocyte cytoadherence mechanisms. In addition to anemia, seropositive dogs (AD-IIandSDgroups)presentedeosinopenia, lymphope-nia and monocytopenia as previously documented by Reis et al. (2006b). It has been shown that reduced white blood cell counts in symptomatic dogs may be associatedwithbone marrowdysfunction, withintense parasitism causing decreased hematopoiesis and redi-recting bone marrow function (Tropia de Abreu et al., 2011).Nevertheless, consideringthatthedogsare from endemicarea,thehematologicchangescannotbeentirely attributedtotheCVL,sinceothercaninevector-borne dis-easesthat present similarlaboratory findings werenot ruledout.

0 1 4

0 7 14 0 2 14

IFN-γ

+

/IL-4

+

Ratio

% I

L

-4

+

% I

F

N

-γ

+

Neutrophils

CD AD-I AD-II SD CD AD-I AD-II SD

Clinical groups

Eosinophils

0 7 14

0 7 14

0 2 4

r= 0.5895 p< 0.0001

r= 0.2007 p= 0.0261

Fig.3.PercentageofneutrophilsandeosinophilsproducersofIFN-␥+_,IL-4+_{andratioIFN-␥}+_/IL-4+_{instimulatedculture(SLAi)ofdogsnaturallyinfectedby}

L.infantumcategorizedbytheirclinicalstatusandlaboratoryresultsasasymptomatic-I(AD-I),asymptomatic-II(AD-II)andsymptomatic(SD).Noninfected

dogswereusedascontrols(CD).Barsrepresentminimumandmaximumvalues;boxesdisplaymedianandinterquartilerange.Significantdifferences

(p<0.05)areindicatedbyconnectinglines.ThePearsoncorrelation(r)andpvalueshowcorrelationbetweenaproductionofIFN-␥+_andIL-4+_{byneutrophils}

withclinicalprogression.

populationinsymptomaticdogs(Bourdoiseauetal.,1997; Reisetal.,2006b;Alexandre-Piresetal.,2010), andthis reductionwasassociatedwithuncontrolledparasitismin theseanimals.

TheCD4+ _{and CD8}+ _{T-cellsubsetsfollowed a similar} profileasobservedfortheCD5+_{Tlymphocytes,with} reduc-tionsintheAD-IIandSDgroups.Decreasedfrequencyof theCD4+_{T-cellsubpopulationinactiveCVLhasbeen} con-firmedbymanygroups(Bourdoiseauetal.,1997;Moreno etal.,1999;Reisetal.,2006b;Guerra etal.,2009).This

reduction foundin seropositiveanimalsappearstobea critical factor for parasite replication, indicating a poor prognosisofdiseaseprogression.Furthermore,CD8+_Tcells havebeenimplicatedinthecontrolofvisceralizingspecies

ofLeishmania(Tsagozisetal.,2003).Someresearchershave

0 7 14

0 7 14 0 7 14

0 7 14

IFN-γ

+

/IL-4

+

Ratio

% I

L

-4

+

% I

F

N

-γ

+

CD AD-I AD-II SD CD AD-I AD-II SD

Clinical groups

CD4

_lymphocytes

0 2 4

0 2 4

r= 0.4341 P< 0.0001

r= 0.6229 P< 0.0001

r= 0.3608 P< 0.0001

r= 0.4406 P< 0.0001

CD8

_lymphoc

_ytes

Fig.4. PercentageofCD4+_andCD8+_{T-lymphocyteproducersofIFN-␥}+_,IL-4+_{andtheIFN-␥}+_/IL-4+_{ratioinstimulatedculture(SLAi)derivedfromdogs}

naturallyinfectedbyL.infantumcategorizedbytheirclinicalstatusandlaboratoryresultsasasymptomatic-I(AD-I),asymptomatic-II(AD-II)and

symp-tomatic(SD).Noninfecteddogswereusedascontrols(CD).Barsrepresentminimumandmaximumvalues;boxesdisplaymedianandinterquartilerange.

Significantdifferences(p<0.05)areindicatedbyconnectinglines.ThePearsoncorrelation(r)andpvalueshowcorrelationbetweenaproductionofIFN-␥+

andIL-4+_byCD4+_andCD8+_{Tlymphocyteswithclinicalprogression.}

Tcellsmediatetheimmuneresponsebycytotoxic mecha-nismsthatassistinprotectionduringtheearlyphaseofthe infectionsasobservedintheAD-Igroup.However,reduced levelsofthiscelltypeintheAD-IIgroupcannotcontrolthe infection,indicatingapoorprognosisasobservedinthe SDgroup.Therefore,theevaluationofT-cellsubsets(CD4+ andCD8+_{)representsanimportantbiomarkerofclinical} progressioninCVL.

maybeabiomarkerforsusceptibilityand/orsevereCVL (Bourdoiseauetal.,1997;Coura-Vitaletal.,2011a).

Recentstudiesshowedthatneutrophilsplayan impor-tant regulatory role in the early stages of Leishmania

infection(PetersandSacks,2009;Ritteretal.,2009). Stud-ieshavealsoshownthatthesecellshaveadirectimpact onthedeathoftheparasiteandthedevelopmentofa pro-tectiveimmuneresponse againstinfections causedbyL.

donovaniandL.infantum(Rousseauetal.,2001;McFarlane

et al.,2008).Thus, thesecells could provide an impor-tantlinkbetweeninnate andadaptiveimmunityduring parasiteinfection, and theyareon of themresponsible for the visceralization of the parasitism for many lym-phoidorgans(Carvalhoetal.,2012).Inthisstudy,itwas observedforthefirsttimethatcaninebloodneutrophils, whenstimulatedinvitrowithSLAi,wereabletoproduce highlevelsofIFN-␥+ inAD-IIandSDanimalscompared withCDandAD-Idogs.Thesefindingswereconfirmedby thecorrelationobservedwhendogsexhibitactiveCVL.In addition,highlevelsofIFN-␥+neutrophilsareanattempt bytheimmunesystemtodecreasetheparasiteload,but IFN-␥alonecannot protecttheanimalsduringinfection (Chamizoetal.,2005;Lageetal.,2007;Reisetal.,2010). Interestingly,theAD-Igroupshowedreducedsynthesisof IL-4byneutrophils compared withtheother groups.In humanpatientswithactiveVL,increasedsynthesisofIL-4 byneutrophilsstimulatedbysolubleLeishmaniaantigens hasbeenreported(Peruhype-Magalhaesetal.,2005).IL-4 appearstoberelatedtotheseverityofCVL(Quinnelletal., 2001),thusthiscytokinecouldbeanimportantbiomarker ofsusceptibilityincaninesnaturallyinfectedbyL. infan-tum.AlthoughPeruhype-Magalhaesetal.(2005)founda highfrequencyofstimulatedeosinophilIFN-␥+inhuman VL,wedidnotobservethisfindingeitherforneutrophilor eosinophilIL-4+_{indogsnaturallyinfectedbyL.infantum.}

Anevaluationofintracytoplasmiccytokineexpression ininvitroexperimentswithT-cellsubsets(CD4+_andCD8+ lymphocytes) showed that both subsets produced high levelsofIFN-␥(mainlybyCD8+_{lymphocyteswithhigh} cor-relation)inseropositiveanimals(AD-IIandSDgroups)in comparisonwiththeCDandAD-Igroups.Previousstudies havereportedthathighexpressionofIFN-␥isassociated withsymptomaticdisease(Lageetal.,2007; Rodriguez-Cortesetal.,2007;Costaetal.,2013).However,Carrillo etal.(2007)foundreducedexpressionofIFN-␥in experi-mentallyinfecteddogspresentingsymptomaticVL.These highlevelsofIFN-␥producedbylymphocytesinAD-IIand SDdogsindicateanattempttocontrolinfectioninthese animals.However,ourresultsalsosuggested thatIFN-␥

wasnotsufficienttopreventdisease,anditcouldnotbe consideredas a marker of resistanceon these animals. Perhaps,successful infectioncontrolcouldnot achieved becausethere wasa balancewithmodulating cytokines suchasIL-10andTGF-␤thatunderminedclinical improve-mentinanimals.Additionalstudiesevaluatingtheprofile ofcytokinemodulatorswillbeimportanttoclarifythisfact. RegardingtheparticipationofIL-4,manyresearchers demonstrated thatthis cytokine is associated with sus-ceptibilitytodiseaseand isassociated withanincrease in parasitic load in CVL (Pinelli et al., 1999b; Quinnell etal.,2001;Alvesetal.,2009).Strauss-Ayalietal.(2007)

reportedthatearlyexpressionofIL-4measuredinspleen cells playsanimportantroleinthepersistenceof para-sitesdespitehighexpressionofIFN-␥(Strauss-Ayalietal., 2007).BasedonthesefindingswesuggestthatAD-I ani-malshaveextremelylowparasitism,withlittlestimulation oftheIL-4+_{synthesisbylymphocytes,whichmaybea} pro-tectivefactorfortheseanimals.Incontrast,theproduction ofIL-4+ _byCD4+ _andCD8+ _{cellsdetectedinseropositive} dogs(AD-IIandSD)maycontributetobetterunderstand thepersistenceandparasitereplicationobservedinsevere CVL,aspreviouslydocumentedbyGuerraetal.(2009).

Inconclusion,thisstudyshowedforthefirsttimethat theAD-Igroupdoesnotdifferfromhealthyanimalsinthat nosignificantalterationoccursinthecellpopulation eval-uated and there isnoactivationof theType2 immune response,whicheffectivelyconfersaresistanceprofilefor them.Incontrast,theanimalsfromtheAD-IIandSDgroups exhibitedamixedimmuneprofile(Type1and2)in paral-lelwiththeimmunosuppressionthataffectedboththeT andBcompartments,withaconcomitantpresenceof out-standingIFN-␥+_andIL-4+_{producedbyneutrophilsandT} lymphocytes,makingthemunabletocontrolparasite repli-cation.

Acknowledgements

Thisstudywassupportedbythefollowinggrants: Fed-eralUniversity ofOuroPreto;DECIT/MS/CNPq/BR/grant: 576062/2008-1;FAPEMIG/BR/grant:CBB-APQ-3073-4.01/ 07, CNPq/BR/grant: 472554/2007-7; PPSUS/MS/CNPq/ FAPEMIG/SES-MG/grant CBB-APQ-00356-10; FAPEMIG/ PPMandPNPD/Institutional/2011.Thefundershadnorole instudydesign,datacollectionoranalysis,thedecisionto publish,orthepreparationofthemanuscript.ABR,CMC, RCG,ATC,OAMF,andMCaregratefulforCNPqfellowships, WCVisgratefultothePNPD/CAPESfellowshipsandFederal UniversityofOuroPreto.Wealsothankthestaffofthe Sec-retariaMunicipaldeSaúdedeBeloHorizonte,MinasGerais, forcooperation,logisticalsupport,andspecialdedication tothiswork.

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