Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis.

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ContentslistsavailableatScienceDirect

Veterinary

Immunology

and

Immunopathology

jo u r n al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / v e t i m m

Short

communication

Cellular

immunophenotypic

proﬁle

in

the

splenic

compartment

during

canine

visceral

leishmaniasis

Alexandre

Barbosa

Reis

a,b,f,∗

_,

_Andréa

_{Teixeira-Carvalho}

c

_,

Rodolfo

Cordeiro

Giunchetti

a,d

_,

_Bruno

_Mendes

_Roatt

b

_,

_Wendel

_Coura-Vital

b

_,

Roney

de

Carvalho

Nicolato

a

_,

_Denise

_{Silveira-Lemos}

b

_,

Rodrigo

Corrêa-Oliveira

e

_,

_Olindo

_de

_Assis

_{Martins-Filho}

c

a_Programa_de_{Pós-Graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Escola_de_Farmácia,_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_CEP

35400-000MinasGerais,Brasil

b_Núcleo_de_Pesquisa_em_Ciências_Biológicas,_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_Minas_Gerais_CEP_35400-000,_Brasil c_Laboratório_de_{Biomarcadores}_de_Diagnóstico_e_Monitorac¸_ão,_Centro_de_Pesquisas_René_Rachou,_Fundac¸_ão_Oswaldo_Cruz,_Belo

Horizonte,CEP21040-360MinasGerais,Brasil

d_Departamento_de_Morfologia,_Universidade_Federal_de_Minas_Gerais,_Belo_Horizonte,_CEP_31270-901_Minas_Gerais,_Brasil e_Laboratório_de_Imunologia_Celular_e_Molecular,_Centro_de_Pesquisas_René_Rachou,_Fundac¸_ão_Oswaldo_Cruz,_Belo_Horizonte,_CEP

21040-360MinasGerais,Brasil

f_Instituto_Nacional_de_Ciência_e_Tecnologia_em_Doenc¸_as_Tropicais,_Salvador,_Bahia,_Brasil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received23June2013 Receivedinrevisedform 30September2013 Accepted22November2013 Keywords:

Caninevisceralleishmaniasis Leishmaniainfantum Spleniccompartment Cellularimmuneresponse

a

b

s

t

r

a

c

t

Todeterminetheroleofthespleeninthepathogenesisofcaninevisceralleishmaniasis (CVL),weanalyzedcellularimmunophenotypicprofilesof52dogsnaturallyinfectedwith Leishmaniainfantum,clinicallyclassifiedasfollows:asymptomaticdogs-I(AD-I), seroneg-ative/PCR+;asymptomaticdogs-II(AD-II),seropositive/PCR+;oligosymptomaticdogs(OD) andsymptomaticdogs(SD).Sevennon-infecteddogs(CD)wereincludedasacontrolgroup. AD-IIpresentedhigherlevelsofCD8+TsplenocytesandlowerTCD4+/TCD8+ratioin com-parisonwithCD.ODandSDshowedlowerpercentagesofCD21+ascomparedwithAD-II. AllseropositivedogspresentedlowerlevelsofCD45RA+thanCD.Regardlessofthestimuli used,theproliferationindexfromsplenocytesinvitrowasinverselycorrelatedwith clini-calstatus.AfterLSAstimulation,therewasahigherpercentageofspecificCD8+TinAD-II thanCDandnon-stimulatedculture.Incontrast,splenocytesfromSDunderinvitroLSA stimulationinduceddecreasedMHC-II+expressionincomparisonwithallgroups,and non-stimulatedculture.Inconclusion,theroleofCD8+Tsplenocytesseemstobeimportantfor aneffectiveimmunologicalresponse,ahallmarkofasymptomaticCVL,whereasthe pro-nouncedlossofMHC-IIexpressionuponLSAstimulationisabiomarkerofsymptomatic CVL.

∗ Correspondingauthorat:ProgramadePós-Graduac¸ãoemCiências Farmacêuticas,EscoladeFarmácia,UniversidadeFederaldeOuroPreto, OuroPreto,CEP35400-000MinasGerais,Brasil.Tel.:+55213135591694; fax:+55213135591680.

E-mailaddresses: [email protected], [email protected]

(A.B.Reis).

1. Introduction

Visceralleishmaniasis(VL)isapotentiallyfatalhuman disease caused by the intracellular protozoan parasite Leishmania infantum(Kayeet al., 2004).Canine visceral leishmaniasis(CVL) represents a problem for both vet-erinarymedicineandpublichealthinapproximately50 countriesinvariousendemicareasoftheworld( Coura-Vital et al., 2011b; Desjeux, 2004). CVL is a systemic,

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chronic,andseverediseasethatisoftenfatalbecauseno efﬁcaciousdrugsexisttocuretheseanimals(Banethetal., 2008; Reiset al., 2006c). According toMancianti et al. (1988),CVLcanbecategorizedintothreedistinctclinical forms,basedonmajorclinicalfeaturesobservedininfected dogs:asymptomatic(AD),withnosuggestivesignsofthe disease;oligosymptomatic(OD),withamaximumofthree clinicalsigns;andsymptomatic(SD),withtypicalclinical signs,showingthemostsevereclinicalsignsofCVL.

Recently, Coura-Vital et al. (2011a) proposed a new classiﬁcationschemeforthecaninediseaseaccordingto serological,molecular, and clinicalfeatures.The asymp-tomaticformwasdividedintwosubgroups:asymptomatic dogs I (AD-I), with negative serological tests for Leish-maniabut positivemolecularresults,and asymptomatic dogsII(AD-II),showingpositiveresultsforbothserology andmolecularanalysesforL.infantum.

VLisadiseaseassociatedwiththeinabilityof lympho-cytestoactivateMtokillLeishmania(NylenandSacks, 2007).Parasitescanbefoundinmononuclearphagocytic cellsinthespleenandliver,whicharethemajoraffected sites (Kaye et al., 2004; Stanley and Engwerda, 2007). In a progressive disease, cellularimmune response are impaired,asindicatedbystudiesshowingthatperipheral bloodmononuclearcells(PBMCs) fromaffectedhumans anddogsfailtorespondtoparasitesolubleantigensboth invitroandinvivo(Boggiattoetal.,2010;GotoandPrianti, 2009).Ontheotherhand,aprotectiveimmuneresponse (observedinasymptomaticVL)ismanifestedbyastrong proliferative response of PBMCs from affected humans anddogs toLeishmaniaantigens,and theproduction of cytokines,suchasIFN-␥andTNF-␣,whicharerequiredfor Mactivationandkillingofintracellularparasites(Cabral etal.,1992,1998;Pinellietal.,1994,1999;SeixasDuarte etal.,2008;Strauss-Ayalietal.,2005).

Somestudieshaveshownthattheproliferation capa-bilityofPBMCsfromdogswithCVLwasdecreasedupon both antigenicand mitogenic stimulation (Cabral etal., 1998;Cardosoetal.,2007;Pinellietal.,1995).However, theinﬂuence of the populations and subpopulations of thesplenocytesderived fromdogswithCVLupon anti-genicstimulationisnotcompletelyunderstood.Because thespleenisoneofthemajororgansaffectedduringCVL, andthecontributionofthecompartmentalizedimmune responseinthegenesisofsplenomegalyduringthis infec-tionremainsunclear,itisrelevanttoinvestigatesigniﬁcant alterationsinthephenotypicfeaturesfrominfecteddogs, performingadetailedexvivoandinvitro immunopheno-typingoftheirsplenocytes.

2. Materialsandmethods

2.1. Dogsandexperimentaldesign

Allproceduresinthisstudywereaccordingtothe guide-lines set by the Brazilian Animal Experimental College (COBEA).ThisstudywasapprovedbytheEthical Commit-teefortheUseofExperimentalAnimalsattheUniversidade FederaldeMinasGerais,Brazil(Protocolno.020/2007).

Fifty-two mixed-breed adult dogs of both genders (agerangingfrom2to6years)wereselected.Theywere

maintained in the kennel at the Instituto de Ciências Biológicas,UniversidadeFederaldeMinasGeraisor pro-vided bytheCentro de Zoonoses–Belo Horizonte/Minas Gerais, Brazil. The dogs used in this study were stray or domiciled mongrel dogs, selected based on their serological results on IFAT (IFI-Leishmaniose-Visceral-Canina-Bio-Manguinhos,RiodeJaneiro,Brazil),which is thegoldstandardimmunologicaltestfordiagnosisofCVL. Animals presenting IFAT titers ≥1:40 were considered positive and includedinto theinfected groups.Animals withIFATnegativeat1:40wereconsiderednon-infected and included as a control group.Positive infectionwas conﬁrmed byPCR in atleast oneskin sample(Degrave et al., 1994), and the species of Leishmania responsible fortheinfectionweredeterminedbyrestrictionfragment lengthpolymorphism–PCR(Volpinietal.,2004).

2.2. Clinicalclassiﬁcation

Thedogswereclinically classiﬁedaccordingto pres-ence/absenceofinfectionsignsserological,andmolecular tests: asymptomatic dogs I (AD-I, n=8), with no sug-gestive signsofdisease,negativeserology, andPCR+for Leishmania;asymptomaticdogsII(AD-II,n=10),withno suggestive signs of disease,positive serology, and PCR+ forLeishmania;oligosymptomaticdogs(OD,n=11),witha maximumofthreeclinicalsignsincludingopaquebristles, and/orlocalizedalopecia,and/ormoderatelossofweight; andsymptomaticdogs(SD,n=16),withcharacteristic clin-icalsignsof CVL,suchasopaque bristles,severelossof weight,onychogryphosis,cutaneouslesions,apathy,and keratoconjunctivitis,andbothgroupswithpositive sero-logy,andPCR+forLeishmania;andcontroldogs(CD,n=7), classiﬁedaccordingtonegativeserologicalandPCR−for Leishmania.

2.3. Spleensamplecollection

The collection of spleen specimens was carried out aftereuthanasiaofthedogswithabarbituricanesthesia (Thiopentalat30mg/kgbodyweight). Spleenfragments (5mm)werestoredoniceupto12hinaPetridishin ster-ileRPMI-1640(Gibco,GrandIsland,NY,USA)priortouse inexvivoandinvitroprocedures.Thetissuewasmincedin atissuegrinderandtransferredto2mLofRPMI-1640.The cellssuspensionwasthenﬁlteredonstainlesssteelgauze toobtainasinglecellsuspension.Themononuclear spleno-cyteswereisolatedbydifferentialcentrifugation(800×g for40minatroomtemperature[RT])onaFicoll-Hypaque cushion(Histopaque1.077,Sigma,St.Louis,MO,USA).The cellsuspensionwaswashedtwiceinRPMI-1640and resus-pendedtoobtain107_cells/mL.

2.4. Determinationofparasiteloadindex–LDU

Following necropsy, fragments of spleen were col-lectedand imprintspreparedontwo microscopeslides. Slides were air-dried, ﬁxed in methanol, stained with Giemsa, and examinedunder an optical microscopy to detectamastigoteformsofLeishmania.Parasitedensities weredeterminedaccordingtoStauber(1955)withsome

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modiﬁcations,andexpressedasLeishmanDonovanUnits (LDUindex),whichcorrespondthenumberofLeishmania amastigotesper1000nucleatedcells.Forcontrolpurposes, sevendogsthatwerenotinfectedwithCVLaccordingtothe IFATassaywerealsosubmittedtoparasitological examina-tion.

2.5. Immunophenotypingbyﬂowcytometry

Immunophenotyping analysisof splenocytes by ﬂow cytometry was undertaken as described by Reis et al. (2005).Brieﬂy,1×107_cells/mL_of_splenocytes_were

sub-jectedtopre-ﬁxationanderythrocytelysisbythegentle addition of 13mL lysis solution (FACS lysing solution, BectonDickinson,MountainView,CA,USA)followedby incubationfor10minatRT.Aftercentrifugation(450×g for 10minatRT),thepelletwasresuspendedin 500␮L phosphatebufferedsalinesupplementedwith10%offetal bovineserum.

Ina96-wellU-bottomplate(LimbroBiomedicals,Inc., Aurora,OH,USA),30␮Lofpre-fixedsplenocyte suspen-sion was incubated at RT for 30min, in the dark, in thepresenceof30␮Lofanti-caninecellsurfacemarker antibodies.Monoclonalantibodies(mAbs),whichdefine canine cell phenotypes, including purified rat anti-dog CD5 (Rat-IgG2a, clone YKIX322.3), anti-dog CD4 (Rat-IgG2a, clone YKIX302.9), anti-dog CD8 (Rat-IgG1, clone YCATE55.9), anti-MHC-II (Rat-IgG2b, clone YKIX334.2), anti-CD45RA (Rat-IgG2b, cloneYKIX753.22.2), and anti-CD45RB (Rat-IgG2b, clone YKIX716.13) were used in anindirectimmunofluorescenceprocedure. FITC-labeled mouseanti-human-CD21(Mouse-IgG1,cloneIOBla)was used in direct immunofluorescence procedures. Unla-beled mAbs used in this study were purchased from Serotec(Oxford,UK)andanti-CD21fromImmunotechCo. (Marselle,France).

During the immunophenotyping the cells were also incubatedunderthesameconditionsinthepresenceof 60␮LofpreviouslydilutedFITC-conjugatedsheepanti-rat IgGantibody.Beforeflowcytometricdatacollectionand analysis,labeledcellswerefixedfor30minwith200␮L ofFACSfixsolution(10.0g/Lparaformaldehyde;10.2g/L sodiumcacodylate,and6.65g/Lsodiumchloride,pH7.2). 2.6. Flowcytometrydatastorageandanalysis

Flowcytometricmeasurementswereperformedona FACScan instrument (Becton Dickinson). The Cell-Quest softwarepackagewasusedinbothdataacquisitionand analysis.Atotalof10,000eventswereacquiredforeach preparation.Caninewhole-bloodleucocyteswere identi-ﬁedonthebasisoftheirspeciﬁcforward(FSC)andside (SSC)light-scatter properties asdescribed byReiset al. (2005).Theresultswereexpressedasthepercentage of positivecellswithintheselectedlymphocytegateforCD5+_,

CD4+_, _CD8+_, _and _CD21+ _cells. _{Semi-quantitative}

analy-seswereperformedtoevaluatedifferentialexpressionof cell surfacemarkers presenting a unimodaldistribution (MHC-II+_,_CD45RA+_,_and_CD45RB+_),_and_the_results_were

expressedasMeanFluorescenceChannel(MFC).

2.7. Splenocytesproliferationassay

Thefunctionalactivityofsplenocyteswasdetermined usingthelymphocyteproliferationassay.Thecell suspen-sionwasadjustedto106_cells/mL_of_RPMI-1640_medium

(Sigma)supplementedwith10%FCS,100,000U/Lpenicillin and0.2g/Lstreptomycin.Twohundredmicrolitersofthe suspensionwastransferredintoeachwellofa96-well ﬂat-bottomedplate(Nunc,Naperville,IL,USA)containing25␮l ofLSA(25␮g/ml)obtainedaccordingReisetal.(2006c),for theantigenicstimulusassays.Themitogeniclectin Con-canavalinA (ConA,Sigma, USA)at 10␮g/mL,and RPMI 1640mediumwereusedascontrols.Allsampleswererun intriplicate.Themicroplateswereincubatedat37◦Cunder a5%CO2atmospherefor3days.Twentyhoursbefore

har-vesting,50␮Lofmediumwith3_H-thymidine₍₅_␮Ci/mL)

wasadded.Theincorporationof3_H-thymidine_was

mea-suredwithaliquidscintillationcounter(TricarbCA1600, PackardBioScienceCompany,Meriden,CT,USA).The stim-ulationindex wascalculated asthe ratio of countsper minute(CPM)instimulatedsamplesversusCPMin non-stimulatedcontrols.

2.8. Statisticalanalysis

Statistical analysis was performed using GraphPad Prism5.0.Thenormalityofthedatawasassessedusingthe Kolmogorov–Smirnofftest.Parametricdatawereanalysis by variance (ANOVA) followed by Tukey’s test. Non-parametric data were analyzed by the Kruskal–Wallis test followed by Dunns’ test. Student’s t-test and the Mann–Whitneytestwereusedtoidentifysignificant dif-ferencesbetweentheratioofstimulatedsamplesversus non-stimulatedcontrolculturesforparametricand non-parametric data, respectively. Correlation analysis was performedbyPearson’sranktestinthecaseofparametric dataandSpearman’sranktestinthecaseofnon-parametric data.Inallcases,significancewasdefinedatp<0.05.

3. Resultsanddiscussion

The role of the anti-Leishmania immune response underlying the susceptibility/resistance during CVL has beenrecognizedinmanyexvivoandinvitroinvestigations (Cardosoetal.,2007;Lageetal.,2007;Pinellietal.,1994, 1995;Reisetal.,2006a).Recently,wedemonstratedthat immunoglobulinlevels,aswellastheparasiteloadare rele-vanthallmarksofdistinctclinicalstatusofCVL(Coura-Vital etal.,2011a;Reisetal.,2009,2006c).Tobetterunderstand the events underlying the compartmentalized immune responseinthespleen,weperformedadetailedanalysis ofclinicalstatusofCVLandexvivoandinvitrophenotypic featuresofthemajorsplenocytesubsets(CD5+_,_CD4+_,_CD8+_,

CD21+_),_and_several_activation_markers_(MHC-II+_,_CD45RA+_,

CD45RB+_)._The_spleen_is_one_of_the_tissues_where_the

par-asite replicates, and is an important organ responsible foradaptiveimmuneresponseduringCVLinfection.Thus, theassessmentofcellularimmunophenotypicproﬁlemay improveourunderstandingof therelationshipbetween themajoreventsofdiseaseprogressionwithuncontrolled parasitereplicationinthespleniccompartment.

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Splenocytes Ratio % of Splenocyte markers Splenocytes (MFC)

Clinical status

0 40 80 CD5+ 0 25 50 CD4+ _CD8+ a CD4+_/CD8+ SD OD AD-II AD-I CD 0 1 2 CD45RB+_/CD45R_A+ c a 0 25 50 CD21+ 0 25 50 a,c 0 1 2 SD OD AD-II AD-I CD 0 500 700 a a a CD45RA+ 300 CD45RB+ 0 500 700 SD OD AD-II AD-I CD 300

Fig.1. ImmunophenotypicproﬁleofsplenocytesindogsnaturallyinfectedwithLeishmaniainfantumcategorizedaccordingtotheirclinicalstatusas asymptomatic(AD),oligosymptomatic(OD),symptomatic(SD),andcontrol(CD)groups.Theresultsareexpressedinbargraphsasmeanpercentageof CD5+_,_CD21+_,_CD4+_,_CD8+_within_gated_lymphocytes,_and_CD4+_/CD8+_ratio_plus_standard_deviation._The_results_of_CD45RA+_and_CD45RB+_are_expressed_as

averageofmeanﬂuorescencechannel(MFC),andCD45RB+_/CD45RA+_ratio_plus_standard_deviation._Signiﬁcant_differences_at_p_<_0.05_are_indicated_by_the

lettersaandcincomparisonwiththeCDandAD-IIgroups,respectively.

Studies have reported a decrease in the percentage ofBcells (CD21+₎_in _peripheral_blood _of_dogs _naturally

infectedwithL.infantum(Bourdoiseau etal.,1997;Reis etal.,2006b).Inthepresentstudy,exvivo immunophe-notypicanalysisofsplenocytesalsoshowedareduction in thepercentage of Blymphocytes(CD21+₎_in _the _OD

group as compared with AD-II, as well as in the SD groupascomparedwithbothCDandAD-II(Fig.1).The decreaseofBcellsindogswithactiveCVLisintriguingand unexpected,mainlybecausetheseanimalsexhibitintense polyclonal B cells, as evidenced by increased produc-tionofimmunoglobulinofvariousclassesandsubclasses (Martinez-Morenoetal.,1995;Reisetal.,2006c; Coura-Vitaletal.,2011a).ResultsobtainedbyGuerraetal.(2009), however,demonstratednodifferenceinthepercentageof thislymphocytesubpopulationinthespleenwhenthe ani-malswerecategorizedbyparasiteload.Thus,wesuggest thatthedecreasedpercentageofBcells(CD21+₎_in

periph-eralblood (Reisetal., 2006b), and spleen aswell from symptomaticdogs(SD)couldreﬂectaninvolvementinthe productionofthesecellsinthebonemarrowandapossible differentiationinplasmacells.

WealsoobservedthatCD8+_T_cells_reﬂect_most

accu-ratelyboththeclinicalstatusandtheoverallbonemarrow parasite density, asincreased levelsof CD8+ _circulating

lymphocytes appeared as themajor phenotypicfeature ofasymptomaticdiseaseanddogsbearingalowparasite load(Reisetal.,2006b).Inthepresentstudy,wedidnot observealterationsinCD5+_and_CD4+_splenocytes_in_ex_vivo

experiments,whereas theAD-IIgrouppresented ahigh percentageofCD8+_T_cells,_and_reduction_in_the_CD4+_/CD8+

ratio(Fig.1).Coura-Vital etal.(2011a)observedsimilar resultswithhigherCD8+_T_cells,_and_lower_CD4/CD8_ratio

inAD-IIandSDgroupsintheperipheralbloodcompared withCD.Inanotherstudythatevaluatedthe immunophe-notypingofleukocytesfromliverandspleenfromnaturally infecteddogsshowedthatthesplenicimmuneresponses in bothsymptomaticand asymptomaticdogswerevery similar(Sanchezetal.,2004).Guerraetal.(2009)showed in naturally infected dogs that, the increase frequency of CD8+ _T _cells _in _peripheral _blood _is _strongly

associ-ated with low splenic parasitism. In ourcurrent study, we observed that low parasite load in spleen is found mainlyinasymptomaticdogs(AD-IandAD-II)conﬁrming

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LDU-L og a,d a a Clinicalstatus SD OD AD-II AD-I 1 10 100 1000 10000

Fig.2.SplenicparasiteloadindogsnaturallyinfectedwithLeishmania infantumcategorizedaccordingtotheirclinicalstatusasasymptomaticI (AD-I),asymptomaticII(AD-II),oligosymptomatic(OD)andsymptomatic (SD)groups.Theresultsareexpressedinbargraphsasaverageof Leish-manDonovanUnits(LDU)inlogscaleplusstandarddeviation.Signiﬁcant differencesatp<0.05areindicatedbythelettersaanddincomparison withtheAD-IandSDgroups,respectively.

thishypothesis.In fact,differentstudieshavecorrelated thelevelofCD8+_T_cells_with_protection_during_L.

infan-tuminfection(Pinelli,1997;Reisetal.,2006b)orduring theimmunogenic response aftervaccine administration against CVL (Giunchettiet al., 2007,2008; Roatt et al., 2012).Thus,ourdataalsosuggestthatCD8+_T_cells_in

sev-eralcompartmentsmaycontroltheparasitereplication. Furthermore,we evaluatedtheexvivo CD45RA+ _and

CD45RB+ _expression_on _splenocytes _in _different_clinical

groups. The results showed a decreased expression of CD45RA+ _in _the_animals_after _{seroconversion.}_However,

nodifferenceswereobservedforCD45RB+_expression_or

theCD45RB+_/CD45RA+_expression_ratio_among_the_groups

(Fig. 1). Guerra et al. (2009) also observed decreased CD45RA+_expression_on_splenocytes_from_animals_infected

withamoderateparasiteload.Thesechangesmaybe asso-ciatedwiththehighsplenicparasitismfrequentlynoticed indogswithhighparasiteloadorexhibitingsevereCVL clinicalstatus.

SpleenparasiteloadwasassessedusingGiemsa-stained microscopy to determine the Leishman Donovan Units (LDU)(Fig.2).Dataanalysisdemonstratedahigherparasite

loadinSD,OD,andAD-IIgroupscomparedwithAD-Idogs. Interestingly,weobservedlowerparasitisminAD-IIthan SDdogsinthespleniccompartment(Fig.2).Sanchezetal. (2004)suggestedthatthestrikingdifferencesinparasite burdenbetweenADandSDanimalscouldbeexplainedby adiminishedinfectionoramoreefﬁcientcontrolofthe parasitereplicationintheformergroup,asweobserved inourstudy.

To evaluate a Leishmania-specific cell-mediated immuneresponse,splenocytesfromdogswithCVLwere stimulated by a mitogen (ConA) (Fig. 3A) or specific antigen(LSA) (Fig.3B). Ourresultsdemonstrated thata lymphoproliferativeresponsedecreasedinallseropositive dogsincomparison withtheCDgroup,when simulated withConAorinallinfecteddogsaftersimulationwithLSA. Furthermore,acorrelationanalysisbetweennon-specific andLSA-specificlymphoproliferativeresponseandclinical statusrevealedthatsevereCVLdiseaseiscorrelatedwith alowproliferationindexduringtheinfection(r=−0.3627, p=0.0113; r=−0.5263, p=0.0001, respectively; Fig. 3A and B).Similarresults havebeen documentedby other researchgroups(Cabralet al.,1998;Pinellietal.,1994; Rodriguez-Cortesetal.,2007).

Moreover,itwasconductedananalysisofthe pheno-typicfeaturesinthespleencompartmentinthepresence or absenceof LSA stimulation(Fig.4). We observed an increasedpercentageofCD8+ _splenocytes_in_OD_and_SD

ascomparedwithCDinnon-stimulatedcultures. More-over,thestimulatedcultureswithLSAantigensshowed increasedpercentageofCD8+_T_cells_in_AD-II_as_compared

withtheCDgroupornon-stimulatedcultureinthesame group(Fig.4A).Similarly,Pinellietal.(1995)alsoobserved thatTlymphocytesfromsymptomaticdogsfailedto prolif-erateafterLeishmaniaantigenstimulationinvitro.Onthe otherhand,theseauthorsreportedthatasymptomaticdogs showedbothCD8+_T_and_CD8+_T_lymphocyte_{proliferation,}

andwerecapableoflysinginfectedmacrophages. Wealsoinvestigatedwhetherdogswithdistinct clin-ical forms of CVL present alterations in the expression ofactivationmarkers(CD45RA+_,_CD45RB+_,_and_MHC-II+₎

on splenocytes (Fig. 4B). The development of a T-cell mediatedspeciﬁcimmune responserequiresLeishmania antigen presentation to Th cells together with major

SD OD AD-II AD-I CD 0 40 80 Clinical status Splenocytes ConA/Control ratio a a a Splenocytes LSA/Control ratio (A) SD OD AD-II AD-I CD 0 3 6 a a a (B) r= -0.3627 p= 0.0113 r= - 0.526p= 0.00013 a

Fig.3. ProliferativeindexofsplenocytesindogsnaturallyinfectedwithLeishmaniainfantumcategorizedaccordingtotheirclinicalstatusasasymptomatic (AD),oligosymptomatic(OD),symptomatic(SD),andcontrol(CD),afterinvitroConcanavalinA(ConA)orL.infantumsolubleantigen(LSA)stimulation. TheresultsareexpressedinbargraphsasscatteringofindividualvaluesandmeanConA/Controlratio(A)orLSA/Controlratio(B).Correlationsbetween proliferationindicesandclinicalstatusarealsoinserted,andPearson’scorrelationcoefﬁcient(r)andp-valuesareshownwithingraphs.

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CD4+ CD8+ CD4+_/CD₈+

Control

LSA

CD4+ CD8+ CD4+_/CD₈+ a_* CD4+ CD8+ CD4+_/CD₈+ a CD4+ CD8+ CD4+_/CD₈+ + + a 0 25 50 0 25 50 CD4+ CD8+ CD4+_/CD₈+

A

B

CD AD-I AD-II OD SD 0 300 410 520 630 a,b,c,d_* 0 300 410 520 630 CD45RA+ CD45RB+ MHC-II+ CD45RA+ CD45RB+ MHC-II+ CD45RA+ CD45RB+ MHC-II+ CD45RA+ CD45RB+ MHC-II+ CD45RA+ CD45RB+ MHC-II+

Control

LSA

Fig.4.ImmunophenotypicproﬁleandactivationstatusofsplenocytesindogsnaturallyinfectedwithLeishmaniainfantumcategorizedaccordingtotheir clinicalstatusasasymptomatic(AD),oligosymptomatic(OD),symptomatic(SD),andcontrol(CD),afterinvitrocultureinthepresenceofmedium(Control) orL.infantumsolubleantigen(LSA)for120h.TheresultsareexpressedinbargraphsasmeanpercentageofCD4+_and_CD8+_within_gated_lymphocytes_and

CD4+_/CD8+_ratio_plus_standard_deviation_(A),_and_average_of_major_{histocompatibility}_complex_(MHC-II+_),_CD45RA+_,_and_CD45RB+_as_mean_{ﬂuorescence}

channel(MFC)withingatedsplenocytesplusstandarddeviation(B).Signiﬁcantdifferencesatp<0.05areindicatedbythelettersa,b,c,anddincomparison withtheCD,AD-I,AD-II,andODgroups,respectively.Signiﬁcantdifferencesatp<0.05betweencontrolandL.infantumsolubleantigen(LSA)culturesare indicatedbyasterisk.

histocompatibility complex (MHC) molecules. However, Leishmania can impair antigen presentation in several ways.Reisetal.(2006b)demonstratedenhancedMHC-II densityas wellasa higher CD45RB/CD45RAexpression indexinperipheralbloodfromasymptomaticdogs,which seemstorepresentakeyelementtocontrolCVL morbid-ity.Inthepresentstudy,weobserveddecreasedMHC-II expressioninsplenocytesafterLSAstimulationinSDgroup ascompared withall groups or non-stimulatedculture inthesamegroup(Fig.4B).CobboldandMetcalfe(1994)

performedpioneeringinvestigationsofMHC-IImolecules indogs.Theyobservedthat,incontrastwithotherspecies, the MHC-II of dogs is expressed constitutively in all circulatinglymphocytes.IncreasedexpressionofMHC-II mayreﬂectan antigenicpriming-relatedimmunological event.Based onthis proposal,ourﬁndings suggestthat symptomatic dogs displayed lower MHC-II expression, probablypromotingmorevulnerabilitytodisease.

In conclusion, ourﬁndingshighlighted the complex-ityofcellularimmunologicaleventsrelatedtothenatural infectionofdogsbyL.infantum.Ourresultsshowedthat therole of CD8+ _T_splenocytes _seems_to _be_key _for _an

effectiveimmunological response,ahallmark of asymp-tomatic CVL, whereas the pronounced loss of MHC-II expressiononsplenocytesuponspeciﬁc-antigen stimu-lationisabiomarkerofsymptomaticCVL.

Acknowledgements

Theauthorsexpresstheirappreciationofthehardwork carriedoutbythekennelstaffattheUniversidadeFederal deOuroPreto,andUniversidadeFederaldeMinasGerais, andfortheirspecialdedicationduringtheexecutionofthis study.WealsothankthepeoplefromFundaçãoNacional daSaúde,MinistériodaSaúde,DistritoRegionaldeBelo Horizonte,MinasGerais,fortheirspecialdedicationtothis work. The authors alsothank the Program for Techno-logicalDevelopmentinToolsforHealth–PDTIS–FIOCRUZ, CEBIO, Universidade Federal de Minas Gerais, Instituto RenéRachou(IRR/FIOCRUZMinas),RedeMineirade Bioter-ismo,FAPEMIG(FundaçãodeAmparoàPesquisadoEstado deMinasGerais).A.B.R.,A.T.C.,R.C.G.,R.C.O.,andO.A.M.F. thankConselhoNacionaldeDesenvolvimentoCientíficoe

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Tecnológico(CNPq),andW.C.V.thankCAPES/PNPDfor fel-lowships.

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