Parasite control and skeletal myositis in Trypanosoma cruzi-infected and exercised rats.

ContentslistsavailableatScienceDirect

Acta

Tropica

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a

Parasite

control

and

skeletal

myositis

in

Trypanosoma

cruzi-infected

and

exercised

rats

Rômulo

D.

Novaes

_,

_Reggiani

_V.

_Gonc¸

_alves

_,

_Arlete

_R.

_Penitente

_,

_Marli

_C.

_Cupertino

_,

Izabel

R.S.C.

Maldonado

_,

_André

_Talvani

_,

_Antônio

_J.

_Natali

a_{InstituteofBiomedicalSciences,DepartmentofStructuralBiology,FederalUniversityofAlfenas,37130-000,MG,Brazil} b_{DepartmentofAnimalBiology,FederalUniversityofVic¸osa,36570-000,MG,Brazil}

c_{SchoolofMedicine,FederalUniversityofOuroPreto,35400-000,MG,Brazil} d_{DepartmentofGeneralBiology,FederalUniversityofVic¸osa,36570-000,MG,Brazil}

e_{DepartmentofBiologicalScienceandNUPEB,FederalUniversityofOuroPreto,35400-000,MG,Brazil} f_{DepartmentofPhysicalEducation,FederalUniversityofVic¸osa,36570-000,MG,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5December2016

Receivedinrevisedform17January2017 Accepted10February2017

Availableonline20February2017

Keywords: Exercisetraining Oxidativestress Pathology Skeletalmuscle

a

b

s

t

r

a

c

t

Non-pharmacologicalstrategieshavebeenrarelydescribedinthetreatmentofinfectiousdiseases. AlthoughexercisetraininghasbeenrecentlyincorporatedintheclinicalmanagementofChagasdisease, therationalebasisthatsupportsthisindicationispoorlyunderstood.Thus,weinvestigatedtheeffect ofanaerobicexerciseontheparasitism,inflammationandoxidativetissuedamageinamurinemodel ofTrypanosomacruzi-inducedskeletalmyositis.Wistarratswererandomizedintofourgroups:trained notinfected(TNI)andinfected(TI),sedentarynotinfected(SNI)andinfected(SI).Arunningtraining programwasadministered5days/weekfor9weeks.Then,infectedanimalswereinoculatedwithT.cruzi andfollowedupforanother9weeks.Exercisetraininginducedbeneficialadaptationsbyincreasingtime tofatigueandlactatethresholdinTNIandTIanimals.SIanimalspresentedhigherparasitemia, skele-talmuscleparasitism,cellnecrosis,leukocyteinfiltration,cytokineslevels,reactiveoxygenspeciesand nitricoxideproduction,thiobarbituricacidreactivesubstances,carbonylproteins,myosinheavychainI depletion,andincreasedcatalase(CAT)andsuperoxidedismutase(SOD)activities.Beyondattenuation inallthesevariables,TIanimalsshowedreducedTNF-␣,CCL-2/MCP-1andCX3CL1,andincreasedIL-10 musclelevels.Furthermore,theseanimalspresentedhigherCATandSODactivitiesandreducedlipid andproteinoxidation.Takentogether,ourfindingsindicatedthatexercisetraininginducedaprotective phenotypeinT.cruzi-infectedmice,enhancinghostdefensesagainsttheparasiteandattenuatingthe pathologicalremodelingassociatedwithskeletalmyositis,aspectspotentiallyassociatedtoanimproved immunologicalandredoxbalanceininfectedanimals.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Chagasdiseaseisaneglectedinfectiousdiseasecausedbythe intracellularprotozoanTrypanosoma cruzi(Santoset al.,2015a; WHO, 2015). Due to high tropism by muscle tissues, skeletal musclesareintenselyparasitizedbyT.cruzi,generatingmyositis, atrophyandnecrosisinanimals(Maldonadoetal.,2004; Vizcaíno-Castilloetal.,2014;Báezetal.,2015)andhumans(MontesdeOca etal.,2004;Torresetal.,2004).Despitethescarceevidence,

skele-∗ Correspondingauthorat:InstituteofBiomedicalSciences,Departmentof Struc-turalBiology,FederalUniversityofAlfenas,RuaGabrielMonteirodaSilva,700, Alfenas,37130−000,MinasGerais,Brazil.

E-mailaddress:romuonovaes@yahoo.com.br(R.D.Novaes).

talmuscledamagehasbeenassociatedwithdirectcellparasitism (i.e.myocytolysis)andunspecificimmune-mediatedmechanisms, in which exacerbated Th1 inflammatory response (Zhang and Tarleton,1999;Vizcaíno-Castilloetal.,2014)andregional over-production ofreactivemetabolites(i.e.free radicals)havebeen implicatedaspivotalmechanismsinvolvedinthepathogenesisof Chagasicmyopathy(Paivaetal.,2012;Báezetal.,2015).

Asexacerbatedimmunologicalandpro-oxidantresponses trig-gered by T. cruzi are coupledmechanisms directly involved in theworseningofChagasdisease,ithasbeenproposedthat anti-inflammatoryandantioxidantagentscouldexertprotectiveeffects againsttissuedamage(Mac¸aoetal.,2007;Novaesetal.,2016a). Furthermore, there is evidence that exercise training induces intensemodulationof theimmunologicalsystemand enhances endogenousantioxidantdefensesincardiacand skeletalmuscle http://dx.doi.org/10.1016/j.actatropica.2017.02.012

inanimals(Niessetal.,1999;Powersetal.,2002;Malm,2004)and humans(Niessetal.,1999;Gleeson2007).Inresponsetoinfections, physicaltrainingprovedtobeeffectiveininducingaTh1 immuno-logical phenotype (Lowder et al., 2005) essential topromoting cell-mediatedimmunityandresistanceagainstT.cruzi(Teixeira et al.,2002; Santoset al.,2015a).Although exercisetraining is effectivein attenuating the severityof myopathies with differ-entetiologies(Bacurauetal.,2016;Hedermannetal.,2016),the applicabilityofexerciseinthetreatmentofinfectiousmyopathies remainsunknown.

Recently, exercise training has emerged as a non-pharmacological strategy for Chagas disease treatment (Lima etal.,2010,2013;Novaesetal.,2016b).Althoughinvestigations inthis areaarescarce, arecent studyindicatedthata 12-week aerobicexercisetraining improvesmaximum oxygen consump-tion,peakheartrate,chronotropicresponseandexercisetolerance in Chagasic patients(Limaet al., 2010,2013). Furthermore,an 8-week pre-infection treadmill training program proved to be effectiveinreducingparasitemiaandweightlossinducedbythe infectioninmice(Schebeleski-Soaresetal.,2009).Whileallefforts havebeendirectedtothecardiovascularsystem,theinfluenceof exercisetrainingoninfectedskeletalmusclesremainsunexplored. Thisstudy wasdesigned toinvestigatetheeffect of anaerobic runningtrainingonparasitism,inflammationandoxidativetissue damageinamurinemodelofT.cruzi-inducedskeletalmyositis.

2. Materialsandmethods

2.1. Experimentaldesign

FortymaleWistarrats(16-weeksold)wererandomizedinto fourgroupscontaining10animalseach.Twogroupsweresubjected totreadmillrunningtraining,andtheothersremainedsedentary. Afterthetrainingperiod,animalsfromonetrainedandone seden-tarygroupwereinfectedwithT.cruzi.Thefourgroupswere:trained not infected (TNI), trained infected (TI),sedentary not infected (SNI),andsedentaryinfected(SI).Theanimalsweremaintainedina controlledenvironment(temperature22_±2◦C,humidity60–70%, and12/12hdark/lightinvertedcycles)withfreeaccesstochow andwater.ThestudywasapprovedbytheAnimalResearchEthics Commissionof theFederalUniversityofVic¸osa,Brazil(protocol 30/2009).

2.2. Runningtrainingandmetabolicadaptationinducedby exercise

All animals in the TNI and TI groups were subjected to an incrementalrunningtraining programona motor-driven tread-mill(InsightInstruments®,RibeirãoPreto,Brazil),5days/week,for 9weeksaspreviouslydescribed(Novaesetal.,2016b).Exercise trainingwasinitiated24hafterdeterminingthebasalindexesof physicalperformance.Toevaluatetheeffectofexercisetraining andinactivityonphysicalperformance,allanimalsweresubjected toatreadmillprogressiverunningprotocoluntilfatigue(Novaes etal.,2011).Thefatiguewasestablishedasthemomentatwhich theanimalswereunabletomaintaintherunningcadenceand inter-ruptedtheracebyover10s.Every3min,peripheralblood(5␮l) wascollectedbytailpuncturetomeasurelactatelevels(Accutrend Lactate®,Roche,Basel,Switzerland).Thetransitionpointbetween theaerobicmetabolismandanaerobicmetabolismwasdetermined byassessingthelactatethreshold(LT).Theprotocolwasrepeated threetimesonalternatingdaysbeforeandaftertheexercise pro-gram.Theresultswereconsideredastheaveragevaluesobtained fromthreeindependentphysicalperformancetests(Novaesetal., 2011).

2.3. Infectionandparasitemia

InfectedanimalswereintraperitoneallyinoculatedwithT.cruzi (150,000trypomastigotes[Ystrain]/100gbodyweight)obtained fromthebloodofinfectedmice(Novaesetal.,2011).Theanimals wereinoculated72hafterthelastphysicalperformanceevaluation. Confirmationofinfection,prepatentperiod, patentperiod,peak and meanparasitemiaweredetermined usingperipheralblood accordingtoBrener’sprotocol(1962).Nineweeksafter inocula-tion,theanimalswereeuthanizedunderanesthesiawithxylazine (2mg/kg,i.p.)andketamine(10mg/kg,i.p.)andthetibialisanterior skeletalmusclesweredissectedandanalyzed.

2.4. Cytokinesimmunoassay

Thecytokinelevelsinmuscletissueweremeasuredby enzyme-linked immunosorbent assay (ELISA).Briefly, musclefragments (100mg)werehomogenizedinphosphatebuffer(pH7.2)and cen-trifugedat10,000gat4◦Cfor10min.ThecytokinesTNF-␣,IFN-␥, IL-6, IL-10andthe chemokinesCCL-2/MCP-1and CX3CL1 were assayedinthehomogenatefollowingthemanufacturer’s instruc-tions(Promega,Madison,WI,USA).Thereactionwasrevealedusing peroxidase-conjugatedstreptavidin(VectorLab.,CA,USA)andthe substratecontaining3,3,5,5”-tetramethylbenzidine(Promega,WI, USA).Thereactionswerereadinspectrophotometerat450nm,and thelevelsofcytokineswerecalculatedbyextrapolatingthe opti-caldensitiesobtainedfromastandard curveusingrecombinant cytokines(Novaesetal.,2016b).

2.5. Histopathologyandstereology

Skeletal muscle fragments were fixed for 48h (10% w/v formaldehydein0.1Mphosphatebuffer,pH7.2).Thefragments wereembeddedin paraffin,verticallysectionedat 4-␮m thick-nessandstainedbyhematoxylin-eosin(H&E).Histologicalsections wereanalyzedbybrightfieldmicroscopy(OlympusBX-60,Tokyo, Japan). Fiftyhistological fields (×400 magnification) were ran-domlysampledandthemuscle(totalarea=1.12×106_␮m2_)was microscopically analyzed in each group (Novaes et al., 2011). Thestereologicalmethodwasusedtoassessthedistributionof skeletal myocytes, tissue necrosis, amastigote nests and inten-sityoftheinflammatoryinfiltrateininfectedanimals.Atestarea (AT)of 3.15×103␮m2 wasused,containing42 test points (Pt) appliedin histological imagesobtainedat×1000magnification acrossfiverandommicroscopicfieldsofeachanimalinatotalof 160×103_␮m2_{ofmuscletissueforeachgroup.Thevolumedensity} ofskeletalmyocytes(VvMY)andnecroticareas(VvNA)were esti-matedas:VvMY/NA=MY/NA/PT,wherePMY/NAisthenumberoftest pointshittingonskeletalmyocytesornecroticareas,andPTisthe totalnumberoftestpoints.Thenumberdensityofinflammatory cells(QAIC)andamastigotenestsofT.cruzi(QATC)wasestimated as:QAIC/TC=(IC/TC)/AT;whereIC/TCisthetotalnumberof inflam-matorycellsorT.cruzinestscountedinthetestarea(AT)(Novaes etal.,2013).

2.6. Oxidative/nitrosativestressmarkers

Insituproductionofreactiveoxygenspecies(ROS)was eval-uated in unfixed5␮m thick muscle cryosections incubated at roomtemperaturewithdihydroethidium(DHE,10␮mol/lin0.01% dimethylsulfoxide) for30min.Sectionswereexaminedby flu-orescencemicroscopy (OlympusBX-60,Olympus, Tokyo,Japan) andtheimagewascapturedat400_×magnification.Fiverandom microscopicfieldswereanalyzedforeachanimal.Redfluorescence produced by DHEoxidation to hydroxyethidiumwasevaluated

byusingImagePro-Plus4.5 software(MediaCybernetics,Silver Spring,MD,USA)(Santosetal.,2015a,b).

Thesamemusclehomogenateusedinthecytokine immunoas-sayswereusedfor assessingoxidativeand nitrosativemarkers. Thiobarbituricacid-reactive substances(TBARS)werequantified todeterminelipidperoxidationinmuscletissue(Gutteridgeand Halliwel,1990).Proteinoxidationwasverifiedbythequantification ofproteincarbonyls(PCN)usingthe2,4-dinitrophenylhydrazine (DNPH)procedure(Levineetal.,1990).Nitricoxide(NO)was indi-rectlydetermined by the quantification of nitrite/nitrate levels usingtheGriessmethod(Ricart-Janéetal.,2002).

2.7. Endogenousantioxidantenzymes

Catalase(CAT)activitywasevaluatedbymeasuringtherateof decompositionofhydrogenperoxide(H2O2)(Aebi,1984). Superox-idedismutase(SOD)activitywasestimatedbyaxanthineoxidase method(Sarbanetal.,2005).Glutathione-s-transferase(GST) activ-itywasestimatedspectrophotometricallyat340nm,asdescribed byHabigetal.(1974),andcalculatedfromtherateofnicotinamide adeninedinucleotidephosphate(NADPH)oxidation.

2.8. Myosinheavychainanalysis

Biochemicalanalysisof myosin heavy chainI (MyHCI) was performedusingsodiumdodecylsulfate-polyacrylamidegel elec-trophoresis(BärandPette,1988).Musclesampleswereplacedin anextractionsolution(10%glycerol,5%2-mercaptoethanol, and 2.3%sodiumdodecylsulfatein a0.9% Tris-HClbuffer[pH 6.8]). Forelectrophoreticseparation,muscleextractswereloadedontoa 7%–10%sodiumdodecylsulfate–polyacrylamidegel electrophore-sisgradientgeloveraperiodof26hat185V.Thegelswerestained withcoomassiebluetoidentifytheMyHCisoformsaccordingto theirmolecularweights.TherelativeMyHCIcontentwasidentified accordingtothemolecularmassandquantifiedbydensitometry usingImageJSoftware(USNationalInstitutesofHealth,USA). 2.9. Statisticalanalysis

Data were reported as mean and standard deviation (mean±S.D.). Data distribution was verified by D’Agostino-Pearsontest. Parametersof exercisetolerance, biochemical and immunological data werecompared using one-way analysis of varianceANOVAfollowedbyTukey’sposthoctest.Parasitological parameterswerecompared usingtheMann–Whitney–Wilcoxon test.MorphologicaldatawerecomparedusingtheKruskal–Wallis test.Aprobabilityofp<0.05wasconsideredstatisticallysignificant.

3. Results

As indicated in Fig. 1, trained animals presented increased exercisetolerance,whichwasevidencedbyprolongationin lac-tatethresholdandtimetofatiguecomparedtosedentaryanimals (p<0.05).

Infectedandtrained(TI)animalspresentedincreasedprepatent period, as wellas reduced patent period, peak and mean par-asitemiacomparedtosedentaryinfected animals(SI)(p<0.05), Table1.

Tissuelevelsofallcytokinesinvestigated(INF-␥,TNF-␣,IL-6, IL-10,andCCL2/MCP-1)weresimilarintheTNIandSNIgroups (p>0.05),exceptCX3CL1,whichpresentedlowlevelsintheSNI group(p<0.05).Ingeneral,bothinfectedgroups(SIandTI) pre-sented increased levels of all cytokines investigated compared touninfectedanimals (SNIand TNI)(p<0.05). WhileIL-10was increased,TNF-␣,CCL2/MCP-1andCX3CL1levelswerereducedin theTIgroupcomparedtotheSIgroup(p<0.05),Fig.2.

Fig.1.Lactatethreshold(LT)andtotaltimetofatigue(TTF)insedentaryandtrained miceprevioustoT.cruziinoculation.TNI,trainednotinfected;TI,trainedinfected; SNI,sedentarynotinfected;SI,sedentaryinfected.Dataareexpressedasmeanand standarddeviation.

SkeletalmusclefromanimalsintheSNIandTNIgroups pre-sentedanormalmicroscopicstructure,whichpresentedmyocytes withwell-definedcross-striation,surroundedbyscarce connec-tivetissue presenting low interstitialcellularity.Both SI andTI animalspresentedevidentskeletalmyositis,tissuenecrosis, con-nectiveexpansionanddiffusedistributionofamastigotenestsofT. cruzi(Fig.3).

StereologicalanalysisindicatedthatbothSIandTIanimals pre-sentedmarkedtissuenecrosis,reducedmyocytedistributionand intense inflammatory infiltration compared tothe SNIand TNI groups(p<0.05).AlltheseparameterswerereducedintheTIgroup comparedtotheSIgroup(p<0.05),Table2.

AnimalsintheSIand TIgroupsshowedintensemuscle pro-ductionofROSandreducedMyHCIlevelscomparedtotheSNI and TNIgroups (p<0.05). Skeletalmusclefrom TIanimals pre-sentedreducedROSandincreasedMyHCIlevelscomparedtothe SIanimals(p<0.05),Fig.4.

TBARS,PCnyandNOmusclelevelsweredrasticallyincreasedin bothSIandTIgroupscomparedtotheSNIandTNIgroups(p<0.05). TheseparametersweremarkedlyattenuatedinTIcomparedtoSI animals(p<0.05),Fig.5.

TheantioxidantenzymesCAT,SODandGSTshowedincreased activitiesinskeletalmusclefrombothSIandTIcomparedtothe SNIandTNIgroups(p<0.05).CATandSOD,butnotGSTactivities, wereincreasedinTIcomparedtoSIanimals(p<0.05),Fig.6.

4. Discussion

While studies withhumans (Limaet al., 2010,2013; Fialho etal.,2012)andanimals(Pretoetal.,2015;Novaesetal.,2016b) are mainly focused on the cardiovascular system, the impact of exercisetherapy on skeletalmuscle duringT. cruzi-infection remains neglected. Corroborating the assumption that skeletal musclesareprimarytargetsofexercisetherapy(Goldspink,2003; Hedermannetal.,2016),ourresultsindicatedthatrunningtraining inducedmetabolicadaptations,improvedparasitologicalcontrol andreducedskeletalmyositisininfectedanimals,aspects poten-tiallyrelatedtomodulationofcytokineproduction,reducedROS production,improvedredoxbalance,attenuationofskeletalmuscle pathologicalremodelingandreductionofMyHCIdepletion.

Consideringthattheparasiticloadiscloselycorrelatedto tis-sueparasitismanddamage,parasitologicalcontrolplaysapivotal roleinthetreatmentofChagasdisease(ZhangandTarleton,1999; Velosoetal.,2008).Parasitologicalcontrolobservedinourstudy hasbeenpotentiallylinkedtotheimmunomodulatory effectof exercisetraining(Novaesetal.,2016b).AsTh1cytokines(especially IFN-␥andTNF-␣)weremarkedlyincreasedininfectedanimals,

Table1

EffectofexercisetrainingonparasitologicalparametersofTrypanosomacruzi-infectedmice.

Prepatentperiod(days) Patentperiod(days) Peakofparasitemia(parasites/0.1mL×103_{) Meanparasitemia(parasites/0.1mL×10}3₎

SI 4.2±0.42 9.7±0.95 21.59±2.84×103 _3.82±1.48

TI 5.5±0.53* _7.4±1.16* _{20.58±2.63×10}3 _1.92±0.63*

SI,sedentaryinfected;TI,trainedinfected.Dataareexpressedasmeanandstandarddeviation.

Fig.2. CytokineandchemokinelevelsintheskeletalmusclefromT.cruzi-infectedmice.TNI,trainednotinfected;TI,trainedinfected;SNI,sedentarynotinfected;SI, sedentaryinfected.Dataareexpressedasmeanandstandarddeviation.*†§Statisticaldifferencesamongthegroups(p<0.05);*vs.TNI,†vs.TI,§vs.SNI.

Table2

EffectofexercisetrainingonskeletalmusclestructureinT.cruzi-infectedmice.

Vvmyocytes(%) Vvnecrosis(%) QAIC(cells/mm2) QATC(nests/mm2)

TNI 92.17±3.68 ND 824.39±236.41 ND

TI 72.26±6.19* 4.28±1.05 1808.11±415.74* 11.38±5.08

SNI 90.52±4.41† ND 958.41±253.60† ND

SI 55.70±7.11*†§ 9.65±3.21§ 2951.80±617.25*†§ 20.25±7.41

Vv,volumedensity;QA,numberdensity;IC,interstitial/inflammatorycells;TC,T.cruzinests;ND,notdetected.TNI,trainednotinfected;TI,trainedinfected;SNI,sedentary

notinfected;SI,sedentaryinfected.Dataareexpressedasmeanandstandarddeviation.*†§Statisticaldifferencesamongthegroups(p<0.05);*vs.TNI,†vs.TI,§vs.SNI.

exercisetrainingdoesnotimpairthepolarizationofthe immuno-logicalresponse,anaspectthatcouldattenuatetheparasitological control and amplify muscle damage. This immunomodulatory effectwasalsoclearlyobservedininfectedtrainedanimals,which presentedsimilarIFN-␥andIL-6levels,reductioninTNF-␣,

CCL-2/MCP-1 and CX3CL1, as well as increased IL-10 tissue levels comparedtoinfectedsedentaryanimals.Asregulatorymolecules, theTh1/Th2cytokinebalanceiscrucialtotheestablishmentofan adjustedinflammatoryprocess,which isabletoensureparasite controlwithoutdeterminingintensesecondaryimmune-mediated

Fig.3. RepresentativephotomicrographsoftheskeletalmusclefromuninfectedcontrolandTrypanosomacruzi-infectedmice(H&Estaining,brightfieldmicroscopy).Trained andsedentaryuninfectedanimalspresentedscarceconnectivestroma(arrow),lowtissuecellularityandevidentmyocytes(asterisk)cross-striation.Trainedandsedentary infectedmiceshowedintenseskeletalmyositis,connectivetissueexpansion(arrow)andintracellularT.cruziamastigotenests(arrowheads).

Fig.4.Reactiveoxygenspecies(ROS)andmyosinheavychainI(MyHCI)levelsintheskeletalmusclefromT.cruzi-infectedmice.TNI,trainednotinfected;TI,trained infected;SNI,sedentarynotinfected;SI,sedentaryinfected.Theimageswereobtainedbyfluorescencemicroscopy.BrighterimagesindicatehighROSproduction,which wasindirectlyquantifiedinA.MyHCIexpressionwasdeterminedopticaldensitometryfrompolyacrylamidegelelectrophoresis.(AandB)Dataareexpressedasmeanand standarddeviation.*†§Statisticaldifferencesamongthegroups(p<0.05);*vs.TNI,†vs.TI,§vs.SNI.

Fig.5. MarkersofreactivestressintheskeletalmusclefromT.cruzi-infectedmice.TBARS,Thiobarbituricacidreactivesubstances;PCN,proteincarbonyl.Nitricoxidewas expressedasnitrite/nitrate(NO2−/NO3−).TNI,trainednotinfected;TI,trainedinfected;SNI,sedentarynotinfected;SI,sedentaryinfected.Dataareexpressedasmeanand

standarddeviation.*†§Statisticaldifferencesamongthegroups(p<0.05);*vs.TNI,†vs.TI,§vs.SNI.

Fig.6.ActivityofendogenousantioxidantenzymesintheskeletalmusclefromT.cruzi-infectedmice.CAT,catalase;GST,glutathione-s-transferase;SOD,superoxide dismutase.TNI,trainednotinfected;TI,trainedinfected;SNI,sedentarynotinfected;SI,sedentaryinfected.Dataareexpressedasmeanandstandarddeviation.*†§Statistical differencesamongthegroups(p<0.05);*vs.TNI,†vs.TI,§vs.SNI.

tissue damage caused byan exacerbatedTh1response (Gomes etal.,2003;Novaesetal.,2016a,b).Takentogetherwiththe para-sitological,histopathologicalandstereologicalresults,ourfindings indicatedthatreductionintypicalTh1molecules(i.e.TNF-␣, CCL-2/MCP-1) do not determine increased host susceptibility to T. cruziinfection.Conversely,thisreductionwaspotentiallyachieved from the improved immunological control and attenuation of skeletalmuscleinflammation.Thispropositionis coherentwith CCL-2/MCP-1reduction,amoleculedirectlyinvolvedinleukocyte chemoattractiontoinfectedorgans(Teixeiraetal.,2002;Yamauchi etal.,2007).Furthermore,asapotentanti-inflammatorycytokine, increasedIL-10levelsalsoreinforcedtheinflammatorycontroland reductioninskeletalmyositisseverity.

Becauseoftheimprovedimmunologicalcontrol,attenuationin theskeletalmusclepathologicalremodelingwasexpected.Beyond directmyocytolysis and necrosisinducedbydirect intracellular parasite proliferation, tissue damage is secondarily determined byexacerbatedregionalinflammatoryprocessdirectedtoT.cruzi (Gomesetal.,2003;Novaesetal.,2016a).Ascytotoxins, includ-ingradical(i.e.O2•−,OH−,ONOO−)andnon-radical(i.e.NO,H2O2, HClO,HNO2)reactivemolecules,producedbyinflammatorycells recruitedtotheinfectedtissuesactindiscriminatelyonparasites andhostcells,theinflammationintensityisdirectlyrelatedto tis-suedamageseverityinChagasdisease(Paivaetal.,2012;Novaes et al.,2015).Thus, reduced cell necrosis,inflammatory infiltra-tionandhigherdistributionofintactmyocytesareconsistentwith reducedtissueparasitism(i.e.tissueamastigotenests)observed in trained animals.Taking into account that skeletal myocytes present limited regenerativepotential (Maldonado etal., 2004; Souzaetal.,2014),thiseffectseemstoberelevantinprotecting thehostagainsttheprogressiveimmunomediatedtissuedamage typicallyobservedinChagasdisease(ZhangandTarleton,1999; Torresetal.,2004).

ExercisetrainingalsopresentedbeneficialeffectsagainstMyHC Idepletionininfectedskeletalmuscle,afindingpotentiallyrelated tothehigherpreservationofintactmyocytes.Conversely, seden-taryanimalspresentedincreasedparenchymadamage(i.e.necrotic

myocytes) and connective expansion, which couldbe partially responsibleforthereducedMyHCIlevelswhenadjustedby mus-clemassortotalmuscleprotein.However,increasedMyHCIlevels couldalsoberelatedtotheabilityofexercisetrainingtoinduce MyHCIgeneexpressionbymechanic-transduction mechanisms (BärandPette,1988;Goldspink,2003).Althoughthismechanism is more relevant in explaining higher MyHC I levels in unin-fectedandtrainedanimals,furtherstudiesarerequiredtoconfirm ifthesepropositionsis coherentininfected animals.Protection againstMyHCIdepletionrepresentsaremarkablefindingin skele-talmyopathies,sincethismoleculeisdirectlyassociatedwiththe productionofenergy,forceandvelocityofcontractionbymyocytes, aspectswithmarkedinfluenceonmusclefunction(BärandPette, 1988;Goldspink,2003).

Theimprovedredoxbalancealsoindicatedtheprotectiveroleof exercisetrainingininfectedanimalscomparedtosedentaryones. ThiseffectwasmediatedbyreducedROSandNOproduction,which wasrelatedtoreducedlipid(i.e.TBARS)andprotein(PCny) oxi-dationinskeletalmusclefromtrainedanimals.Althoughreactive stresshasbeendirectlyimplicatedinheartmorphofunctional dam-ageandChagascardiomyopathyprogression(Mac¸aoetal.,2007; Guptaetal.,2009),theimpactofthisprocessonskeletal myosi-tisispoorlyunderstood.Apparently,mitochondrialdysfunctionin parasitizedskeletalmyocytesisanintrinsicsourceofROSandRNS (Báezetal.,2015).However,theinflammatoryprocessand “res-piratoryburst”ofrecruitedleucocyteshasbeenproposedasthe mainsourceofreactivemediators,especiallyNO,H2O2,O2•−,and OH−(Paivaetal.,2012;Santosetal.,2015b).Thispropositionis supportedgiven thatanti-inflammatorystrategies are accompa-niedbyamarkedreductioninROSandRNSproductioninChagas disease(Novaesetal.,2016a,b).Thus,byreducingleucocyte migra-tionandinflammationseverity,exercisetrainingregulatesapivotal sourceofreactivemetabolites,reducingROSandRNStissuelevels andsecondaryreactivetissuedamage.Althoughthismechanism seemstobesimilartothoseobservedinChagascardiomyopathy (Novaesetal.,2016b),theROSandRNSproductionpathwaysand

theroleofthesemoleculestothedevelopmentofskeletalmyositis wasnotcompletelyelucidated,requiringfurtherstudies.

Inthepresentstudy,reducedlipidandproteinoxidationcannot beexclusivelyattributedtotheattenuationinmuscle inflamma-tionandreducedreactivemetaboliteproduction.Thus,increased antioxidantenzymeactivity(i.e.CATandSOD)observedininfected and trained animals is also a remarkable adaptation that con-tributes to counteract ROS and RNS, restricting reactivetissue damage.Improvementsinredoxmetabolismhavebeen systemati-callydescribedinanimalsandhumans,anadaptationthattogether withimmunologicalmodulationhasprovidedtherationalebasis that supports the indication of exercise training to treat sev-eralhealthconditions,includingmetabolic(Bacurauetal.,2016; Hedermannet al.,2016)and infectious diseases(Lowderet al., 2005;Schebeleski-Soaresetal.,2009).

Takentogether, ourfindings indicatedthat exercise training inducedaprotectivephenotypeinT.cruzi-infectedmice,enhancing thehostdefensesagainsttheparasiteandattenuatingtheextent ofpathologicalremodelingassociatedwithskeletalmyositis. Tak-ingintoaccountthatthereductionofskeletalmuscledamagewas accompaniedbyanattenuationoftheinflammatoryprocess,ROS andNOproductionandreactivetissuedamage,aswellasthe upreg-ulationofantioxidantenzymeactivity,thebeneficialadaptations inducedbyexercisewerepotentiallyassociatedwithanimproved immunologicalandredoxbalanceininfectedanimals.

Conflictofintereststatements

Therearenoconflictsofinterest.Allauthorscontributedtodata collectionandanalysis,articlepreparationandhaveapprovedthe finalmanuscript.

Acknowledgments

ThisworkwassupportedbytheBrazilianfundingagencies Con-selhoNacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq) (454901/2014-3)andFundac¸ãodeAmparoàPesquisadoEstadode MinasGerais(FAPEMIG)(APQ-02309-14).TheauthorsAndré Tal-vaniandAntônioJ.NatalihaveresearchfellowshipsfromConselho Nacional deDesenvolvimento Científico e Tecnológico (CNP-q), Brazil.

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