RESEARCHARTICLE HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis AkilHammami,BelmaMeldaAbidin,TaniaCharpentier,AymericFabie´,Annie- PierDuguay,KristaM.Heinonen,SimonaSta¨ger* a1111111111 INRS-InstitutArmand-FrappierandCenterforHost-Parasiteinteractions,531BoulevarddesPrairies,Laval (QC),Canada a1111111111 a1111111111 *[email protected] a1111111111 a1111111111 Abstract Leishmaniadonovaniisknowntoinducemyelopoiesisandtodramaticallyincreaseextrame- dullarymyelopoiesis.Thisresultsinsplenomegaly,whichisthenaccompaniedbydisruption OPENACCESS ofthesplenicmicroarchitecture,achronicinflammatoryenvironment,andimmunosuppres- Citation:HammamiA,AbidinBM,CharpentierT, Fabie´A,DuguayA-P,HeinonenKM,etal.(2017) sion.Chronicallyinflamedtissuesaretypicallyhypoxic.Theroleofhypoxiaonmyeloidcell HIF-1αisakeyregulatorinpotentiatingsuppressor functionsduringvisceralleishmaniasishasnotyetbeenstudied.HereweshowthatL.dono- activityandlimitingthemicrobicidalcapacityof vanipromotestheoutputfromthebonemarrowofmonocyteswitharegulatoryphenotype MDSC-likecellsduringvisceralleishmaniasis. thatfunctionassafetargetsfortheparasite.Wealsodemonstratethatsplenicmyeloidcells PLoSPathog13(9):e1006616.https://doi.org/ 10.1371/journal.ppat.1006616 acquireMDSC-likefunctioninaHIF-1α-dependentmanner.HIF-1αisalsoinvolvedindriving thepolarizationtowardsM2-likemacrophagesandrenderingintermediatestagemonocytes Editor:DavidSacks,NationalInstituteofHealth, UNITEDSTATES moresusceptibletoL.donovaniinfection.OurresultssuggestthatHIF-1αisamajorplayerin theestablishmentofchronicLeishmaniainfectionandiscrucialforenhancingimmunosup- Received:August25,2017 pressivefunctionsandloweringleishmanicidalcapacityofmyeloidcells. Accepted:August29,2017 Published:September11,2017 Copyright:©2017Hammamietal.Thisisanopen accessarticledistributedunderthetermsofthe Authorsummary CreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,and TheprotozoanparasiteLeishmaniadonovanicauseschronicinfectioninthespleen,whichis reproductioninanymedium,providedtheoriginal accompaniedbyachronicinflammatoryenvironment,anenlargementoftheorgan,and authorandsourcearecredited. immunosuppression.Theenvironmentofchronicallyinflamedtissuesischaracterizedby DataAvailabilityStatement:Allrelevantdataare lowoxygenlevelsandtissuedisruption,whichinducetheexpressionofthetranscriptionfac- withinthepaperanditsSupportingInformation torHIF-1αinallcells.Thekineticsofmonocyteproductionanddifferentiationinthebone files. marrowandthespleen,andtheroleofhypoxiainmyeloidcellfunctionsduringvisceral Funding:Thisworkwassupportedbythe leishmaniasishavenotyetbeenstudied.HereweshowthatL.donovanipromotestheoutput CanadianInstituteofHealthResearchgrantMOP- fromthebonemarrowofmonocyteswitharegulatoryphenotypethatfunctionassafetar- 123293(toSS)andPJT-148614(toKMH),the getsfortheparasite.WealsodemonstratethatHIF-1αpotentiatesinhibitoryfunctionsof FondsderechercheduQue´bec–Sante´grant myeloidcellsandisinvolvedindrivingthepolarizationtowardsM2-likemacrophagesand #32598(toKMH),andtheCanadaFoundationfor InnovationJohnEvansLeaderFundgrant#31377 renderingthemmoresusceptibletoL.donovaniinfection.OurresultssuggestthatHIF-1αis (toKMHandSS).KMHisChercheur-Boursier PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 1/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection (Junior1)oftheFondsderechercheduQue´bec– Sante´.AHwaspartlysupportedbyanImperial amajorplayerintheestablishmentofchronicLeishmaniainfectionandiscrucialfor TobaccoscholarshipfromtheFondation enhancingimmunosuppressivefunctionsandloweringleishmanicidalcapacityofmyeloid UniversitaireArmand-FrappierInstitutNationalde cells. laRechercheScientifique.Thefundershadnorole instudydesign,datacollectionandanalysis, decisiontopublish,orpreparationofthe manuscript Competinginterests:Theauthorshavedeclared Introduction thatnocompetinginterestsexist. Eliminationofintracellularpathogensrequirestheinductionofpro-inflammatorycytokines andcytotoxicmoleculessecretion.Unfortunately,thisprocessalsoleadstolocaltissuedisrup- tionandinflammation.Inflamedtissuesrepresentachallengingmicroenvironment,charac- terizedbyhypoxia,acidosisandhypoglycemia.Thismicroenvironmenttypicallycausesthe stabilizationofthetranscriptionfactorHIF-1α,themasterregulatoroftheresponsetohypoxia [1,2].HIF-1αhaspleiotropicfunctionsaimedatprotectingtissuesfrominjuryandhelping cellstoadapttoadifficultmicroenvironment.However,stabilizationofHIF-1αinsomecells oftheimmunesystem,suchasmyeloidcells,mayalsohaveunwantedconsequences.For instance,HIF-1αisresponsibleforthepolarizationtowardstheM2-likephenotypeoftumor- associatedmacrophages(TAM)[3],promotingthereforetumorgrowth.HIF-1αwasalso showntoenhancefunctionanddifferentiationofmyeloidderivedsuppressorcells(MDSC)in thetumormicroenvironment[4].Moreover,wehavereportedthatHIF-1αstabilizationin dendriticcellsinhibitedtheirfunctionandconsequentlylimitedtheexpansionofprotective CD8Tcellresponsesduringexperimentalvisceralleishmaniasis(VL)[5]. TheHIF-pathwayisalsoexploitedbysomepathogensfortheirreplicationand/orsurvival insidethehost’scell[6–9].OneexampleofsuchapathogenisLeishmania.Theprotozoanpar- asiteLeishmaniaisthecausativeagentofleishmaniasis,adiseasewithmultipleclinicalmani- festationsrangingfromself-healingcutaneousandmucocutaneouslesionstopotentiallylethal visceralinfections.Thepromastigoteformoftheparasiteistransmittedtothehostbyasandfly vector.Onceinsidethehost,promastigotestransformintoamastigotes.Macrophagesarethe maintargetcellsoftheparasite.However,tosurviveinsidemacrophages,Leishmanianeedsto attenuatetheirmicrobicidalpotential[10].Oneofthemanystrategiesisthestabilizationof HIF-1α[11],whichappearstobeessentialforthesurvivalofthepromastigoteforminsidethe cell[6,11].HIF-1αstabilizationcanoccurfollowingmassiveinfiltrationbypro-inflammatory cellsinthetissueand/orasaconsequenceofpathogeninvasion.Thesetwophenomenaare associatedwithincreasedoxygenconsumption,whichcausesalocalhypoxicenvironment [12].Duringvisceralleishmaniasis,HIF-1αstabilizationisalsoinducedinuninfectedcellsby theinflammatoryenvironmentandappearstohamperDCfunctions[5].Todate,theroleof HIF-1αinothermyeloidcellsduringinvivoLeishmaniainfectionshasnotyetbeenexplored. Dendriticcellsandneutrophilshavebeenextensivelystudiedinvariousmodelsofleish- maniasis;however,thecontributionofmonocytestosusceptibilityand/orresistanceto infectionisstillunclear.Theearlyliteratureproposesapossibleroleof“undifferentiated macrophage-granulocytes”assafetargetsforLeishmania,contributingthereforetodisease susceptibility[13].Passosetal.[14]demonstratethatintermediatemonocytesareinvolved inmediatingimmunopathologyinpatientsinfectedwithL.braziliensis.Anotherstudy reportstheupregulationofA adenosinereceptorsonhumanmonocytesandtheassocia- 2B tionofthisupregulationwithpathogenicityinpatientsexposedtoL.donovani[15].Incon- trast,monocyte-derivedDCappeartobeessentialforprimingprotectiveTh1responsesin L.majorinfectedmice[16]andclassicalmonocytesarethoughttobeabletokillL.major [17]andL.braziliensisviareactiveoxygenspecies[18]. PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 2/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection Inthisstudy,wewantedtoinvestigatetheroleofHIF-1αstabilizationinmyeloidcells,par- ticularlymonocytes,duringexperimentalchronicVL.Wefoundthatmyeloidcellsareincreas- inglyrecruitedtothespleenduringchronicinfection.SplenicmyeloidcellsupregulateHIF-1α anddisplayHIF-1α-dependentinhibitoryfunctiononprotectiveTh1responses.Moreover, HIF-1αlimitstheirleishmanicidalfunctionsandregulatesthedifferentiationandoutputof inflammatorymonocytesfromthebonemarrow. Results Myeloidcells,particularlyLy6ChiandLy6Clo/intmonocytes,accumulate inthespleenofL.donovaniinfectedmiceoverthecourseofinfection Theliteratureabouttheroleofmonocytesduringexperimentalvisceralleishmaniasisisscarce. Hence,wewantedtohaveafullpictureofthemonocytesandneutrophilsrecruitmentkinetics tothespleenoverthecourseofexperimentalL.donovaniinfection,beforeassessingtheroleof HIF-1αinsplenicmyeloidcells.WefirstmonitoredthefrequencyofCD11bhiLy6Ghineutro- phils.AsshowninFig1A,thepercentageofneutrophilspresentinthespleengradually increasedduringthefirst4weeksofinfection.Similarlytoneutrophils,Ly6Chimonocytes wereincreasinglyrecruitedtothespleenoverthecourseofinfection(Fig1B).Incontrast,the frequencyofLy6Clo/intmonocytesdidnotvarysubstantiallyasdiseaseprogressed(Fig1B). Interestingly,thetwomonocytepopulationswerelesseasilydistinguishableduringthechronic phaseofinfection. WenextexaminedwhethersplenicmyeloidcellsexpressedCD11catvarioustimepointsof infection.Atd14p.i.about55%ofallCD11b+cellsinthespleenexpressedCD11c;theper- centageofCD11c+cellsincreasedoverthecourseofinfectionandatd35p.i.80%ofthesplenic CD11b+cellswerealsoCD11c+(Fig1C).Asexpected,allLy6ChimonocyteswereCD11c+and about85%oftheLy6Clo/intmonocytesexpressedCD11c(Fig1D). BecauseLysM-specificHIF-1α-deficientmicearenotagoodmodeltostudytheroleof HIF-1αinmonocytes/macrophagesinthespleen[19]andthevastmajorityofsplenicCD11b+ cellsduringVLwereCD11c+,wedecidedtouseCD11c-specificHIF-1αdeficientmice[5]to investigatetheroleofHIF-1αinmyeloidcells,particularlymonocytes,duringchronicVL.To note,neutrophilsdidnotexpressCD11c,hencetheyareHIF-sufficientinbothgroupsofmice. HIF-1α-deficientmiceinCD11c+cellsshowincreasedfrequencyand numbersofinflammatorymonocytesinthespleen WehavepreviouslyreportedthatHifflox/flox–Cd11c-Cre+micearehighlyresistanttoL.dono- vaniinfection([5]andS1AFig).Duringtheacutephaseofinfection,HIF-1αimpairsdendritic cellfunctionsandlimitsCD8Tcellexpansion[5].Atthisstageofdisease,parasiteclearancein thesemiceismainlyCD8Tcell-dependent[5];however,itisstillunclearhowthesemicecon- trolL.donovanigrowthduringchronicVL,whenCD8Tcellsareexhausted[20].CD8+den- driticcellsarethoughttoberesponsibleforCD8Tcellcross-priming[21].TheseDC subpopulation,unlikeCD4+DCs,mainlyexpressesDNGR1(S1BFig)andthusdirectly descendsfromDCprecursorsratherthanbeingmonocyte-derived[22].Hence,wedecidedto extendourinvestigationontheroleofHIF-1αtoothermyeloidcells,particularlymonocytes andmonocytes-derivedcells.Becausemonocytescontributetoparasiteclearanceinother modelsofleishmaniasis[16–18],wefirstcomparedtherecruitmentofmonocytestothespleen inHifflox/flox–Cd11c-Cre+mice(HIF-1α-deficient)andtheirCre-littermates(HIF-1α-suffi- cient)atvarioustimepointsofinfection.Before,though,wemonitoredHIF-1αexpressionin purifiedCD11b+cellsfrombothmousegroupstoconfirmthatHIF-1αwasindeeddeletedin PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 3/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection Fig1.Myeloidcells,particularlyLy6ChiandLy6Clo/intmonocytes,accumulateinthespleenofL.donovaniinfectedmiceoverthe courseofinfection.MicewereinfectedwithL.donovaniandsacrificedatvarioustimepointsafterinfection.Neutrophilswereexcludedfromall analysisinvolvingmonocytes.(A)RepresentativeFACSplotsdepictingthegatingstrategyusedtoidentifyneutrophils(left)andpercentageof neutrophilsinthespleenofinfectedmice(right).(B)GatingstrategyusedtoidentifyLy6C+monocytes(left)andpercentageofsplenicLy6Chi (uppergraph)andLy6Clo/int(lowergraph)monocytes.(C)PercentageofCD11c+myeloidcells.(D)PercentageofCD11c+Ly6C+(lefthistogram rawanduppergraph)andLy6Clo/int(righthistogramrawandlowergraph)monocytes.Alldatarepresentmean±SEMofoneof4independent experiments,n=4. https://doi.org/10.1371/journal.ppat.1006616.g001 PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 4/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection Hifflox/flox–Cd11c-Cre+myeloidcells(S2AandS2BFig).AsobservedinC57BL/6mice,thefre- quencyandthenumberofLy6Chimonocytesincreasedoverthecourseofinfectioninthe Cre-andCre+group(Fig2Aand2B).However,asignificantlyhighernumberofinflammatory monocyteswaspresentinthespleenofCre+mice.Non-classicalLy6Clo/intmonocytes(Fig2A and2C)andneutrophils(Fig2DandS3AFig)displayedsimilarfrequenciesinbothmouse groups,butcellnumberswerehigherinCre+mice,reflectingaslightlymorepronounced splenomegalyinHIF-1αconditionalknockouts.Similarresultswereobtainedwhenweexam- inedF4/80expressioninmyeloidcells(Fig2EandS3BFig). Next,wefurthercharacterizedsplenicmonocytesbymonitoringtheexpressionofCCR2 andF4/80,andMHCIIonLy6Clo/intandLy6Chicells.85%ofLy6Chimonocytesco-expressed CCR2andF4/80atd14and21p.i.;thefrequencythendecreasedto50%atlatertimepointsof infection(Fig2FandS4AFig).NodifferenceswereobservedbetweenHIF-1α-sufficientand deficientmonocytes.ThefrequencyofCCR2+F4/80+Ly6Clo/intmonocytessteadilyincreased overthecourseofinfectiontoreachaplateauofabout70%atd21p.i.(Fig2FandS4BFig)in bothgroupsofmice.Thesemonocytespossiblyrepresentanintermediatestageinthedifferen- tiationprocesstowardsmacrophages. Surprisingly,50%ofCre-Ly6ChimonocyteswerepositiveforMHCII;byd21p.i.,thefre- quencyofMHCII+inflammatorymonocytesincreasedto80–90%andwasmaintainedatthis levelduringchronicinfection(Fig2GandS4CFig).ThepercentageofMHCII+Ly6Chimono- cyteswasslightlyhigherinHIF-1α-deficientmiceatd14,d28,andd35p.i.Recently,Ly6Chi monocyteswitharegulatoryphenotypehavebeendescribed[23].Thesemonocytesare inducedbyIFNγinthebonemarrowandexpressMHCIIandSca-1.Hence,weassessedSca-1 expressiononmonocytes.Fromd21p.i.on,themajorityoftheLy6Chimonocytesexpressed Sca-1,suggestingthatinflammatorymonocytesmayalsodisplayaregulatoryphenotypedur- ingchronicVL(Fig2H). Basedonoursurfacemarkeranalysis,splenicmonocytesresembledmonocyticmyeloid- derivedsuppressorcells(M-MDSC)[24]and/ormonocytewitharegulatoryphenotype[23]. TheotherknownsubsetofMDSCoriginatesfrompolymorphonucleatedcells(PMN-MDSC) andischaracterizedbytheco-expressionofLy6GandLy6C(Ly6G+Ly6Clo)[24].Todetermine whetherPMN-MDSCwerealsopresentinthespleenofL.donovaniinfectedmice,wemoni- toredthesurfaceexpressionofLy6ConCD11bhiLy6Ghineutrophils.100%oftheneutrophils wereLy6C+alreadyatd14p.i.(Fig2IandS4DFig);Ly6Cexpressionwasmaintainedduring thechronicphase.ThissuggeststhatneutrophilsexpresssimilarmarkerstoPMN-MDSCand couldpotentiallyexhibitimmunesuppressiveproperties. HIF-1αinducesanM2-likephenotypeandlimitsleishmanicidalcapacity inmyeloidcells Inthefollowing,wesoughttocharacterizemyeloidcellfunction.Tothisend,CD11b+cells werepurifiedfromthespleenofinfectedCre-andCre+miceatvarioustimepointsofinfec- tion;theexpressionofseveralgeneswasassessedbyqPCR.Interestingly,CD11b+cellsfrom Hifflox/flox–Cd11c-Cre+miceshowedalowerexpressionofTNF(Fig3A),arginase(Fig3B), Fizz1(Fig3C),Mgl1,andMgl2(Fig3Dand3E);incontrast,theyexpressedhigheriNOS mRNAlevels(Fig3F).Hence,HIF-1αseemstosustainthedifferentiationtowardstheM2-like macrophagesubtype. Betweend14and21p.i.,splenicstromalcellsarekilledbyexcessiveTNFproduction[25]; consequently,thesplenicmicroarchitectureisaltered[26].Disruptionofthemicroarchitec- tureistypicallyaccompaniedbytheprogressivelossofBcellGerminalCenters[27].Interest- ingly,thesplenicmicroarchitectureininfectedCre+miceappearedtobemoreintactthanin PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 5/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 6/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection Fig2.HIF-1α-deficientmiceinCD11c+cellsshowincreasedfrequencyandnumbersofinflammatorymonocytesin thespleen.Hifflox/flox-Cd11c-Cre+andCre-micewereinfectedwithL.donovaniandsacrificedatvarioustimepointofinfection. Neutrophilswereexcludedfromallanalysisinvolvingmonocytes.(A)RepresentativeFACSplotsdepictingL6C+monocytesin Cre-(uppergraphs)andCre+(lowergraphs)miceoverthecourseofinfection.(B-F)Percentage(uppergraph)andabsolute numbers(lowergraph)ofsplenicLy6Chimonocytes(B),Ly6Clo/intmonocytes(C),Ly6G+neutrophils(D),F4/80+cells(E), CCR2+F4/80+Ly6Chimonocytes(F).(G)PercentageofsplenicMHCII+Ly6Chimonocytes.(H)PercentageofsplenicSca-1+ Ly6Chimonocytes.(I)PercentageofsplenicLy6C+Ly6G+neutrophils.Alldatarepresentmean±SEMofoneof4independent experiments,n=4.*denotesp<0.05. https://doi.org/10.1371/journal.ppat.1006616.g002 theCre-controlsatd28p.i.(Fig3G).ThismaybeaconsequenceofthelowerTNFproduction bymyeloidcells(Fig3A).Notably,myeloidcells(Fig3G,blue)wereincreasinglypresentin thesplenicredpulpofinfectedmiceafterd14p.i. HIF-1αhasbeenreportedtopromoteiNOSexpression[28–30]. Hence,weweresur- prisedtoobserveanincreaseiniNOSmRNAlevelsinmyeloidcellsfrominfectedCre+ mice(Fig3F).Toverifyourinvivoobservation,weinfectedHIF-1α-sufficientanddefi- cientbonemarrow-derivedmacrophages(BMM)withL.donovaniamastigotesandana- lyzediNOSproductionbyflowcytometry.CD38wasusedasanM1marker.Asexpected, stimulationofBMMwithIFNγincreasedthepercentageofCD38+cells(Fig4Aand4B) andtheproductionofiNOS(Fig4Aand4C),whichwasslightlyhigherinHIF-1α-defi- cientcells.Incontrast,treatmentwithIL-4failedtopromoteiNOS(Fig4C)andreduced thefrequencyofCD38+cells(Fig4B),independentlyfromthepresenceorabsenceof HIF-1α.However,whenweinfectedBMMwithL.donovaniamastigotes,adramatic increaseiniNOSproductionwasobservedinHIF-1αdeficientBMMbutnotinHIF-1α sufficientcells(Fig4Aand4C),confirmingourinvivoobservation(Fig3F).Wealsoana- lyzedtheexpressionofM2markersArg-1(Fig4D),Fizz-1(Fig4E),andIL-10(Fig4F).A slightdecreaseinthelevelsofArg-1andFizz-1mRNAwasdetectedinCre+comparedto Cre-cells;moreover,IL-10mRNAwasnotupregulatedinHIF-1αdeficientBMMfollow- inginfectionwithL.donovani. TobesurethatHIF-1αwasindeeddeletedinBMMfrom conditionalknockouts,weassessedtheexpressionofHIF-1αandtwoHIF-1αdownstream targets,Pgk-1andGlut-1incytokine-treatedandinfectedBMM.HIF-1α(Fig4G),Pgk-1 (Fig4H)andGlut-1(Fig4I)werenotinducedinHIF-1αdeficientBMMfollowingL. donovaniinfectionorcytokinetreatment,suggestingthatrecombinationoccurredin BMMfromCre+mice. BecauseHIF-1αisknowntoregulatecellmetabolism,wenextmeasuredintracellularlac- tate(Fig5A)andglucoselevels(Fig5B).Interestingly,HIF-1α-sufficientmyeloidcellshada higherintracellularlactateconcentrationcomparedtoHIF-1α-deficientcells(Fig5A),reflect- ingthemetabolicswitchtowardsanaerobicglycolysis[31,32].Cre-cellsalsodisplayeda slightlyhigherintracellularglucoseconcentration(Fig5B).Wealsoassessedtheproductionof reactiveoxygenspecies(ROS),whicharetypicallynotgeneratedbyM2macrophages[32]. HIF-1α–deficientsplenocytesexpressedhigherlevelsofROS(Fig5CandS5AFig).Neutro- phils(Fig5DandS5BFig)andinflammatorymonocytes(Fig5EandS5CFig)lackingHIF-1α contributedtothisdifference. ToruleoutthepossibilitythatmyeloidcellsacquiredanM2-likephenotypebecauseof higherlevelsofIFNγpresentintheenvironment,weassessedtheexpressionoftheINFγ receptorbyFACS.AsshowninFig5F,thefrequencyofCD11bhiLy6C+cellsexpressingIFNγR wassimilarinbothgroupsofmice,withexceptionofd21p.i.,whentheexpressionwaslower inCre+mice. Takentogether,theseresultssuggestthatHIF-1αmaybeinvolvedinthedifferentiation towardsmacrophageswithanM2-likephenotype,whichisunabletokillLeishmania[31,33]. PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 7/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 8/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection Fig3.HIF-1αinducesanM2-likephenotypeandlimitsleishmanicidalcapacityinmyeloidcells.Hifflox/flox-Cd11c-Cre+andCre-mice wereinfectedwithL.donovaniandsacrificedatvarioustimepointofinfection.(A—F)Real-timePCRanalysisofmRNAexpressionlevelsin splenicCD11b+cellspurifiedfrominfectedmiceatvarioustimepointsafterinfectionfor(A)Tnf,(B)Arg1,(C)Fizz1,(D)Mgl1,(E)Mgl2,and (F)iNOS.(G)Immonohistochemicalanalysisofsplenicsectionsfromnaïveandinfectedmiceatd14and28p.i.;CD169(red),B220(green), CD11b(blue);magnification:10x. https://doi.org/10.1371/journal.ppat.1006616.g003 HIF-1αenhancestheinhibitoryfunctionsofmyeloidcellsduringchronic VL Wenextinvestigatedtheinhibitorypotentialofsplenicmyeloidcells.CD11b+cellswerepuri- fiedfromthespleenofL.donovaniinfectedmiceatd14and28p.i.andco-culturedata1:1 ratiowithnaïveCD4Tcellsstimulatedwithplate-boundanti-CD3andwithanti-CD28and rIL-12.MyeloidcellspurifiedfrominfectedCre-miceatd14p.i.onlyslightlyinhibitedthedif- ferentiationtowardsIFNγ-producingCD4Tcells(Fig6Aand6B);asimilarresultwas obtainedwithHIF-1α-deficientmyeloidcellspurifiedatthesametime.Remarkably,d28p.i. CD11b+cellsfrominfectedHIF-1αsufficientmicestronglyinhibitedTh1differentiation(Fig 6Aand6B);asignificantlylowerdegreeofinhibitionwasobservedinsamplescontainingd28 p.i.HIF-1α-deficientmyeloidcells. Takentogether,ourresultssuggestthatmyeloidcellspurifiedduringchronicinfection inhibitTcellresponses,implyingthatthesecellsarephenotypicallyandfunctionallysimilarto MDSC.ThisinhibitoryfunctionrequiresHIF-1α.Thus,thistranscriptionfactorisnotonly involvedinattenuatingtheleishmanicidalcapacityofmyeloidcells,butalsoinenhancing theirinhibitoryfunction. HIF-1αdeficientintermediatestagemonocytesaremoreresistanttoL. donovaniinfectionunderhypoxicconditions TodeterminewhetherHIF-1α-deficientmonocytesweremoreresistanttoinfectionbyL. donovani,weinfectedbonemarrow-derivedmonocytesinvitrowithfluorescentlylabelled amastigotesandmonitoredtheinfectionfor24hbyFACSandImageStream.Monocyteswere eitheractivatedornotwithIFNγ2hpriortoinfection;cellswerekeptunderhypoxiccondi- tionsatalltimetomimicthebonemarrow[34]andthesplenicenvironment(S6AFig).We firstconfirmedthatCre+cellshadareducedHIF-1αexpression(S6BFig).About25–30%of HIF-1α–sufficientLy6Chi/intmonocytescontainedparasitesafter12hofinfection;at24h,40– 45%ofthecellsharboredparasites(Fig7A).Interestingly,whenmonocyteswereexposedto IFNγpriortoinfection,thepercentageofparasitizedcellsdramaticallyincreasedto60%at 12hand80%at24hofinfection(Fig7AandS6CFig).Thisisprobablyduetothefactthat IFNγinducesregulatoryLy6Chi/intmonocytes[23]andthatthesemaybemorepermissivefor L.donovaniamastigotes.Incontrast,HIF-1α-deficientinflammatorymonocytesweresignifi- cantlylessparasitizedat12and24hintheabsenceofIFNγ(Fig7AandS6CFig);asfortheir HIF-1α-sufficientcounterparts,theadditionofIFNγdramaticallyincreasedtherateofinfec- tion(Fig6A),suggestingthatHIF-1αisnotinvolvedininducingregulatorymonocytes.Nev- ertheless,IFNγ-pulsedHIF-1α-deficientLy6Chi/intmonocyteswereslightlymoreresistantto infectionthanwildtypeinflammatorymonocytes.Similarresultswereobtainedwhenweana- lyzedthedegreeofinfectionofLy6Clomonocytes(Fig7BandS6DFig).Wenextdetermined whetherthenumberofparasitespercellwasequalinbothgroupsofmiceusingtheImage- Streamtechnology(examplesofanalysisaredepictedonFig7C).AsshowninFig7Dand7E, nomajordifferenceswereobservedinthepercentageofcellsharboringvariousnumbersof parasitesbetweenHIF-1α-deficientandHIF-1α-sufficientmonocytes. PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 9/27 HIF-1αexacerbatesMDSC-likeinhibitoryfunctionsduringinfection PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006616 September11,2017 10/27