HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of

27 Pages·2017·8.25 MB·English

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