Infection-adapted emergency hematopoiesis promotes visceral leishmaniasis

26 Pages·2017·26.11 MB·English

RESEARCHARTICLE
Infection-adapted emergency hematopoiesis
promotes visceral leishmaniasis
BelmaMeldaAbidin1,AkilHammami1,SimonaSta¨ger1,2,KristaM.Heinonen1,2*
1 INRS-InstitutArmand-Frappier,Universite´duQue´bec,Laval,Que´bec,Canada,2 CentreforHost-Parasite
interactions,Laval,Que´bec,Canada
*[email protected]
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Abstract
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a1111111111 Cellsoftheimmunesystemarederivedfromhematopoieticstemcells(HSCs)residingin
a1111111111 thebonemarrow.HSCsbecomeactivatedinresponsetostress,suchasacuteinfections,
whichadaptthebonemarrowoutputtotheneedsoftheimmuneresponse.However,the
impactofinfection-adaptedHSCactivationanddifferentiationonthepersistenceofchronic
infectionsispoorlyunderstood.Wehaveexaminedherethebonemarrowoutcomeof
chronicvisceralleishmaniasisandshowthattheparasiteLeishmaniadonovaniinduces
OPENACCESS
HSCexpansionandskewstheirdifferentiationtowardsnon-classicalmyeloidprogenitors
Citation:AbidinBM,HammamiA,Sta¨gerS,
HeinonenKM(2017)Infection-adaptedemergency witharegulatoryphenotype.Ourresultsfurthersuggestthatemergencyhematopoiesis
hematopoiesispromotesvisceralleishmaniasis. contributestothepathogenesisofvisceralleishmaniasis,asdecreasedHSCexpansion
PLoSPathog13(8):e1006422.https://doi.org/
resultsinalowerparasiteburden.Conversely,monocytesderivedinthepresenceofsolu-
10.1371/journal.ppat.1006422
blefactorsfromtheinfectedbonemarrowenvironmentaremorepermissivetoinfectionby
Editor:ChristianR.Engwerda,QueenslandInstitute
Leishmania.OurresultsdemonstratethatL.donovaniisabletosubverthostbonemarrow
ofMedicalResearch,AUSTRALIA
emergencyresponsestofacilitateparasitepersistence,andputforwardhematopoiesisasa
Received:January17,2017
noveltherapeutictargetinchronicinfections.
Accepted:May22,2017
Published:August7,2017
Authorsummary
Copyright:©2017Abidinetal.Thisisanopen
accessarticledistributedunderthetermsofthe
Hematopoieticstemcells(HSCs)areresponsibleforthegenerationofallbloodcellsand
CreativeCommonsAttributionLicense,which
thusplayanimportantbutoftenunderappreciatedroleinthehostresponsetoinfections.
permitsunrestricteduse,distribution,and
HSCsarenormallydormant,buttheycanbecomeactivatedinresponsetostress,suchas
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. infections.Thisstressresponseismeanttogeneratemorebloodcellsandhelpthebodyto
eliminatetheinvadingpathogen.WehavestudiedheretheactivationofHSCsinamouse
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation modelofchronicinfectionwiththeparasiteLeishmaniadonovani.Wefoundthatthepar-
files. asiteefficientlyactivatesHSCsandsteersthemtoproducelargenumbersofspecificblood
cellsthatareamongthepreferredtargetsoftheparasiteandbecomeevenmoresuscepti-
Funding:ThisworkwassupportedbytheNatural
bletoinfectionwhenproducedwithinthediseasedenvironment.Usingamousestrainin
SciencesandEngineeringResearchCouncilof
Canada(grant#419226-2012toKMH)http://www. whichHSCactivationcannotbesustained,wefoundthatdiminishedHSCactivitycorre-
nserc-crsng.gc.ca,theFondsderecherchedu latedwithdecreasedparasitenumbers.WethereforeproposethatHSCactivationbythe
Que´bec–Sante´(grant#32598toKMH)http:// parasitepromotestheinfectionandcouldbeusedasanewtargetfortreatment.
www.frqs.gouv.qc.ca,CanadianInstitutesofHealth
Research(grant#PJT-148614toKMHand#MOP-
123293toSS)http://www.cihr-irsc.gc.ca,andthe
CanadaFoundationforInnovation(CFILeaders
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006422 August7,2017 1/26
Emergencyhematopoiesisinvisceralleishmaniasis
Fund#31377toKMH&SS)https://www. Introduction
innovation.ca.Thefundershadnoroleinstudy
Emergencyhematopoiesisinresponsetoseverebacterialinfectionisassociatedwithanexpan-
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. sionofhematopoieticstem/progenitorcells(HSPCs)andtheirprogeny,mediatedbyacombi-
nationofenvironmentalcues,includinginflammatorycytokinesandmicrobialproducts[1–
Competinginterests:Theauthorshavedeclared
4].Activated,proliferatingHSPCsandmyeloidprogenitorcells(MPCs)leavethebonemar-
thatnocompetinginterestsexist.
rowandmigratetothespleen,liver,andotherinflamedtargetorganswheretheycandirectly
contributetohematopoiesis[1,5,6].Inthecaseofacutebacterialinfection,theresulting
increaseinmyeloiddifferentiationpromotestheimmuneresponsethatisrequiredforclear-
anceofthepathogen[3,4].Similarly,increasedmyelopoiesisinresponsetoanacuteviral
infectioncanbebeneficialforhelpingtomounttheappropriateTcellresponsestoeliminate
infectedcells[7].Bonemarrowhomeostasisisusuallyrestoredaftertheinfectioniscleared.
However,thepotentialimpactofinfection-adaptedhematopoiesisontheabilityofpathogens
topersistandestablishchronicinfectionshasnotbeenwidelystudied.
Persistentinflammatoryconditions,includingchronicparasiticinfectionssuchasvisceral
leishmaniasismayleadtoanacquiredbonemarrowfailure,wheretheHSPCsandbonemar-
rowstromaarenolongerabletosupportbloodhomeostasis[8–10].Theunderlyingmecha-
nismsarepoorlyunderstood,butthepathologyclearlysuggeststhattheparasiteinfection
profoundlychangesnormalhematopoiesis:visceralleishmaniasisisgenerallyassociatedwith
hepatosplenomegaly,extramedullaryhematopoiesis,pancytopenia,andimmunosuppression.
PreviousstudiesusingtheBalb/cmousemodelofinfectionwithLeishmaniadonovanisuggest
thattheparasitedoesnotdirectlyinfectHSPCsorMPCs.Instead,L.donovaniestablishesaper-
sistentinfectioninbonemarrowmacrophages,whichcorrelateswithanenhancedMPCoutput
bybonemarrowandspleen,andtheproductionofmyeloidgrowthfactors[10–12].However,
thecontributionofenhancedmyelopoiesistothecourseofinfectionisnotwellunderstood.
HSPCactivationcorrespondstochangesinWntsignalingactivity,withthevariousintra-
cellularsignalingpathwayspromotingeitheractivationorquiescence[13–15].Wehaveprevi-
ouslyshownthattheabsenceoftheWntsignalingreceptorFrizzled6(Fzd6)resultsin
defectivestemcellself-renewal,completelyabrogatingtheirabilitytoreconstituteanirradiated
hostaftertransplant,anddampensHSPCexpansionduringLPS-inducedemergencyhemato-
poiesis[15].WntsignalinggenerallycontributestotheestablishmentofTcellmemoryand
regulateseffectorTcellresponses[16,17]andleukocytetrafficking[18],butitsroleininflam-
mation-inducedmyelopoiesisandtheregulationofchronicinfectionsisnotknown.
WeshowherethatexperimentalL.donovaniinfectioninducestheexpansionofhematopoi-
eticstemcell(HSC)-likecellsandSca1+emergencyMPCsinthebonemarrow.Themyeloid
progenyoftheseemergencyMPCsconsistspredominantlyofLy6Chimonocyteswitharegula-
tory,suppressorcell-likephenotype.Wefurtherdemonstratethattheexpansionisfunctionally
important,asmonocytesgeneratedinthepresenceofsolublefactorsextractedfromthe
infectedbonemarrowaremorepermissivetoinfection,andastuntedemergencyresponse
suchasseeninFzd6-/-miceresultsindecreasedparasiteburden.Collectivelyourresultssup-
portthehypothesisthatLeishmaniasubvertsthehostbonemarrowemergencyresponseto
promoteitsownproliferationandtoallowforcontinuedpersistenceoftheinfection.
Results
L.donovaniinducestheexpansionofHSC-likecellsinthebonemarrow
andspleen
L.donovaniinfectionresultsinenhancedmyelopoiesisinthebonemarrowandspleenof
Balb/cmice[11,12];however,itisunclearwhichHSPCstheparasitetargetsandwhat
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Emergencyhematopoiesisinvisceralleishmaniasis
functionalconsequencesstemfromtheincreasedmyeloidoutput.Weinitiallyfollowedthe
progressionofL.donovaniinfectioninthebonemarrowofmiceonC57Bl/6backgroundand
observedasharpincreaseinparasiteburdenbeginninginthethirdweekafterinfection(Fig
1A).Inparallelwithparasiteburden,theproportionandnumberofbonemarrowLin-Sca-
1+c-Kit+(LSK)cellsandCD150+CD16/CD32-LSKs,whichcorrespondtoanHSC-likepheno-
typeinuninfectedmice,alsoincreased,reachingaplateaubetweenday21andday28,depend-
ingonthestrengthoftheinfection(Fig1Band1C,S1Fig).Asimilarexpansionwasalso
observedinthespleen:bothLSKsandHSC-likecellswerevirtuallyundetectableinthenaïve
spleen,buttheirnumberscontinuedtoaugmentininfectedmicethroughday35(Fig1D,S1
Fig).TheseresultsindicatethatthemostimmatureHSPCsareindeedaffectedbyinfection
withL.donovani,andthatthiseffectpersistsintime.
AdultHSCsareusuallydormantinthebonemarrow,withmorethantwothirdsofthecells
residinginaquiescentstateintheG0phaseofthecellcycle.However,theybecomereadily
activatedunderstressorinresponsetoinflammatorycytokines[4,19,20].Chronicinfection
withL.donovaniinducedHSC-likecellstoentercellcycle,resultinginagraduallossofquies-
centcells(Fig1E).Thiswasaccompaniedbydifferentiation,astheproportionofCD150+
HSC-likecellsthathadacquiredCD48andthusrepresentedmultipotentprogenitorswith
myeloidbias[19,21]alsoincreasedfrom25%to75%(Fig1Fand1G);however,therewasa
significantexpansionoftheCD48-populationaswell.HSCactivationandcellcycleentryhave
beenshowntocorrelatewiththeinductionofβ-catenin-dependentWntsignalinginnon-
infectioussettings[13].Weobservedasignificantincreaseinintracellularlevelsofactiveβ-
cateninthatwasspecificforCD150+HSC-likecells(Fig1H),suggestingthatWntsignaling
couldcontributetoregulatingL.donovani-inducedHSCexpansion.
InductionofmyelopoiesisduringL.donovaniinfectionresultsinthe
generationofalteredprogenywitharegulatoryphenotype
Leishmaniasisisaccompaniedbyanincreaseincirculatingmonocytes,andL.donovani
inducestheexpansionandexporttospleenofMPCsinBalb/cmice[11,22,23].Tobetter
definethekineticsandthetypesofprogenitorsthatwererespondingtotheinfection,weana-
lyzedthebonemarrowMPCcompartmentandobservedthatthenumberandproportionof
granulocyte-monocyteprogenitors(GMPs;CD16/CD32+cKit+CD41-CD150-Lin-)remained
stableovertime(Fig2Aand2B,S2Fig).Therewasnospecificchangeintheproportionof
activelycyclingGMPs(Fig2C,S2Fig),andincontrasttoHSC-likecells,theincreaseinactive
β-cateninlevelswasverymodest(Fig2F).However,therewasastrikingupregulationofSca1
expressiononGMPs(Fig2DandS2Fig),whichtranslatedintoadeclineinthenumbersof
Sca1-GMPsthatarenormallyassociatedwithsteady-statehematopoiesis.Thesteadyincrease
inthenumbersofSca1+“emergency”GMPs(Fig2E;[24])togetherwiththestablelevelsof
totalGMPs(Fig2Aand2B)suggestedthatthemajorchangebroughtaboutbyL.donovanidid
notimpactasmuchthenumbersofMPCsasthetypeofprogenytheygenerated.Blympho-
poiesiswasnearlycompletelyabrogatedatlaterstages(S3Fig),indicativeofprofoundalter-
ationstothebonemarrowenvironment.
GMPsasapopulationgiverisetobothgranulocytes(mostlyneutrophils)andmonocytes.
Duringrestinghematopoiesis,approximatelyhalfofthebonemarrowmyeloidcellsaregranu-
locytes(SSChiGR1hiLy6G+),withtheremaininghalfdividedintoLy6Chimonocytesreadyto
enterthecirculationandaLy6Clo/-populationthatcomprisesalternativemonocytesandmac-
rophagesinadditiontoimmaturestagesofbothmonocytesandgranulocytes.Ly6Chimono-
cytesrepresentedtheonlymyeloidcelltypewhoseproportionsteadilyincreasedovertimein
theinfectedmarrow(Fig3A).Onday28,weobservedinfectedcellswithmonocyteaswellas
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Emergencyhematopoiesisinvisceralleishmaniasis
Fig1.Parasiteexpansioncoincideswithproliferationandaccumulationofbonemarrowhematopoieticstem/progenitor
cells(HSPC).(A)Bonemarrowparasiteburdenwasassessedusingtheseriallimitingdilutiontechnique.Graphshowsparasite
burdenperonefemurandonetibia.(B)RepresentativeflowcytometrydataandgatingstrategyofBMHSPCs.BMcellswerefirst
gatedonLin-(B220-CD3ε-CD11b-GR1-Ter119-)andidentifiedaccordingtoSca1andcKit(CD117)expression.HSCsweredefined
asCD150+CD16/CD32-withintheLin-cKithiSca1+(LSK)population.NumberswithinflowcytometryplotsrepresentmeanLSK
percentagewithintotalbonemarrowandmeanHSCpercentagewithinLSKs.SeealsoS1Fig.(C)Histogramsshowpercentageand
absolutenumbersofLSKsandHSCspertwofemoraandtwotibiae.Dataarepooledfromthreeindependentexperiments,witheach
individualdotrepresentingonemouse.Horizontallinesrepresentthesamplemean.(D)AbsolutenumbersofLSKsandCD150+
HSCsinspleensofinfectedmice.SeealsoS1Fig.(E)Ki-67/Hoechstco-stainingwasusedtodistinguishtheG0,G1,andS/G2/M
cellcyclephasesofCD150+HSC-likecellsduringinfection.(F)AnalysisofCD48expressionwithinBMCD150+HSC-likecells.
NumberswithinflowcytometryplotsrepresentCD48+andCD48-HSCsubsetswithinCD150+HSClikecells.(G)Graphsdepictthe
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Emergencyhematopoiesisinvisceralleishmaniasis
numbersofcellsforthetwosubsetsatvarioustimepointsafterinfection.(H)Intracellularactiveβ-cateninlevels(MFI)withinCD150+
HSC-likecells.SeealsoS1Fig.Allbargraphsrepresentmean+SEMwith4micepergroupcomingfromonesingleinfection.Similar
resultswereobtainedintwoadditionalindependentexperiments.*P<0.05;**P<0.01;***P<0.001.
https://doi.org/10.1371/journal.ppat.1006422.g001
myeloblastmorphologiesinbonemarrowsmears(Fig3B).Allmonocytescontainedasingle
parasite,whilethemyeloblast-likecells,similartomaturemacrophages,couldbefoundto
houseseveral.Theselatter,productivelyinfectedcellscouldrepresenteitherimmaturemye-
loidcellsorpotentiallyanintermediatestageinmonocytematurationtowardsamoremacro-
phage-likemorphology.
Fig2.BonemarrowHSCsswitchtheirdifferentiationtowardsnon-classicalmyeloidprogenitors.(A)Representative
flowcytometrydataandgatingstrategyofgranulocyte-monocyteprogenitors(GMPs)inthebonemarrow.Steadystate
myeloidprogenitor(MP)cellswerefirstgatedonLin-Sca1-c-kithiandthensubdividedaccordingtotheexpressionofCD41,
CD150andCD16/CD32.GMPswereidentifiedasCD16/CD32+CD41-CD150-.Duetotheinflammation-inducedshiftinSca1
expression,thetotalLin-c-KithiHSPCpopulationwasincludedforanalysisduringinfection.SeealsoS2Fig.(B)Graphsshow
percentageandabsolutenumbersofGMPsatvarioustimepoints.Dataarepooledfromthreeindependentexperiments,with
eachindividualdotrepresentingonemouse.Horizontallinesrepresentthesamplemean.(C)PercentageofGMPsinS/G2/M
phasesofcellcycle.(D)PercentageofSca-1+emergencyGMPswithinallGMPs.(E)NumbersofcellswithinSca-1+andSca-
1-GMPsubsets.(F)Intracellularactiveβ-cateninlevels(MFI)inGMPs.
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Emergencyhematopoiesisinvisceralleishmaniasis
Fig3.Leishmaniaparasiteexpansionpromotesmyeloidoutputinthebonemarrow.(A)Representativeflowcytometrydatato
demonstrategatingstrategyformyeloidcellsubsetsinthebonemarrow.Graphsshowpercentageandnumbersofgranulocytes
(GR1hiSSChi),maturemonocytes(Ly6ChiCD11b+)andremainingimmature/residentmyelo-monocytes(Ly6Clo/-GR1lo/-CD11b+).
Dataarepooledfromthreeindependentexperiments,witheachindividualdotrepresentingonemouse.Horizontallinesrepresentthe
samplemean.SeealsoS3Fig.(B)Giemsa-stainedinfectedbonemarrowmonocytes,myeloblast-likecellsandmacrophages
bearingintracellularLeishmaniaamastigotes(100Xunderoilimmersionlens).Scalebar=5μm.(C)Sca-1andMHC-IIexpression
onLy6ChiandLy6Clo/-monocytesubsets.(D)GalectinandLy6Cexpression(MFI)onLy6Chimonocytes.Allbargraphsrepresent
mean+SEMwith4micepergroupcomingfromonesingleinfection.Similarresultswereobtainedinasecond,independent
experiment.*P<0.05;**P<0.01;***P<0.001.
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Tobetterevaluatemonocytedifferentiationandmaturation,wesoughttodetermineifthe
newlygeneratedmonocyteshadacquiredanaltered,regulatoryphenotype,similartowhathas
beenreportedinresponsetotrypanosomalinfection[25].Ly6Chimonocytesrapidlyacquired
Sca1expression,similartotheemergencyGMPs(Fig3CandS3Fig).Incomparison,onlya
smallpercentageofLy6Clo/-cellsupregulatedSca1.Ly6Chimonocytesalsoupregulated
MHCII(Fig3CandS3Fig)anddown-regulatedLy6Cexpression(Fig3D),whichsuggested
thattheyhadbeenexposedtoIFN-γ[25].Tofurthersupportthehypothesisofmonocyte
developmentalskewing,theLy6ChimonocytesgeneratedinL.donovani–infectedmicealso
upregulatedGalectin-3(Fig3DandS3Fig),whichisassociatedwithalternativemacrophage
activation[26],IL-10production[27],andpro-fibroticresponses.Thesedatacollectivelyshow
thatL.donovanisubvertsbonemarrowhematopoiesis,enhancingthegenerationofemergency
GMPsandthedifferentiationofmonocytesthat,duetotheirregulatoryorimmaturepheno-
type,couldrepresentsafetargetsandpromoteparasiteexpansion[22].
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Emergencyhematopoiesisinvisceralleishmaniasis
Fzd6promotesbonemarrowresponsetoL.donovani
WehavepreviouslyshownthattheabsenceoftheWntsignalingreceptorFrizzled6(Fzd6)
dampensHSCexpansionduringLPS-inducedemergencyhematopoiesis[15].GiventhatWnt
signalingwasinducedinL.donovani–activatedHSCs(Fig1H),wesoughttoevaluateifFzd6
wasalsoimportantforthebonemarrowresponsetotheparasite,andifwecoulduseFzd6-de-
ficientmiceasatooltoinvestigatetheimportanceofthebonemarrowresponseinpromoting
parasiteexpansion.TherewasnosignificantdifferenceinbonemarrowLSKorHSCnumbers
betweenFzd6-/-andFzd6+/+miceonday14,beforetheexpansion(Fig4A)oronday21(S4
Fig).However,expansionwasbluntedinFzd6-/-miceonday28post-infection(Fig4A),which
correspondstothepeakofexpansion(Fig1G).ThedifferenceinHSPCaccumulationwasnot
duetodecreasedproliferation,asFzd6-/-HSCsenteredthecellcycleatleastasefficientlyas
theirFzd6+/+counterparts(Fig4B).Ifanything,therewasadecreaseintheproportionofqui-
escentHSCsinFzd6-/-bonemarrowonday28(Fig4B),correlatingwiththeirpreviously
reportedself-renewaldefect[15],andindicatingthattheFzd6-/-bonemarrowrespondedto
theinfection.However,theresponsedidnotresultinasgreatanaccumulationofHSPCs.
Therewasalsonosignificantdifferenceinβ-cateninactivation(Fig4C),similartowhatwe
hadpreviouslyreportedatsteadystate[15].TheseresultssuggestedthatFzd6-/-micecould
provideamodeltoinvestigatetheimpactofL.donovani–adaptedhematopoiesisonparasite
expansion.
Fzd6-/-micedisplaynospecificdefectsinthebonemarrowMPCcompartmentatsteady
state(S5Fig)oronday14post-infection,priortotheparasite-inducedchangesinhematopoi-
esis(Fig4Dand4E).However,onday28weobservedatwo-folddecreaseinGMPs(Fig4D)
andmorespecificallyinSca1+emergencyGMPs(Fig4E).SimilartoHSCs,therewasnodiffer-
enceincellcycle(Fig4F)orβ-cateninactivity(Fig4G)betweenFzd6-/-andFzd6+/+GMPs;
norwasGM-CSFreceptorexpressionaltered(S6Fig).Wealsoevaluatedthepresenceofcom-
monmonocyteprecursors(cMoPs;Lin-CD115+cKithiFlt3-Ly6C+CD11b-,S5Fig),andfound
thatthenumberofcMoPswasslightlyhigherinL.donovani–infectedbonemarrowwhen
comparedtouninfectedcontrols(Fig4H).cMoPsacquiredSca1expressionintheinfected
marrow,similartoGMPsandLy6Chimonocytes(Figs2D,2Eand3C).However,Fzd6-/-
cMoPswerenotexpandedinresponsetoL.donovaniandalowerproportionhadacquired
Sca1(Fig4H).Myeloidcolonyformationinculturewasalsodecreased,withHSPCsisolated
frominfectedFzd6-/-bonemarrowgenerating3-timesfewerCD11b+myeloidcellsthan
Fzd6+/+controls(Fig4I).TogetherthesedatashowthatthepresenceofFzd6promotespara-
site-inducedHSPCexpansionandskewingtowardsanemergencyphenotype.
Enhancedmyelopoiesiscorrelateswithincreasedparasiteburden
TofurtherinvestigatetheimpactofHSPCexpansionondownstreammyeloiddifferentiation
duringinfection,weevaluatedtheacquisitionofregulatorymarkersbyFzd6-/-Ly6Chimono-
cytes.Therewasnodifferenceintheproportionofgranulocytes(SSChiGR1hi/Ly6G+),mature
Ly6Chimonocytes,orLy6Clomonocytes/macrophagesatsteadystate(S7Fig)oronday14
(Fig5A)orday21(S4Fig).However,therewasaspecificdecreaseintheoutputofbothGR1hi
andLy6Chimyeloidcells(Fig5A)andF4-80+CD11bintmacrophages(Fig5B)intheFzd6-/-
bonemarrowonday28.IncontrasttoFzd6+/+cells,Fzd6-/-Ly6Chimonocytesdidnotdown-
regulateLy6CorCcr2(Fig5CandS8Fig)andexpressedhigherlevelsofCxcr4(Fig5DandS8
Fig).Ccr2andLy6Cdownregulationaregenerallyassociatedwithmonocytedifferentiation
intomacrophages[28–30],whileCxcr4expressionhasbeenrecentlyshowntocorrespondtoa
transitionalstagebetweencMoPsandmaturemonocytes[31].Together,thesedatasuggest
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Fig4.Frizzled-6isrequiredforparasite-inducedexpansionandmyeloiddifferentiationofHSPCs.(A)AnalysisofbonemarrowLSK
andHSCcompartmentsininfectedFzd6-/-(KO)andFzd6+/+(WT)mice.MeanpercentageforLSKsandHSC-likecellswithintotalbone
marrowareindicatedinthecornerofeachhistogram.GraphsshowthenumbersofLSKsandHSCsondays14and28.Day28dataare
pooledfromthreeindependentexperiments,witheachindividualdotrepresentingonemouse.Horizontallinesrepresentthesamplemean.
(B)Representativeflow-cytometryplotsforcellcycleanalysisofWTandKOHSCsonday28.GraphshowspercentCD150+HSClikecells
intheG0,G1andS-G2-Mphasesofcellcycle.(C)Intracellularactiveβ-cateninlevels(MFI)inWTandKOCD150+HSCsondays14and
28.(D)FlowcytometryanalysisofBMGMPs.NumbersinthehistogramsrepresentmeanpercentageforWTandKOGMPs.Graphsshow
thenumbersofHSPCsandGMPsatday14and28.Day28dataarepooledfromthreeindependentexperiments,witheachindividualdot
representingonemouse.SeealsoS4,S5andS6Figs.(E)PercentageandnumbersofSca-1-andSca-1+GMPsonday14and28pi.(F)
CellcycleanalysisofWTandKOGMPsonday28.Similarresultswereobtainedfromsixindividualmiceforeachgroup.(G)Intracellular
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activeβ-cateninlevels(MFI)indifferentGMPsubsetsonday28.(H)Gatingstrategyandrepresentativeflowcytometryplotsforcommon
monocyteprogenitors(cMoPs).BMcellswerefirstgatedonLin-(CD3ε−B220−NK1.1−Ly6G−)CD115+andthensubdividedaccordingto
Sca1.cMoPswereidentifiedascKit+CD135-Ly6C+CD11b-withinLin-CD115+cellpopulations.MeanpercentageforcMoPswithintotal
bonemarrowisdepictedinflowcytometryplots,andthegraphshowsabsolutenumbers(mean+SEMfromsixmicepergroup).Tonote,
therewerenoSca1+cMoPsinnaïvemice.(I)Flowcytometryanalysisofcellsrecoveredfrommyeloidcolonyformingassays.Numbers
shownindifferentquadrantsindicatethemeanpercentageinCD11b+cells.Histogramrepresentspooleddatafromonesingleinfectionfor
atotaloffivemice.Similarresultswereobtainedinasecond,independentexperiment.Allbargraphsrepresentmean+SEMwith10mice
pergroupforday28pooledfromtwoindependentexperimentsand3micepergroupforday14unlessotherwisenoted.*P<0.05;
**P<0.01;***P<0.001.
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thatFzd6-/-Ly6Chimonocytesweremoreimmatureandperhapslesslikelytodifferentiate
locallyintomacrophages.
BothFzd6-/-andFzd6+/+Ly6ChimonocytesexpressedelevatedSca1andMHCII(Fig5D
andS8Fig),indicativeofaninflammatorymicroenvironment.Infurthersupportoftheir
potentialregulatoryfunction,Ly6ChimonocytesfrombothgroupsupregulatedArginase1
expression,andexpressedlowlevelsofIL-10(Fig5EandS8Fig).Furthermore,therewasno
upregulationofiNOSbyLy6ChimonocytesorF4-80+bonemarrowmacrophagesfromeither
Fzd6-/-orFzd6+/+mice(Fig5FandS8Fig).Thus,Fzd6-/-Ly6Chimonocytesareproducedin
lowernumbers,presumablyduetodecreasedexpansionofupstreamprogenitors,buttheydo
notsignificantlydifferintheexpressionofactivationmarkers,suchasMHCII,Sca1,oriNOS.
However,Fzd6-/-micepresentwithlowerparasiteburdeninthebonemarrow(Fig5G),sug-
gestingthatthedecreasedproductionof“safetargets”intheformofLy6Chimonocytesmight
besufficienttodampenparasiteexpansion,independentoftheirfunctionalcapacity.Infur-
thersupportofthishypothesis,weestablishedastatisticalcorrelationbetweenbonemarrow
parasiteburdenandthenumberofCD150+HSCs(Fig5H)aswellasbonemarrowLy6Chi
monocytes(Fig5I),independentofmousegenotypeorthestageofinfection(D14-D28).
Todeterminewhetherthedifferencesinbonemarrowwerealsoreflectedinperipheral
organs,weevaluatedtherecruitmentofmyeloidcellsalsotoliverandspleen,twomajortarget
organsinvisceralleishmaniasis.Similartothebonemarrow,spleenmyeloidcompartment
wasstillunchangedonday14,butthenumbersofgranulocytesandmonocyteswereallsub-
stantiallyincreasedbyday28(Fig6Aand6B).Oncemore,therewasnodifferencebetween
Fzd6-/-andFzd6+/+miceatsteadystate(S7Fig),orearlierduringtheinfection,beforethe
onsetofalteredhematopoiesis(Fig6BandS4Fig).WeobservedaspecificdecreaseinFzd6-/-
GR1hiandLy6Chimyeloidcells(Fig6Aand6B)withnodifferenceinLy6Clomonocytesor
F4-80+CD11bintredpulpmacrophages(Fig6A–6C).Therewasalsoacorrespondingdecrease
inLy6Chimonocytesintheliver(S9Fig).Thedecreaseinmyeloidcellscorrelatedwith
decreasedparasiteburdenonday28inFzd6-/-spleen(Fig6D)andliver(S9Fig).Wntsignal-
inghasbeenreportedtoaffectleukocyteinfiltration[18];however,therewasnodifferencein
theproportionofLy6Chimonocytesinperipheralblood(S4Fig)orintherelativeratioof
Ly6Chimonocytespresentinspleenascomparedtobonemarrow(Fig6E),suggestingthatthe
decreaseinFzd6-/-peripheralmonocyteswasmainlyduetodifferencesinhematopoiesis.
TherewasnodifferenceintheexpressionofSca1,MHCII,Ly6C,orCcr2betweenFzd6-/-and
Fzd6+/+Ly6Chimonocytesinspleen(Fig6Fand,6G)orliver(S9Fig).Fzd6+/+Ly6Chimono-
cytesexpressedIL-10(Fig6HandS8Fig)butdidnotupregulateiNOS(Fig6H),thussupport-
ingthetheorythattheymayhaveregulatoryfunctions.AsmallerproportionofFzd6-/-Ly6Chi
monocyteswereIL-10+.Wnt5ahasbeenreportedtodirectlyinduceIL-10productioninden-
driticcellsthroughanon-canonicalpathway[32],whileβ-cateninactivationpromotesTLR-
mediatedIL-10secretionbyhumanmonocytes[33].Overalltheseresultssuggestthatthe
decreasedmonocyteoutputbytheFzd6-/-bonemarrowtranslatesintodecreasedmonocyte
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006422 August7,2017 9/26
Emergencyhematopoiesisinvisceralleishmaniasis
Fig5.DiminishedmyeloidoutputinFzd6-/-micecorrelateswithareducedparasiteburdenduringthe
chronicphaseofinfection.(A)AnalysisofbonemarrowmyeloidsubsetsinfectedFzd6-/-(KO)andFzd6+/+
(WT)mice.Meanpercentageforeachcellsubsetisindicatedwithinflowcytometryplots.Graphsshow
numbersofgranulocytesandmonocytesonday14and28.SeealsoS4,S6andS7Figs.(B)Numberswithin
flowcytometryplotsindicatemeanpercentageofLy6Clo/-F4-80+bonemarrowmacrophages.Histograms
showtotalnumbersofmacrophagesandpercentF4-80+withinLy6Chimonocytes(mean+SEMfromseven
micepergroup).(C)Ly6CandCCRexpression(MFI)onLy6Chimonocytesatday28pi.(D)Percentageof
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006422 August7,2017 10/26