MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis

26 Pages·2016·6.74 MB·English

RESEARCHARTICLE
MIF-MediatedHemodilutionPromotes
Pathogenic Anemia in Experimental African
Trypanosomosis
BenoîtStijlemans1,2*,LeaBrys1,2,HannelieKorf3,PawelBieniasz-Krzywiec4,
AmandaSparkes1,2,LieseVansintjan1,2,LinLeng5,NeleVanbekbergen6,
MassimilianoMazzone4,GuyCaljon7,8,JanVanDenAbbeele8,StevenOdongo1,9,CarlDe
Trez1,9,StefanMagez1,9,10,JoA.VanGinderachter1,2,AlainBeschin1,2‡,RichardBucala5‡,
PatrickDeBaetselier1,2‡
a11111
1 LaboratoryofCellularandMolecularImmunology,VrijeUniversiteitBrussel(VUB),Brussels,Belgium,
2 LabofMyeloidCellImmunology,VIBInflammationResearchCenter,Ghent,Belgium,3 Translational
ResearchCenterforGastrointestinalDisorders(TARGID),DepartmentofClinicalandExperimental
Medicine,KULeuven,Leuven,Belgium,4 VIBVesaliusResearchCenter,UniversityofLeuven(KUL),
Leuven,Belgium,5 DepartmentofInternalMedicine,YaleUniversitySchoolofMedicine,NewHaven,
Connecticut,UnitedStatesofAmerica,6 LaboratoriumCellulaireGenetica(CEGE),VrijeUniversiteit
Brussel(VUB),Brussels,Belgium,7 LaboratoryforMicrobiology,ParasitologyandHygiene(LMPH),
UniversityofAntwerp,Wilrijk,Belgium,8 UnitofVeterinaryProtozoology,DepartmentofBiomedical
OPENACCESS Sciences,InstituteofTropicalMedicineAntwerp(ITM),Antwerp,Belgium,9 DepartmentofStructural
Biology,VrijeUniversiteitBrussel,Brussels,Belgium,10 LaboratoryforBiomedicalResearch,Ghent
Citation:StijlemansB,BrysL,KorfH,Bieniasz-
UniversityGlobalCampus,Yeonsu-Gu,Incheon,SouthKorea
KrzywiecP,SparkesA,VansintjanL,etal.(2016)
MIF-MediatedHemodilutionPromotesPathogenic ‡Theseauthorsarejointlastauthorsonthiswork.
AnemiainExperimentalAfricanTrypanosomosis. *[email protected]
PLoSPathog12(9):e1005862.doi:10.1371/journal.
ppat.1005862
Abstract
Editor:KentL.Hill,UniversityofCalifornia,Los
Angeles,UNITEDSTATES
AnimalAfricantrypanosomosisisamajorthreattotheeconomicdevelopmentandhuman
Received:March11,2016
healthinsub-SaharanAfrica.Trypanosomacongolenseinfectionsrepresentthemajorcon-
Accepted:August11,2016
straintinlivestockproduction,withanemiaasthemajorpathogeniclethalfeature.The
Published:September15,2016 mechanismsunderlyinganemiadevelopmentareilldefined,whichhampersthedevelop-
Copyright:©2016Stijlemansetal.Thisisanopen mentofaneffectivetherapy. Here,thecontributionoftheerythropoieticanderythrophago-
accessarticledistributedunderthetermsofthe cyticpotentialaswellasofhemodilutiontothedevelopmentofT.congolense-induced
CreativeCommonsAttributionLicense,whichpermits
anemiawereaddressedinamousemodeloflowvirulencerelevantforbovinetrypanoso-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare mosis.Weshowthatininfectedmice,splenicextramedullaryerythropoiesiscouldcompen-
credited. sateforthechroniclow-gradetypeIinflammation-inducedphagocytosisofsenescentred
DataAvailabilityStatement:Allrelevantdataare bloodcells(RBCs)inspleenandlivermyeloidcells,aswellasfortheimpairedmaturation
withinthepaperanditsSupportingInformationfiles. ofRBCsoccurringinthebonemarrowandspleen.Rather,anemiaresultedfromhemodilu-
Funding:Thiswork,performedinframeofan tion.Ourdataalsosuggestthatthehemecatabolismsubsequenttosustainederythropha-
InteruniversityAttractionPoleProgram(PAI-IAPN. gocytosisresultedinironaccumulationintissueandhyperbilirubinemia.Moreover,
P7/41,http://www.belspo.be/belspo/iap/index_en.
hypoalbuminemia,potentiallyresultingfromhemodilutionandliverinjuryininfectedmice,
stm),wassupportedbygrantsfromtheFWO(KaN
impairedtheeliminationoftoxiccirculatingmoleculeslikebilirubin.Hemodilutionalthrombo-
1515813N)andNIHAI042310.BSwassupportedby
theStrategicResearchProgram(SRP3,VUB).RB cytopeniaalsocoincidedwithimpairedcoagulation.Combined,theseeffectscouldelicit
andLLweresupportedbyNIHRO1AI42310and multipleorganfailureanduncontrolledbleedingthusreducethesurvivalofinfectedmice.
R01AI110452grants.Thefundershadnorolein
MIF(macrophagemigratinginhibitoryfactor),apotentialpathogenicmoleculeinAfricantry-
studydesign,datacollectionandanalysis,decisionto
panosomosis,wasfoundhereintopromoteerythrophagocytosis,toblockextramedullary
publish,orpreparationofthemanuscript.
PLOSPathogens|DOI:10.1371/journal.ppat.1005862 September15,2016 1/26
MIFandPathogenicAnemia
CompetingInterests:Theauthorshavedeclared erythropoiesisandRBCmaturation,andtotriggerhemodilution.Hence,thesedataprompt
thatnocompetinginterestsexist. consideringMIFasapotentialtargetfortreatmentofnaturalbovinetrypanosomosis.
AuthorSummary
BovineAfricantrypanosomosisisaparasiticdiseaseofveterinaryimportancethat
adverselyaffectsthepublichealthandeconomicdevelopmentofsub-SaharanAfrica.Ane-
miaisamajorcauseofdeathassociatedwiththisdisease.Yet, themechanismsunderlying
anemiadevelopmentarenotelucidated,whichhampersthedesignofeffectivetherapeutic
strategies.We showherethatinaTrypanosomacongolenseinfectionmousemodelrele-
vantforbovinetrypanosomosis,redbloodcells(RBCs)aregeneratedinthespleen.This
compensatesfortheimpairedmaturationofRBCsoccurringinthebonemarrow,thenor-
malsiteofRBCgeneration,andforthedestructionofRBCstakingplaceintheliverand
thespleen.Instead,anemiaresultsfromanincreaseinbloodvolume(hemodilution).The
immunemoleculeMacrophageMigrationInhibitoryFactor(MIF)wasfoundtodrive
RBCdestruction,toblockRBCmaturation,aswellastotriggerhemodilution.Ironaccu-
mulationintissueduetosustainedRBCdestructionandhemodilutioncausestissuedam-
age,whichculminatesinthereleaseoftoxicmoleculeslikebilirubin,inimpaired
productionofblooddetoxifyingmoleculeslikealbumin,andindefectivecoagulation.
Combined,theseeffectsinitiatemultipleorganfailurethatcanreducethesurvivalof
infectedmice.Giventheunmetmedicalneedforthisparasiteinfection,ourfindingsoffer
promiseforimprovedtreatmentprotocolsinthefield.
Introduction
Africantrypanosomosis(AT)isaneglectedtropicaldiseaseofmedicalandveterinaryimpor-
tancethatadverselyaffectshumanhealthandwelfare,aswellastheeconomicdevelopmentin
sub-SaharanAfrica[1,2].ATiscausedbyblood-bornehemoflagellatedprotozoanparasites
fromtheTrypanosomagenusthataretransmittedbythehematophagoustsetsefly(Glossina
spp.)vector[3].Trypanosoma-induceddiseasesinmammalsincludesleepingsicknessin
humansornaganaindomesticlivestock,withfatalconsequencesunlesstreated[4,5].Given
thatsofarnoeffectivevaccineisavailable,thatcertaintrypanosomestrainshavebecomeresis-
tanttocurativeandpreventivetreatments,andthateradicationoftsetsefliesremainsimpossi-
bleinmostregions[1,6],strategiesfocusingatreducingAT-associatedpathogenicitymightbe
analternativeapproachtoreducetheeconomiclossesincattleproduction.
Incaseofbovinetrypanosomosis,themaindifferencebetweenso-calledtrypano-suscepti-
bleand-tolerantanimalsreliesintheircapacitytocontrolanemiadevelopment,themajor
causeofdeathassociatedwiththedisease[7,8],andherebytoremainproductive[9].Differ-
encesinerythropoieticpotentialhavebeensuggestedasacontributingfactortoanemiadevel-
opment[8].Yet, themechanismsunderlyingthisphenomenonremainpoorlyunderstood,
whichhampersthedesignofeffectivetherapeuticstrategies.Giventhesimilaritiesbetweenthe
anemicresponsesofcattleandC57Bl/6miceupontrypanosomeinfection[8],theunderlying
mechanismsweremainlyscrutinizedinmurinemodels[10,11].Thedatacollectivelysuggest
thatthepro-inflammatorytypeIimmuneresponse,involvingTNF, IFN-γandM1-type(clas-
sicallyactivated)myeloidcells,contributestopathogenicityingeneralandanemiainparticu-
lar,andincombinationwithimpairedB-cellfunctionality,resultsinreducedsurvivalofthe
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MIFandPathogenicAnemia
mice[10,12,13].Thus,identificationofgene-productsregulatingpro-inflammatorysignals
duringthecourseofthediseasemightpavethewaytodevelopnovelinterventionstrategies.In
thiscontext,wepreviouslyidentifiedmacrophagemigrationinhibitoryfactor(MIF)asa
potentialsusceptibilitymarkerforAfricantrypanosomosis.Thisubiquitouslyproducedcyto-
kineisaprominentinducerofsystemicinflammationinmanyinflammatorydiseases[14,15]
thatactsbyrecruitingandactivatingmyeloidcellstowardsM1-typecellstothesiteofinflam-
mation[16,17],andbysuppressingapoptosisofinflammatorycells[18].We haveshownusing
atrypanosusceptiblemodelbasedonC57Bl/6miceinfectedwithT.bruceibrucei,thatMIF
contributestotissuepathogenicitybysustainingthroughoutinfectionapersistenttypeIpro-
inflammatorychemokine(CXCL1,CCL2)andcytokine(IFN-γ,TNF, IL-6)response,andby
enhancingtherecruitmentofLy6C+monocytesandneutrophils(PMNs)intheliverwithcon-
comitanthepatomegaly[19].Moreover,PMN-butnotmonocyte-derivedMIFwasmainly
responsibleforliverdamage.Inaddition,MIFpromotesthedevelopmentofso-calledanemia
ofinflammationintrypanosusceptiblemicebyenhancingredbloodcell(RBC)clearancefrom
theblood,andbytriggeringthestorageofironinlivermyeloidcellsthatdeprivesironfrom
erythropoiesisandimpairsthegenerationofmatureRBCs.However,despitereducedliver
injuryandanemialevelsinT.b.brucei-infectedMif-/-miceormicetreatedwithaneutralizing
anti-MIFantibody,thehostsurvivaltimewasnotaffected[19].
ComparedtoT.b.brucei,T.congolenseinfectioninC57Bl/6miceisconsideredatrypanoto-
lerantmodelmorerelevantforbovinetrypanosomosis[20,21].IncontrasttoT.b.brucei,T.
congolensecausesachronicinfection(3–4monthsversus1month),duetothecapacityofT.
congolense-infectedmicetorestrainthetypeIimmuneresponseandtoswitchtoanIL-
10-mediatedtissueprotectiveanti-inflammatoryresponse[21,22].Thismodelcouldthus
allowathoroughanalysisofthemechanismsunderlyinganemiadevelopmentinanimmune
environmentdifferentfromthatofanemiaofinflammation.Here,weevaluatedtheroleof
MIFinT.congolenseinfection-associatedanemiadevelopment,byfocusingonthemodulation
oftheerythropoieticanderythrophagocyticpotentialintissuesincludingthebonemarrow,
thespleenandtheliver.Additionally,thecontributionofhemodilutiontoanemiawas
addressed.
Results
1.MIFdeficiencycorrelateswithprolongedsurvivalandreducedtissue
pathogenicityduringT.congolenseinfection
ThesystemicproductionofMIFincreasedprogressivelyduringthecourseofT.congolense
infection(S1Fig).Hence,thepotentialroleofMIFintheoutcomeofi.p.infectionwasevalu-
atedbycomparingwildtype(WT)andMIF-deficient(Mif-/-)C57Bl/6mice.Althoughparasi-
temiadevelopmentwassimilarinWTandMif-/-mice,aprolongationofmediansurvivaltime
occurredinMif-/-mice(Fig1Aand1B).Similarobservationswereobtainedusinganatural
route,tsetsefly-mediatedinfectionmodel(S2AandS2BFig).Consideringthesimilarcapacity
ofWTandMif-/-micetocontrolparasitegrowth,theincreasedsurvivalofMif-/-micecould
resultfromlowertissuepathogenicity[23,24].Inagreement,ascomparedtoWTmice,Mif-/-
miceexhibitedreducedserumAST(alanineaminotransferase,reflectingsystemictissueinjury)
andALT(aspartateaminotransferase,reflectingliverinjury)levels(Fig1Cand1D),aswellas
reducedhepato-andsplenomegalythatcoincidedwithalowerincreaseinthenumberofwhite
bloodcells(WBC)intheliverandthespleenofMif-/-mice(Figs1Eand1FandS3).NoWBC
accumulationwasobservedinthebonemarrowofinfectedWTandMif-/-mice(Fig1F).
ThedifferencesinpathogenicitybetweenWTandMif-/-infectedmicewereclearlyestab-
lishedfrom3monthspostinfection(p.i.)(Figs1C–1FandS3),hencewefocusedatthistime
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MIFandPathogenicAnemia
Fig1.MIFdeficiencyconferspartialprotectionandreduceshepatosplenomegalyandwhitebloodcellaccumulation
duringT.congolenseinfection.(A)Parasitemia,(B)survival,serum(C)ASTand(D)ALTlevelsininfectedmice.Duringthe
courseofinfection,(E)liverandspleenweightaswellas(F)liver,spleenandbonemarrowwhitebloodcell(WBC)numbers.
ForbonemarrowWBCs,onlydatafrom3monthsp.i.areshown.Wildtype(WT,blacksymbol);Mif-/-(whitesymbol)mice.
Resultsarerepresentativeof2(E)or3independentexperimentsandpresentedasmean(A,C,D,E,F)ormedian(B)of3–5
individualmice±SEM.*:p(cid:20)0.05,**:p(cid:20)0.01,***:p(cid:20)0.001.
doi:10.1371/journal.ppat.1005862.g001
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Fig2.MIFcontributestopathogenicchemokineandcytokineproductionduringT.congolenseinfection.At3monthsp.i.,ex-vivoprotein
levelsofMIF,CXCL1,CCL2,TNF,IL-6,IFN-γandIL-10in(A-C)supernatantfromtheliver,spleen,andbonemarrowcellcultures,and(D)inthe
blood.IL-12p70levelsinbloodareshownforWT(blackbar)andMif-/-(openbar)mice.Proteinlevelsinnon-infectedmice(dashedline)were
similarinbothmousestrains.(N.D.:notdetectable).Resultsarerepresentativeof3independentexperimentsandpresentedasmeanof3
individualmice±SEM.*:p(cid:20)0.05,**:p(cid:20)0.01,***:p(cid:20)0.001.
doi:10.1371/journal.ppat.1005862.g002
point.At3monthsp.i.,MIFsecretionwasenhancedintheliver,spleenandbonemarrowof
infectedWTmice(Fig2),mirroringtheincreasedMIFlevelmeasuredintheblood.Inagree-
mentwiththereducedtissueinjuryininfectedMif-/-mice,thelevelsofneutrophil(CXCL1)
andmonocyte(CCL2)chemoattractants,aswellasofpro-inflammatorycytokinesdocu-
mentedtocontributetoT.congolense-inducedtissuedestruction(TNF, IL-6,IFN-γ)[25,26],
wereincreasedtoalesserdegreeintheliver,spleen,bonemarrowandbloodofMif-/-thanWT
mice(Fig2A–2D).ThiswasalsotrueforIL-12p70intheblood(Fig2D)[27].However,the
systemicandtissuelevelsofIL-10,whichincreasesuponT.congolenseinfectionandiscrucial
tolimittissuedestruction[28],didnotdifferbetweenWTandMif-/-mice(Fig2).
Inadditiontodifferencesinpro-inflammatorycytokineproduction,thedecreasedtissue
pathogenicityandincreasedsurvivalofMif-/-micealsocouldbeduetoasuperiorabilityofthe
Mif-/-micetomountaparasite-specificantibodyresponse[13].AsshowninFigs3AandS4,
similarserumlevelsofparasite-specificIgGantibodieswererecordedinWTandMif-/-mice
until1.5monthsp.i.;thereafterandfrom3monthsp.i.,theIgGlevelsdeclinedinWTmicewhile
theyremainedelevatedinMif-/-mice.ThedropinIgGlevelsininfectedWTmicedidnotcorre-
latewithadecreaseinthenumberoftotal(B220+)orgerminalcenter(GL-7+Fas+B220+)splenic
B-cells(Figs3Band3C,S5AandS5B),butcouldbeduetoanincreaseinB-cellapoptosis(Fig
3D).TheincreasedIgGlevelsobservedininfectedMif-/-miceascomparedtoWTmicewasasso-
ciatedwithanincreasednumberoftotalandgerminalcenterB-cellsaswellaswithlowerB-cell
apoptosis(Fig4A–4D).
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Fig3.MIFdeficiencypreservesparasite-specificIgGresponses,maintainsgerminalcenterformationand
preventsB-cellapoptosisduringT.congolenseinfection.(A)Parasite-specificbloodIgGlevel(1/500dilution)
inWT(blackbar)andMif-/-(openbar)miceat1.5,3and4monthsp.i..At3monthsp.i.(B)absolutenumbersof
B220+MHC-II+B-cellsand(C)ofgerminalGL-7+Fas+B-cellsand(D)percentageofAnnexin-V+B220+cellsinthe
spleenofWT(blackbar)andMif-/-(openbar)miceweredeterminedusingthegatingstrategydescribedinS5Aand
S5BFig.Resultsarerepresentativeof2independentexperimentsandpresentedasmeanof3individual
mice±SEM.*:p(cid:20)0.05,**:p(cid:20)0.01,***:p(cid:20)0.001.
doi:10.1371/journal.ppat.1005862.g003
Collectively,inT.congolense-infectedmice,theabsenceofMIFresultsinareducedpro-
inflammatoryimmuneresponse,whichinturncouldcontributetoalowerhepato-splenomeg-
alyandanenhancedB-cellresponsethatcollectivelycouldenhancethesurvivaltime.
2.MIFdeficiencycorrelateswithreducedanemiaduringT.congolense
infection
AnemiaistheprominentpathogenicfeatureofanaturalT.congolenseinfection,whichis
mediatedbyhematopoieticcells,butnotbyTlymphocytesorantibodies[10,29].SinceMIF(i)
contributestotheaccumulationofWBC(includingphagocytes;Fig1F)intissuesthatare
potentiallyinvolvedinerythrophagocytosisandextramedullaryerythropoiesis,and(ii)stimu-
latestheproductionoftheerythroidlineagedevelopment-blockingcytokineIL-6[30]inthe
twomainerythropoietictissues(bonemarrow,spleen;Fig2Band2C),weinvestigatedMIF’s
roleinanemiadevelopmentduringT.congolenseinfection.
Anemiaischaracterizedbytwodistinctphases:(i)arapiddeclineinRBClevelsfollowedby
partialrecoveryduringtheearlyphaseofinfectionand(ii)amoreprogressivedeclineinRBC
levelsduringthechronicphaseofinfection(Fig4A).Duringtheearlyphaseofinfection(i.e.
day5–10p.i.),theRBCpercentagesinitiallydroppedtoabout50%ofnon-infectedmicein
bothWTandMif-/-mice(Fig4A).Betweenday10–14p.i.,apartialrecoverythatreaches
about75%oftheRBClevelinnon-infectedmiceoccurredinWTmice,whileinMif-/-mice
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Fig4.MIFdeficiencypartiallyreducesanemiaduringT.congolenseinfection.(A)AnemiadevelopmentininfectedWT(closedsymbol)andMif-/-
(opensymbol)mice.NumberofRBCsinnon-infectedmicewassetas100%.At1.5,3and4monthsp.i.,(B)serumhemoglobinlevels,(C)serumiron
levels,and(D)numbersofsplenicwhitebloodcells(WBC,blackbar)andRBCs(whitebar)weredetermined.Resultsarerepresentativeof2independent
experimentsandpresentedasmeanof3individualmice±SEM.**:p(cid:20)0.01,***:p(cid:20)0.001.
doi:10.1371/journal.ppat.1005862.g004
thisrecoveryreachedabout95%(Fig4A).Subsequently,duringthechronicphaseofinfection,
theRBClevelsdeclinedprogressivelyandremainedsignificantlylowerinWTthanMif-/-mice.
Mif-/-micealsoexhibitedreducedanemiainanaturaltsetsefly-mediatedinfection(S2CFig).
TheserumhemoglobinandironlevelswerereducedinWTmiceascomparedtonon-infected
animalsduringthecourseofinfection(Fig4Band4C).IninfectedMif-/-mice,thesereductions
werelesspronouncedwhencomparedtoinfectedWTmice.Inbothgroupsofmice,theserum
hemoglobinandironlevelsreachednadirlevelsat3monthsp.i.,thetimepointwhenthedif-
ferencesintissuepathogenicitywereestablished(Figs1Cand1Dand4A).
Collectively,duringthechronicstageofT.congolenseinfection,MIFpartiallyimpairedrecov-
eryfromearlystageanemiaandcontributedtothedeclineinserumhemoglobinandironlevels.
3.Mif-/-micerecoverfromdyserythropoiesisduringT.congolense
infection
Typically, inresponsetochronicanemia,splenomegalyandincreasedbloodreticulocytecon-
tentareindicativeofinefficienterythropoiesisinthebonemarrowandextramedullary
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MIFandPathogenicAnemia
erythropoiesisinthespleen[31].Comparedtonon-infectedmice,thenumbersofsplenic
Ter119+RBCsincreasedmoreprominentlyinT.congolense-infectedWTthanMif-/-mice
from3monthsp.i.(Fig4D).Remarkably,thelargestincreaseincellnumberinthespleensof
infectedmicewasfoundintheRBCandnottheWBCcompartment(Fig4D),showingthe
expansionoftheerythroidcompartmentasamaincauseforT.congolense-associated
splenomegaly.
TherelativeabundanceofTer119+CD71+reticulocytesandmatureTer119+CD71-RBCs
(identifiedasdescribedinS6AFig)wasquantifiedinthebloodofWTandMif-/-miceat3
monthsp.i.,whichcorrespondswithatimepointwhenmaximalcellnumbers,levelofhepato-
splenomegalyandtissuepathogenicitywerereachedinbothgroupsofmice(Figs1C–1Fand
S3).TheconcentrationofreticulocytesincreasedinthebloodofinfectedWTmicewhencom-
paredtonon-infectedanimals,andthisincreasewaslesspronouncedinMif-/-mice(Fig5A).
Concomitantly,thereductionintheconcentrationofmatureRBCswasmorepronouncedin
thebloodofWTthanMif-/-mice.WithintheerythropoietictissuesofinfectedWTmice,retic-
ulocyteandmatureRBCaccumulationwasnotaffectedinthebonemarrow,butwasdramati-
callyincreasedinthespleen(Fig5Band5C),suggestingextramedullaryerythropoiesis.A
comparisonofinfectedWTandMif-/-micerevealedadetrimentalcontributionofMIFto
matureRBCaccumulationinthebonemarrow,andtoreticulocyteaccumulationinthespleen
(Fig5Band5C).
Next,weassessedthestageatwhicherythropoiesiscouldbeaffected(fromnucleatederyth-
roblasts(I)untilenucleatederythrocytes(VI)),bygatingforTer119+RBCsinaCD44versus
FSC-Aplot[32](S6BFig).InthebonemarrowofinfectedWTmice,ablockadeinthetwolast
stepsofRBCdifferentiationresultedindecreasedpercentage(S6CFig)andnumbers(Fig5D)
ofnucleatedreticulocytes(stageV)andenucleatedreticulocyte/erythrocyte(stageVI)cells.In
thespleen,infectedWTmiceexhibitedincreasedaccumulationoftheRBCdifferentiation
stageIIIpolychromaticerythroblaststostageVImatureRBCs(Fig5E),confirmingextrame-
dullaryerythropoiesisinthisorgan.Theerythropoiesisefficacyinthebonemarrowandthe
spleenwasimprovedininfectedMif-/-ascomparedtoWTmice.Indeed,Mif-/-miceexhibited
amoreefficientRBCmaturationmainlyduringthetransitionfromorthochromaticerythro-
blastsandnucleatedreticulocytes(stageIVandV)tothelaststepofdifferentiationi.e.enucle-
ationoferythrocytes(stageVI,Fig5Dand5E).Ofnote,thegeneexpressionlevelsofVcam1
(Vascularcelladhesionmolecule-1)andMaea(EMP:erythroblast-macrophageprotein),two
moleculescrucialforerythroblast—macrophageinteractionintheterminalstageoferythro-
poiesis,i.e.duringtheerythroblastenucleation[33,34],werehigherinMif-/-thaninWTmice
(Fig5F),suggestinganegativeeffectofMIFonthereticulocyteterminalmaturation.
Collectively,thesedataindicatedthatmiceexhibitedreticulocytosisduringthechronic
stageofT.congolenseinfection,whichwaslikelyduetoanincreasedproductionofRBCs,
mainlyinthespleen,toovercomethechroniclossofmatureRBCsobservedintheblood.
Moreover,RBCswereimpairedintheirterminalstagesofmaturationbothinthebonemarrow
andspleenofinfectedmice.Finally,MIFcontributedtothereticulocytosisandtothe
impairmentoftheterminaldifferentiationofRBCsfromtheorthochromaticerythroblastto
theenucleatedreticulocyte/erythrocytestage.
4.Mif-/-miceexhibitreducederythrophagocytosis,hemecatabolismand
tissueironaccumulationduringT.congolenseinfection
ThepercentageofbloodAnnexin-V+RBCsincreasedinT.congolense-infectedWTmice,and
thisincreasewaslowerinMif-/-thaninWTmice(Fig6A).Phosphatidylserineexposure,
whichformsthebasisoftheAnnexin-Vstainingassay,isan“eat-me” signalobservedduring
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Fig5.MIFcontributestodyserythropoiesisduringT.congolenseinfection.At3monthsp.i.,(A-C)numbersof
matureRBCs(erythrocytes,Ery.)andimmatureRBCs(reticulocytes,Ret.)definedasdescribedinS6AFigin(A)blood,
(B)bonemarrowand(C)spleenofWTandMif-/-mice.WT(blackbar),Mif-/-(openbar)andnon-infected(greybar)mice.
Resultsarerepresentativeof3independentexperimentsandshownasmeanof3individualmice±SEM.(D,E)Numbers
ofthedifferenterythroidpopulations(definedasdescribedinS6BFig)in(D)bonemarrowand(E)spleen.(F)Gene
expressionlevelsofVcam1andMaeaintotalspleenfromWT(blackbar)andMif−/−(whitebar)miceat3monthsp.i.
GeneexpressionlevelswerenormalizedusingS12andexpressedrelativelytoexpressionlevelsinnon-infectedmice.
Resultsarerepresentativeof2independentexperimentsandpresentedasmeanof3–5individualmice±SEM.*:
p(cid:20)0.05,**:p(cid:20)0.01,***:p(cid:20)0.001.
doi:10.1371/journal.ppat.1005862.g005
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Fig6.MIFcontributestoerythrophagocytosisduringT.congolenseinfection.(A)RepresentativeAnnexin-Vgating
strategyandpercentageofAnnexin-V+RBCsinbloodofWT(blackbar)andMif-/-(openbar)mice.(B,Cleftpanel)At3
monthsp.i.,109pHrodolabelledRBCsisolatedfromnon-infectedWTmicewereinjectedi.v.inWT(blackbar)orMif-/-
(openbar)mice.18hlater,miceweresacrificed,liver(B)andspleen(C)myeloidphagocyticcells(MPC),namely
CD11b+Ly6CintLy6G+PMNs,CD11b+Ly6ChighLy6G-monocytesandCD11b+Ly6C-Ly6G-F4/80+macrophages(identified
asdescribedinS7AFig)weretestedfordeltamedianfluorescentintensity(MFI)oftheintracellularpHrodosignal
determinedbysubtractingthePEsignalofcellsfrommicereceivingunlabeledRBCsfromthePEsignalofcellsfrommice
receivingpHrodo-labeledRBCs.(B,Crightpanel)NumbersofMPCinthe(B)liverand(C)spleenofWT(blackbar)or
Mif-/-(openbar)mice.Resultsarerepresentativeof2independentexperimentsandpresentedasmeanof3individual
mice±SEM.*:p<0.05;**:p<0.01.
doi:10.1371/journal.ppat.1005862.g006
apoptosisofsenescentcells.Thus,weinvestigatedwhetheranincreasedRBCelimination
throughphagocytosisintheliverandthespleencontributedtoanemiainT.congolense-
infectedmice.Anassayconsistingofi.v.injectionofpHrodo-labeledRBCsininfectedWTor
Mif-/-micefollowedbyanalysisoftheappearanceofafluorescentsignalinphagocytesfrom
theliverandthespleenthathaveengulfedlabeledRBCs[35],wasused.IninfectedWTmice,
PMNs,monocytesandmacrophages(gatedasinS7AFig)exhibitedRBCphagocyticactivity.
ThisactivitywasmorepronouncedandMIF-dependentforPMNsintheliverandformono-
cytesandmacrophagesinthespleen(Fig6Band6C).Inparallel,ahighernumberofphago-
cyticPMNs,monocytesandmacrophageswasobservedintheliverofinfectedWTmicewhen
comparedtoMif-/-mice(Fig6Band6C).Thesameheldtrueformacrophagesinthespleen.
Together, thesedatasuggestthatthereducedanemiainT.congolense-infectedMif-/-miceisa
combinedeffectofareducedapoptosis/senescenceofRBCs,areducednumberofphagocytic
cellsandareducedphagocyticcapacityofthesecells.
PLOSPathogens|DOI:10.1371/journal.ppat.1005862 September15,2016 10/26