Inflammatory Monocytes Orchestrate Innate Antifungal Immunity in the Lung

13 Pages·2014·4.59 MB·English

Inflammatory Monocytes Orchestrate Innate Antifungal
Immunity in the Lung
Vanessa Espinosa1,2., Anupam Jhingran3.¤, Orchi Dutta1,2, Shinji Kasahara3¤, Robert Donnelly4,
Peicheng Du4, Jeffrey Rosenfeld4, Ingrid Leiner5, Chiann-Chyi Chen6, Yacov Ron6, Tobias M. Hohl3¤*,
Amariliz Rivera1*
1Rutgers,NewJersey Medical School,DepartmentofPediatrics,CenterforImmunity and Inflammation, Newark, NewJersey, United StatesofAmerica, 2Rutgers,
GraduateSchoolofBiomedicalSciences,Newark,NewJersey,UnitedStatesofAmerica,3FredHutchinsonCancerResearchCenter,VaccineandInfectiousDisease
Division, Seattle, Washington, United States of America, 4Rutgers, New Jersey Medical School, Molecular Resource Facility and High Performance and Research
ComputingGroup,OfficeofInformationTechnology,RutgersUniversity,Newark,NewJersey,UnitedStatesofAmerica,5MemorialSloanKetteringCancerCenter,Sloan
KetteringInstitute,NewYork,NewYork,UnitedStatesofAmerica,6Rutgers,RobertWoodJohnsonMedicalSchool,DepartmentofPharmacology,Piscataway,New
Jersey,UnitedStatesofAmerica
Abstract
Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients.
Although-CC-chemokinereceptor-2(CCR2)andLy6C-expressinginflammatorymonocytes(CCR2+Mo)andtheirderivatives
initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain
poorlydefined.Usingconditionalandantibody-mediatedcellablationstrategies,wefoundthatCCR2+Moandmonocyte-
derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent
Aspergillusreporter(FLARE)conidiathatreportfungalcellassociationandviabilityinvivo,weidentifytwomechanismsby
whichCCR2+MoandMo-DCsexertinnateantifungalactivity.First,CCR2+MoandMo-DCsconditionthelunginflammatory
milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2+Mo temporally coincided with their
differentiationintoMo-DCs,aprocessthatresultedindirectconidialkilling.Ourfindingsillustratebothindirectanddirect
functionsfor CCR2+Moand theirderivatives ininnateantifungalimmunityin thelung.
Citation:EspinosaV,JhingranA,DuttaO,KasaharaS,DonnellyR,etal.(2014)InflammatoryMonocytesOrchestrateInnateAntifungalImmunityintheLung.PLoS
Pathog10(2):e1003940.doi:10.1371/journal.ppat.1003940
Editor:DonC.Sheppard,McGillUniversity,Canada
ReceivedSeptember13,2013;AcceptedJanuary8,2014;PublishedFebruary20,2014
Copyright:(cid:2)2014Espinosaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Thestudieswereperformedwithsupportfromthefollowingfundingagenciesandgrants:NIHgrantK22CA160874toAR,NIHgrantR21CA167238-
01A1toAR,NIHgrantF31AI098408-01A1toVE,andNIHgrantRO1AI093808toTMH(http://www.nih.gov/).ARreceivedcareerdevelopmentsupportfromthe
HispanicCenterofExcellenceatNJMSwhichisfundedbygrantD34HP16048(http://www.hrsa.gov/index.html).TMHreceivedsupportfromtheRobertA.Sinskey
Foundation(http://www.guidestar.org/organizations/95-4628223/robert-m-sinskey-foundation.aspx).Thefundershadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestexist.
*E-mail:[email protected](TMH);[email protected](AR)
.Theseauthorscontributedequallytothiswork.
¤Currentaddress:DepartmentofMedicine,InfectiousDiseaseService,MemorialSloan-KetteringCancerCenter,NewYork,NewYork,UnitedStatesofAmerica.
Introduction depletionofneutrophilsleadstouncontrolledfungalgrowthinthe
lung and to mortality from IA [19,20,21,22,23]. In addition to
The incidence of fungal infections has been on the rise for neutrophils, protective immune functions have been ascribed to a
several decades due to increased use of immunosuppressive and varietyofinnatecellsthatincludemacrophages,NKcells,myeloid
myeloablativetherapiesformalignantandnon-malignantdiseases DCs and plasmacytoid DCs [17,19,21,24,25]. While alveolar
[1,2,3]. Invasive aspergillosis (IA), most commonly caused by A. macrophages are capable of conidial killing in vitro [26] and in
fumigatus,isafrequentcauseofinfectiousmorbidityandmortality vivo [27], and likely contribute to innate defense, clodronate-
in patients with leukemia and in allogeneic hematopoietic cell mediated alveolar macrophage ablation did not lead to IA,
transplant (HCT)recipients [4,5,6,7]. suggestingthatAMfungicidalactivitycanbefunctionallycompen-
Previous studies have determined that innate and adaptive satedbyotherleukocytesinvivo[21].Similarly,thecontributionsof
components of the immune system play essential roles in defense NKcellsandmyeloidDCstoantifungaldefenseagainstaspergillosis
againstIA[2,4,8,9,10,11,12,13,14,15,16,17].Neutrophilshavelong have been examined only in neutropenic mouse models of IA
beenrecognizedasanessentialinnatecellindefenseagainstIA,as [24,25]. Thus, despite the important contributions of other innate
neutropeniarepresentsanimportantclinicalriskfactor[18].Human cells subsets in antifungal immunity, previous studies suggest that
susceptibility to IA in patients with defective neutrophil function neutrophils are the sole indispensable innate effector cell in host
(e.g. patients with chronic granulomatous disease) underscores the defenseagainstIA[19,20,21,22].
functional role of neutrophils in host defense. These findings are In contrast to their essential role against respiratory fungal
recapitulated in animal models of IA in which antibody-mediated infection, neutrophils have been found to be dispensable for
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VitalRoleforMonocytesinAntifungalImmunity
influx, as suggested in LPS-induced models of pulmonary
Author Summary
inflammation [49]. A second model of CCR2+Mo antifungal
Despite the significant impact of fungal infections to activityduringrespiratoryfungalinfectionmayinvolvetherelease
human health our understanding of immunity to these of pro-inflammatory mediators [25] to enhance the fungicidal
pathogens remains incomplete. Human mycoses are activity of resident or recruited leukocytes. A third model of
associated with high morbidity and mortality, even with antifungal activity involves direct antimicrobial effects of
modern antifungal therapies. Aspergillus fumigatus is the CCR2+Mo andderivative cells.
mostcommonetiologicagentofinvasiveaspergillosis(IA),
Inthepresentstudywesetouttoelucidatethemechanismsby
a serious infection that develops in immunodeficient whichCCR2+Mocontributetoinnateantifungalimmunityinthe
patients. In this study we employ a combination of cell
lung. To this end, we employed genetically engineered mice that
ablation strategies to examine the role of CCR2+Ly6C+
express a diphtheria toxin receptor (CCR2 depleter mice) or a
inflammatory monocytes (CCR2+Mo) in innate responses
GFP transgene (CCR2 reporter mice) under the control of the
against a pulmonary infection with A.fumigatus conidia.
We find that CCR2+Mo and their derivative dendritic cells endogenous CCR2 promoter [29,38] and fluorescent Aspergillus
reporter (FLARE) conidia that trace the outcome of CCR2+Mo
(Mo-DCs)arerequiredfordefenseagainstIAandthatmice
lackingthesecellssuccumbtoinfection withA.fumigatus. and Mo-DC interactions with conidia in the lung with single-
Our studies indicate that CCR2+Mo and Mo-DCs exert encounter resolution [27]. We found that sustained depletion of
crucial innate antifungal defense by two main mecha- CCR2+Mo and Mo-DCs led to the development of IA and a
nisms:1)CCR2+MoandMo-DCsareasignificantsourceof reductioninneutrophilconidiacidalactivity.Beyondtheirimpact
inflammatory mediators thataugment the killing capacity on neutrophil conidiacidal responses, CCR2+Mo and Mo-DCs
of neutrophils and 2) conidial uptake by CCR2+Mo is formedaTNFandiNOS-producingeffectorcellpopulationinthe
coincident with their differentiation into Mo-DCs that lungthatexertedrapidandeffectiveconidiacidalactivitysimilarin
directly kill fungal conidia via partially NADPH oxidase- magnitude to neutrophil fungicidal activity. In aggregate, our
dependent mechanisms. In aggregate, our studies find a studies suggest that CCR2+Mo and their derivatives mediate an
novel essential function for CCR2+Mo in innate defense essential role in antifungal defense in the lung by directly
against a pulmonary fungal pathogen by mediating containing conidial germination and by enhancing neutrophil
indirect and direct containment of fungal cells at the
antifungal activity.
portal ofinfection.
Results
defenseagainsttheintracellularpathogensListeriamonocytogenesand
Toxoplasmagondii[28,29].Inbothinfectionmodels,CCR2+Ly6Chi CCR2+ inflammatory monocyte-depleted mice develop
inflammatorymonocytes(CCR2+Mo,throughoutthismanuscript invasive aspergillosis
CCR2+Mo is used as an abbreviation for inflammatory mono- To examine the contributions of CCR2+ Mo and their
cytes,definedasCD45+CCR2+Ly6ChiCD11b+Ly6G2leukocytes) derivatives to respiratory fungal defense, we monitored the
were identified as essential innate effector cells that mediate outcome of intratracheal A. fumigatus conidial challenge in CCR2
bacterialandparasiticeradication[28,29,30,31].Inthesemodels, depleter mice [38] that express a functional diphtheria toxin
the formation of monocyte-derived TNF- and inducible nitric receptor (DTR) under control of the CCR2 promoter. CCR2
oxidesynthase-producingdendriticcells(Tip-DCs)correlatedwith depletermiceweretreatedwithdiphtheriatoxin(DT)onday21,
microbialclearance[31,32,33].SincebacterialuptakebyTip-DCs +1, and +3 to ablate CCR2-expressing cells during respiratory
during systemic listeriosis and salmonellosis appears to be an fungal infection. We included twocontrol groups:non-transgenic
infrequent event (,1% of Tip-DCs) [34,35,36], it remains C57BL/6J (B6) littermates that received the same DT adminis-
unknown whether inflammatory monocytes and their derivatives tration regimen as CCR2 depleter mice and B6 mice that were
exert relevant antimicrobial activity by pathogen engulfment and depletedofneutrophilsbyadministrationofanti-Ly6Gantibodies.
killingat theportal of infection. Consistent with previous studies using a different neutrophil-
In fungal infection models, the role of CCR2+Mo has depleting antibody [20,21,22], anti-Ly6G-treated mice rapidly
largely been understood in the context of adaptive CD4 T cell succumbedtoIA(Figure1A).Non-transgenicB6controlanimals
responses. Inarespiratory A.fumigatusinfectionmodel CCR2+Mo treatedwithDTdidnotdevelopdiseasesymptomsthroughoutthe
are rapidly recruited to the lung and differentiate into duration of the experiment. Strikingly, CCR2 depleter mice
CCR2+CD11c+MHCII+CD11b+CD1032 monocyte-derived DCs treated with DT uniformly succumbed to infection when
(Mo-DC)that are essential for the induction and maintenance of challenged with inocula that ranged from 4–86107 conidia
A.fumigatus-specific Th1 CD4 T cell responses [37,38]. Mo-DCs (Figure 1A and 1B). To determine whether mortality was
have also been found to be important for initiation of fungus- associatedwithfungaltissueinvasion,Gomorimethenaminesilver
specific T cell responses in the context Blastomyces vaccination (GMS)-stainedlungsectionswereexaminedfromCCR2depleter
and Histoplasma capsulatum infection in the lung [39,40,41,42]. In mice and control animals at various time points post-infection.
vivo studies with human blood monocytes have shown that Lung sections from CCR2 depleter mice showed extensive and
these cells have fungistatic activity ex vivo and elaborate progressive hyphal growth (Figure 1C) starting at day +3 post
cytokines and chemokines following stimulation with A. fumigatus infection (p.i). Extensive lung parenchymal destruction and
conidia [43,44,45,46]. Although emerging evidence indicates obliterationofbronchoalveolararchitecturewasapparentatlater
that CCR2+Mo and their derivatives contribute to innate time points. In contrast, lung sections from DT-treated B6 mice
defense against systemic candidiasis [47,48], it remains unclear onlyshowedevidenceofconidiathatfailedtogerminateatalltime
whether CCR2+Mo act to control the influx and activity of points examined. This is consistent with our previous studies
other effector cell populations or directly contribute fungicidal in which B6 mice were able to effectively prevent conidial
capacity at sites of infection. germination [37,50,51]. In aggregate these findings demonstrate
One possible model is that CCR2+Mo and their derivatives that CCR2+ cells are essential for early host defense against
control antifungal activity in the lung by regulating neutrophil A.fumigatusandthattheirablationleadstothedevelopmentofIA.
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VitalRoleforMonocytesinAntifungalImmunity
SusceptibilityofCCR2depletermiceisnotduetolackof 2D). Despite a total lack of NK cells, RAG2/2cC2/2 mice
NK cells controlledA.fumigatusconidialinoculaat24and48hp.i.,asjudged
by the recovery of viable fungal cells from the lungs of RAG2/2
Previous studies have shown a protective role forNK cells in a
cC2/2comparedtocontrolmice(Figures2Eand2F).Inaddition,
neutropenic model of IA [24]. Since a subset of NK cells express
we did not observe invasive fungal growth in infected RAG2/2
CCR2, we explored whether the phenotype observed in CCR2
cC2/2 mice by lung histopathology (Figure 2G) and RAG2/2
depletermicecouldbelinkedtoadefectinNKcells.Weexamined
cC2/2micedidnotdevelopdiseasesymptomswithintheoneweek
therecruitmentofNKcellstothelungofCCR2depletermiceand
observation period. In contrast, CCR2 depleter mice showed a
to control non-transgenic littermates during respiratory fungal
significantincreaseinthenumberofviablefungalcellsinthelungat
infection.WeobservedthatDTtreatmentsignificantlydepletedNK
24and48hp.i.(Figures2Eand2F)whichprecededinvasivefungal
cellsinthelungofinfectedCCR2depletermiceat24and48hp.i.
growthat3daysp.i.(Figure1C).Inaggregate,theseresultsindicate
(Figures2Aand2B).ToexaminewhetherthisreductioninNKcells
that the development of IA in CCR2-depleter mice cannot be
couldbelinkedtothedevelopmentofIAinCCR2depletermice,we
explainedbyDT-inducedablationofCCR2+NKcells.
examinedtheprogressionofA.fumigatusinfectioninmicethatlackall
lymphocytes, including NK cells, iNKT cells, and innate lympho-
cytes(recombinationactivatinggene[RAG-2]andcommongamma CCR2+Mo depletion and neutrophil recruitment during
chain [cC] double deficient mice; RAG2/2cC2/2). NK1.1+ cells respiratory fungal infection
wereabsentfromthelungsofA.fumigatus-infectedRAG2/2cC2/2 Given the crucial role of neutrophils in defense against IA, we
mice (Figure 2A) but the mice showed normal neutrophil and examined the impact of CCR2+Mo ablation on neutrophil
enhancedmonocyterecruitmenttoinfectedlungs(Figures2Cand chemotactic responses and recruitment to the lung. Although
Figure1.CCR2+cellsprotectagainstInvasiveAspergillosis.A–B)CCR2depleter(solidgrayline)andcontrolB6non-transgeniclittermates
(solidblackline)weretreatedwith250ngofDTi.p.onday21,+1,and+3.Neutrophildepletedmice(dashedblackline)wereB6miceinjectedwith
1A8(anti-Ly6Gantibodies)daily.(A)Allanimalswereinfectedwith86107liveA.fumigatusconidia.ThegraphshowsKaplan-Meiersurvivalofindividual
groupspooledfromtwoindependentexperimentswith4–5micepergroupperexperiment.Statisticalanalysiswasperformedwithlog-ranktestand
Bonferronicorrectionformultiplecomparisons:WTvs.CCR2depleterP=0.0002,WTvsanti-Ly6GtreatedP=0.0003.(B)Kaplan-MeiersurvivalofDT-
treatedB6 (solid blackline,inoculum66107 conidia)andCCR2 depletermice(66107 conidia, dashedblackline;46107 conidia,solidgreyline).
Statisticalanalysiswasperformedasdescribedin(A).WTvs.CCR2depleter66107p=,0.0001,WTvsCCR2depleter46107p=0.001.Datashownis
forfivemicepergroup.(C)Representativephotomicrographsofformalin-fixedGMS-stainedlungsectionscollectedattheindicatedtimesp.i.fromDT-
treatedCCR2depleter(toprow)andB6mice(bottomrow).Na¨ıveanimalsweresacrificedatday+6andreceived3dosesofDT.Sectionsshownarefor
onemousepergroupandarerepresentativeof3–5micethatwereexaminedpergrouppertimepointintwoindependentexperiments.
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VitalRoleforMonocytesinAntifungalImmunity
previous studies have clearly established that neutrophils are not Although DT administration clearly eliminated all CCR2+Mo
directlyeliminatedbyDTadministrationinCCR2depletermice ininfectedmice(Figures3Cand3D),DTadministrationdidnot
[29,38] we hypothesized that CCR2+Mo ablation could interfere deplete lung neutrophils (identified as CD45+CD11b+Ly6-
withlungneutrophilrecruitmentduetotheirroleasproducersor G+Ly6C+ cells) (Figure 3C). Furthermore, similar numbers of
amplifiers of chemotactic mediators, as has been observed in a neutrophilswerepresentinthelungofCCR2depleterandcontrol
LPS-induced model of lung inflammation [49]. To test this mice at various times after infection (Figure 3E). Although
possibility, CCR2 depleter mice were treated with DT, infected there was a modest trend towards lower numbers of neutrophils
with A. fumigatus conidia, and euthanized at various time points in CCR2 depleter mice these differences did not reach statistical
after infectiontomeasure theproductionof neutrophil-recruiting significance. In contrast, B6 mice treated with anti-Ly6G anti-
chemokines and to enumerate and analyze lung homogenates by bodieshadpreservedlungCCR2+Morecruitment(Figure3F),but
flowcytometry.CCR2depletermicetreatedwithDThadsimilar were depleted of neutrophils (Figure 3G). In aggregate, these
lung levels of chemokine (C-X-C) motif ligand 1 (CXCL1) and findings indicate that CCR2 depleter mice produce wild-type
CXCL2 as control non-transgenic littermates treated with DT, levelsofCXCL1andCXCL2duringrespiratoryfungalinfection
suggestingthatCCR2+cellsarenotrequiredfortheproductionof and display preserved neutrophil recruitment to the site of
these chemokines during respiratory fungal infection (Figures 3A infection, though these processes per se are insufficient to prevent
and 3B). thedevelopment ofIA.
Figure2.CCR2+NKcellsandinnatelymphocytesaredispensableforinnatedefenseagainstIA.(A)RepresentativeplotsofCD45+lung
cellsobtainedfromcontrolB6,DT-treatedCCR2depletermice,andRAG2/2cC2/2miceonedayp.i.with86107A.fumigatusconidiaandanalyzedfor
NK1.1expression.B–D)Thebargraphsshowthetotalnumberoflung(B)NK1.1+cells,(C)CD11b+Ly6G+Ly6C+neutrophils,or(D)CD11b+Ly6G2Ly6C+
monocytes(CCR2+Mo)inDT-treatedCCR2depleter(graybars),controlmice(whitebars),orRAG2/2cC2/2(blackbars)atday+1and+2p.i.(E–F)The
scatterplotsshowthemean6SEMoflungCFUsrecoveredfromcontrol(whitecircles),DT-treatedCCR2depletermice(graycircles)orRAG2/2cC2/2
(blackcircles)atday+1and+2p.i.(B–F)Datashownisformean6SEMfor4–5micepergroupfromoneoftworepresentativeexperiments.Mann-
Whitneytestusedforstatisticalanalyses,*p,0.05,**p,0.01.G)ThephotomicrographshowsGMS-stainedlungtissuefromarepresentativeRAG2/2
cC2/2mouseonday+3p.i.
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Removal of CCR2+Mo impacts neutrophil conidiacidal receptor Dectin-1 was similar in CCR2 depleter and in control
activity mice at 36p.i.(data not shown).
Since neutrophil recruitment was not affected by CCR2+Mo To extend these observations, we compared bone marrow
neutrophil conidiacidal activity in vitro in the absence and
depletion, we hypothesized that neutrophil function may be
presenceofCCR2+Mo,usingbonemarrowcellsharvestedfrom
altered,resultinginareductioninneutrophilfungicidalactivityin
DT-treated CCR2 depleter and non-transgenic littermate con-
CCR2depletermice.Totestthishypothesis,weutilizedarecently
trols.WhenCCR2+Mowereabsentfromneutrophil–conidiaco-
developed fluorescent Aspergillus reporter strain (FLARE) to
cultureexperiments,neutrophilconidialviabilitywashigherthan
monitor and quantify neutrophil-mediated uptake and killing of
in co-cultures that included CCR2+ Mo, though neutrophil
A.fumigatus conidia in vivo [27]. The FLARE strain distinguishes
conidial uptake was similar in both cases (Figures 4D–4F).
live and dead conidia by incorporation of a tracer (Alexa Fluor
Addition of flow-sorted bone marrow monocytes (identified as
633;AF633)andaviability(DsRed)fluorophore.Hostleukocytes
that engulf live DsRed+AF633+ conidia emit two fluorescent CCR2(GFP+),CD11b+CD11c2NK1.12cells)restoredtheconidia-
cidal function of neutrophils to baseline levels (Figure S1). These
signals,oneofwhich(DsRed)isextinguishedwhenleukocyteskill
findings indicate that CCR2+ Mo and derivative cells enhance
engulfed conidia. Using the FLARE strain, we quantified
neutrophil conidiacidal activity when these leukocytes are com-
neutrophil conidial uptake and killing in CCR2 depleter and
binedaspurifiedcellularcomponentsinthetesttubeorarefound
control mice.
inthecomplex inflammatory contextwithinthelungs.
Infection of DT-treated CCR2 depleter mice with FLARE
conidia revealed that CCR2+Mo ablation did not alter the
CCR2+Mo differentiate into Mo-DCs that produce a
frequency of neutrophils with engulfed conidia at 12 or 36hours
p.i. compared to non-transgenic, DT-treated littermate controls variety of protective factors during respiratory fungal
(Figure 4B and data not shown), indicating that ablation of infection
CCR2+Mo does not decrease neutrophil conidial uptake. How- To determine additional mechanisms by which CCR2+Mo
ever, the frequency of neutrophils with live conidia was and/or Mo-DC mediate protection against A.fumigatus, we
substantially increased in DT-treated CCR2 depleter mice performed a transcriptome analysis on sorted cell populations
compared to control mice (Figures 4A and 4C). In other words, withRNA-seq.TothisendweinfectedCCR2reportermicewith
conidia engulfed by neutrophils were more likely to be killed in A.fumigatus and sorted CCR2+Mo and Mo-DC (identified as
controlmicethaninCCR2depletermice(Figure4C).Neutrophil CCR2(GFP+),CD11b+CD11c+NK1.12)48hp.i.to.97%purity.
expression of Toll-like receptor 2 and 4 and of the C-type lectin CCR2+Mopresentinthelungofna¨ıveCCR2reportermicewere
Figure3.CCR2+cellsaredispensablefortheproductionofneutrophilchemokinesandneutrophilrecruitment.(A–E)Controland
CCR2 depleter mice were treated with DT and infected with 66107 conidia on day 0 and euthanized at the indicated times for ELISA of lung
homogenatesandFACSanalysisoflungsinglecellsuspensions.(A–B)Thescatterplotsshowmean6SEMlung(A)CXCL1and(B)CXCL2levelsat
48hp.i.inCCR2depleter(whitecircles)andcontrolB6mice(blackcircles).(C–E)RepresentativeFACSplots(day+1p.i.)fromCCR2depleter(C,top
row)andcontrolB6mice(C,bottomrow)gatedonlungCD45+CD11b+cellsandanalyzedforLy6CandLy6G.Monocytes(Mo)areidentifiedas
Ly6C+Ly6G2cellswhileneutrophils(Ne)areidentifiedasLy6G+Ly6C+cells.(D)Thegraphshowsmeannumber(6SEM)ofmonocytesrecoveredfrom
thelungofDT-treatedB6mice(blackcircles)orCCR2depletermice(whitetriangles)attheindicatedtimepointsp.i.Pooleddatashownfromthree
independentexperiments(3–5micepergroupandperexpt.).(E)Thescatterplotsshowmean6SEMofnumberofneutrophilsrecoveredfromthe
lungofCCR2depletermice(whitecircles)orcontrollittermates(blackcircles)atvarioustimesafterinfection.Eachsymbolrepresentsonemouse.
Dataiscumulativefortwoorthreeindependentexperimentswith3–5micepergrouppertimepoint.(F–G)Thebargraphsshowthemeannumber
(6SEM)oflungmonocytes(F)andneutrophils(G)recoveredfromanti-Ly6G-treatedandcontrolmiceasdescribedinFigure1.Statisticalanalyses
wereperformedusingMannWhitneytests,n.s(notsignificant),*p,0.05.
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VitalRoleforMonocytesinAntifungalImmunity
Figure4.DiminishedneutrophilconidiacidalactivityinCCR2depletermice.CCR2depleterandcontrolmiceweretreatedwith10ng/gm
DTonday21andday0andinfectedwith36107FLAREconidia.(A)RepresentativeFACSplotsoflungneutrophilsisolatedfromCCR2depletermice
andcontrolmiceandanalyzedfordsRedandAF633fluorescence.Plotsshowthefrequenciesofneutrophilsthatcontainlive(redgate)orkilled
conidia(bluegate)at36hp.i.(B)Thescatterplotspooledfrom2experimentsshowtheaveragefrequency(6SEM)oflungneutrophilconidial
uptake (R1+R2) and (C) lung neutrophil conidial viability (R1/(R1+R2) in CCR2 depleter and control mice. *p,0.05 by Mann-Whitney test. (D)
Representative FACS plots of bone marrow neutrophils isolated from control or CCR2 depleter mice and cultured in vitro with FLARE conidia.
NeutrophilswereidentifiedasCD45+CD11b+Ly6G+cellsandanalyzedfordsRedandAF633fluorescenceasshown.(EandF)Thescatterplotspooled
from2experimentsshowtheaveragefrequency(6SEM)ofbonemarrowinvitroneutrophilconidialuptake(R1+R2)(E)andinvitroconidialviability
(R1/(R1+R2)inbonemarrowneutrophilsisolatedfromCCR2depleterandcontrolmice(F).**p,0.01byMann-Whitneytest.
doi:10.1371/journal.ppat.1003940.g004
sorted as a control population. We performed three independent GFP+ cells isolated from FLARE-infected CCR2 reporter mice.
experimentsandfoundconsistentupregulationofmultiplecytokines We found evidence of GFP+ cells that contain viable DsRe-
and chemokines in response to fungal infection (Figure 5A), with d+AF633+conidiaaswellasGFP+cellsthatcontainkilledAF633+
the highest expression of these genes in the Mo-DC subset, as conidia (Figure 6A). To define the relative contribution of
confirmed by qRT-PCR (Figure 5B). Cells isolated in the CCR2+Mo and their derivative Mo-DCs to conidial killing in
GFP+CD11b+CD11c+ fraction expressed genes identified as part vivo, we determined the kinetics of cell recruitment (Figure 6B),
of the core DC signature (Figure 5A) [52], consistent with their conidial uptake (Figure 6C), and killing by flow cytometry
designation as dendritic cells (Mo-DCs). CCR2+Mo and Mo- (Figures 6Dand6E).This analysisrevealed that althoughsimilar
DCs were not only capable of producing IL-12, Nos2 and TNF numbers of CCR2+Mo and Mo-DCs were present in the lung at
upon infection but appeared to act as essential sources for these 36h p.i. (Figure 6B), Mo-DCs were far more likely to engulf
inflammatory mediators during respiratory fungal infection, conidia and contain killed conidia compared to CCR2+Mo
since ablation of these cells in CCR2 depleter mice resulted in (Figures 6Cand6E).
significantly diminished production of these factors (Figures 5C– Analysisofconidiacidalactivityonapercellbasisrevealedthat
E). These findings thus suggest that CCR2+Mo and Mo-DC onceconidiawereinternalized,CCR2+MoandMo-DCswereas
recruited to the lung during A.fumigatus infection express soluble efficient in mediating conidial killing as neutrophils (Figures 6F–
factors, including cytokines (e.g. TNF) and effector molecules 6H). The efficiency of conidial killing was determined by
(e.g. pentraxin-3) that enhance neutrophil antifungal activity. examining different fungus-engaged leukocyte populations (neu-
trophils,CCR2+Mo,Mo-DCs)andbycomparingthefrequencies
CCR2+Mo and Mo-DCs are required for direct fungal offungus-engagedleukocytesthatcontaineitherviableconidiaor
spore elimination killed conidia. To examine the requirement for NADPH oxidase
ToexaminewhetherCCR2+MoandMo-DCsplayadirectrole inMo-DCconidiacidalactivity,wegeneratedmixedbonemarrow
inconidialkillingweinfectedCCR2reporterwithFLAREconidia chimeric mice that contained equal numbers of congenically
totrackthedynamicsofpulmonaryCCR2+Morecruitment,their marked NADPH oxidase-deficient (p47phox(2/2) and –sufficient
differentiation into Mo-DC, and their conidiacidal activity. (p47phox(+/+)) hematopoietic cells. In this host setting, NADPH
CCR2+ cells in the lung are comprised primarily of oxidase-deficient and –sufficient leukocytes are isolated from and
CCR2+CD11b+Ly6C+inflammatorymonocytes(CCR2+Mo)that analyzedinthesameinflammatorycontext.Similartoneutrophils,
arepresentinthena¨ıvelung(FigureS2)andarerapidlyrecruited Mo-DCs employ reactive oxygen species (ROS) as a conidiacidal
frombonemarrowstoresduringrespiratoryfungalinfection[38]. mechanism, since NADPH-deficient Mo-DCs kill conidia less
CCR2+Mo rapidly upregulate CD11c and MHC class II effectively than NADPH-oxidase sufficient counterparts (Figure
expression levels intheinflamed lung (Figure S2,[38]). S3). Analysis of FLARE killing by Mo-DCs showed that the
TodeterminewhetherCCR2+MoandMo-DCsarecapableof frequency of viable conidia in p47phox2/2 Mo-DCs was higher
conidial killing in vivo, we first performed imaging cytometry of comparedtop47phox+/+Mo-DCs(FigureS3).Despitethesuperior
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VitalRoleforMonocytesinAntifungalImmunity
Figure 5. Inflammatory responses of CCR2+Mo and Mo-DC during respiratory fungal infection. Lung CCR2+Mo
(GFP+CD45+CD11b+CD11c2Nk1.12)andMo-DC(GFP+CD45+CD11b+CD11c+NK1.12)wereFACSsorted48hp.i.fromCCR2reportermice(purity.
97%forallsorts)fortranscriptomeanalysisbyRNA-seq(A)orforquantitativeRT-PCR(B).ControlCCR2+Mowerealsoisolatedfromthelungof
uninfectedCCR2reportermice(na¨ıvesample)to.97%purity.(A)GeneexpressiondatashowninAisforoneexperimentandrepresentativeof3
independentbiologicalreplicatesandthreeidependentsequencingreactionsusingSOLiDsequencingplatform.Differencesingeneexpressionare
shownasfragmentsperkilobase(FPKM)ascalculatedusingCufflinksandRsoftware.(B)Thegraphsshowexpressionofspecifictranscriptsinthe
indicated cell populations by qRT-PCR using Taq-Man probes normalized to GAPDH. Data shown is mean 6SEM pooled from two separte
experiments.(C)ThegraphshowspulmonaryNos2inductioninDT-treatedCCR2depleterandcontrolmiceattheindicatedtimepointsp.i.Data
shownismean6SEMpooledfromtwoseparteexperimentswith3micepergrouppertimepoint.(D–E)Thescatterplotsshowmean6SEMlung(D)
IL-12p70and(E)TNFlevelsat48hp.i.inCCR2depleter(greycircles)andcontrolB6mice(blackcircles)asinFigure3A.
doi:10.1371/journal.ppat.1003940.g005
conidiacidalacitivityinp47phox+/+Mo-DCs,therewassignificant Discussion
killing preserved in p47phox2/2 cells, indicating that conidial
killing by Mo-DCs is only partially dependent on NADPH In this study, we uncover novel and essential functions for
oxidase. When neutrophils and Mo-DCs were analyzed side-by- CCR2+inflammatorymonocytesandtheirderivativeMo-DCsin
side, neutrophil conidiacidal activity was more dependent on innate antifungal defense in the lung. The protective role of
NADPH oxidase activity than Mo-DC conidiacidal activity (data CCR2+Mo and their derivatives against A. fumigatus is not
notshownand[27]).ThesefindingsindicatethatMo-DCs,similar compensated by neutrophil antifungal activity. Similarly, our
toneutrophils,employNADPHoxidaseactivityasaconidiacidal findingsconfirmthelong-standingtenetthatneutrophilfunctionis
mechanism. essential for host defense against IA [19,20,21,22]. Thus,
The total number of viable fungal cells in the lung of CCR2 CCR2+MoandderivativeMo-DCaswellasneutrophilsrepresent
depleter mice was significantly elevated at (Figure 6I), demon- essentialinnateimmunecellsthatpreventtheformationoftissue-
strating that lung conidiacidal activity is significantly reduced at invasivehyphaeandIAinthemurinelung.Incontrast,NKcells
early time pointsp.i. whenCCR2+MoandMo-DCs areablated, and other common gamma chain-dependent innate lymphocyte
consistentwithanessentialroleininnateantifungaldefenseinthe populations were not essential to mediate innate defense against
lung. Although essential, CCR2+Mo and Mo-DCs per se are not inhaledA.fumigatusconidia,sincemicedeficientintheseleukocyte
sufficient for conidial containment since monocyte-sufficient, populations contained conidial germination and did not develop
neutropenicmice(anti-Ly6Gtreatedmice)alsoshowedenhanced invasive disease.
conidial survival and fungal germination in the lung (Fig. 6I). In Previous studies showed that neutrophil depletion leads to
aggregate our findings are consistent with a model in which increased pulmonary recruitment of CD11b+CD11c+ TNF-pro-
CCR2+MoandMo-DCderivativesareessentialinpreventingIA ducingDCs[25].TheTNF-producingDCpopulationdescribedby
development via a non-redundant role in conidial clearance Park et al. [25] was recruited in response to enhanced CCL2
by direct killing and by regulation of neutrophil conidiacidal productionandappearssimilartoMo-DCsdescribedinourstudy.
activity. The finding that ablation of CD11c-expressing cells diminished
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VitalRoleforMonocytesinAntifungalImmunity
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VitalRoleforMonocytesinAntifungalImmunity
Figure6.CCR2+ModifferentiateintoMo-DCandefficientlykillA.fumigatus.CCR2reportermicewereinfectedwithFLAREconidiaandlung
cellsuspensionswereenumeratedandexaminedby(A)imagingcytometryand(B–H)flowcytometry.(A)ImagingcytometryoflungGFP+(CCR2+)
cellsfromFLARE-infectedCCR2reportermice36hp.i.ThemicrographdepictsdsRed+AF633+anddsRed2AF633+monocytesthatcontainliveand
killedconidia,respectively.BF,bright-field.(B–E)Thegraphsshowthetotalnumber(mean6SEM)oflungCCR2+Mo(whitecircles)andMo-DCs
(blackcircles)attheindicatedtimesp.i..CCR2+Mo(whitecircles),andMo-DC(blackcircles)wereidentifiedasshowninFigureS1.(B)Datashows
totalrecrutimentofeachsubsetovertime.(C)ThegraphshowsthetotalnumberofCCR2+Mo(whitecircles)orMo-DCs(blackcircles)thatcontain
engulfedconidia.(D–E)ThegraphshowsthetotalnumberofCCR2+Mo(whitecircles)orMo-DCs(blackcircles)thatcontain(D)liveor(E)killed
conidia.(F–H)ComparisonofCCR2+Mo,Mo-DCandneutrophilconidiacidalactivity.Thescatterplotsshowthefrequencyoffungus-engaged(F)
CCR2+Mo,(G)Mo-DC,and(H)neutrophilsthatcontainlive(redcircles)orkilled(bluecircles)FLAREconidiaattheindicatedtimesp.i.Resultsare
pooledfromtwoexperiments.(I)ThegraphshowslungCFUsfromDT-treatedB6controls(whitecircles),DT-treatedCCR2depleter(blacksquares),
andanti-Ly6G-treatedB6mice(greytriangles)atday+1p.i..Eachsymbolrepresentsonemouse.Resultsareforoneexperimentrepresentativeoftwo
individualexperimenstforalldatashowninthisfigure.StatisticalanalysiswasperformedusingaMannWhitneytest.
doi:10.1371/journal.ppat.1003940.g006
fungal clearance in this model was consistent with a protective TNF early during respiratory A. fumigatus infection, the precise
role of one or several CD11c-expressing myeloid cell subsets in function of TNF during conidial clearance remains to be
the context of neutropenia [25]. Similarly, the accumulation of established. It is unclear whether TNF-producing CCR2+Mo
CD11b+CD11c+ myeloid DCs in the lung was greater in represent an important target of TNF signaling to enhance cell-
CCR7(2/2) neutropenic mice than in CCR7(+/+) neutropenic intrinsic conidiacidal activity. In an ocular model of fungal
mice. This finding correlated with reduced susceptibility to IA, keratitis,iNOSactivitywasdispensableforhostdefenseagainstA.
consistent with a protective role of CD11b+CD11c+ DCs at the fumigatusinthecornea.TheroleofCCR2+Mo-derivednitricoxide
site of respiratory fungal infection in neutropenic animals [53]. during pulmonary fungal infection remains undefined. In both
In our experiments, we examined the relationship between instances, the development of cell type-specific gene knockout
CCR2+Moandtheirderivativesandneutrophilrecruitmentand strategies [57] willenableresearchers toaddressthese questions.
functioninthelung.DT-treatedCCR2depleterandcontrolmice Besides their role as producers of inflammatory mediators our
showed similar kinetics and magnitude of neutrophil recruitment data shows that CCR2+ Mo and Mo-DCs are crucial for direct
duringtheearlyphasesofinfection.IntherespiratoryA.fumigatus conidial containment. Although both populations kill conidia
infection model, conidial clearance is a hallmark of the first efficiently, the frequency of Mo-DCs with engulfed conidia is far
24hours post-infection. In both mouse strains, the number of higher than that of CCR2+Mo. Thus, Mo-DCs kill a significant
viablefungalcellsisreducedbyafactoroffivetotenduringthis larger number of conidia than CCR2+ Mo. Unlike alveolar
timeperiod,withamoreeffectivereductioninmonocyte-sufficient macrophages [58], the conidiacidal activity of CCR2+Mo and
mice compared to monocyte-depleted mice. This early difference their derivatives was partially dependent on NADPH oxidase.
in conidial clearance occurs despite the preserved synthesis of Thus, CCR2+Mo and their derivatives contribute to ROS-
neutrophil chemotactic factors in the lung and the rapid dependent mechanisms that are implicated in human defense
accumulation of neutrophils at the site of infection. Thus, the againstAspergillussp.,i.e.thesusceptibilityofpatientswithchronic
difference in fungal CFUs among the groups likely reflects three granulomatous disease to IA. [23,26,59]. These findings are
factors: the early absence of CCR2-Mo and derivative cell similar to observations in leishmaniasis, in which CCR2+Mo
conidiacidal activity, the reduction in neutrophil conidiacidal mediate elimination of parasites via the production of reactive
activityonaper-cellbasis,andneutrophilrecruitmentthatmaybe oxygen species (ROS) [55]. During secondary responses to L.
considered suboptimal since the number of viable fungal cells in monocytogenes infection, inflammatory monocytes also represent a
thelungofCCR2depletermiceisonaveragetwiceashighasin significant sourceof protective ROS[60].
control mice. Our work extends previous studies on the role of CCR2+Mo
Essentialprotectivefunctionsformonocyte-derivedDCssubsets andtheirderivativesintraffickingfungalantigentolung-draining
have been demonstrated in other infection models lymph nodes, priming Aspergillus-specific CD4 T cells, and in
[28,30,31,54,55]. In the context of systemic Listeria monocytogenes inducing the development of Th1 effector cells. Taken together,
infection CCR2-dependent, TNF- and iNOS-producing DCs our findings suggest that CCR2+Mo are required in antifungal
(Tip-DC) were found to play an essential role in innate defense defense as innate conidiacidal effectors and precursors of
against intracellular bacteria [31], a finding that has been inflammatory Mo-DCs; the latter cells provide a significant
extended to other intracellular pathogens, for example Leishmania reservoir of conidiacidal activity in the lung and elicit Th1
and Toxoplasma [28,30,55,56]. Our studies now show that the responses [37,38] that perpetuate a protective immune response
protectivefunctionforCCR2+Moandtheirderivativecellsisnot [37].Whether lung-resident Aspergillus-elicited Tip-DCs described
restrictedtointracellularbacteriaandparasitesbutisalsoessential in this study are identical to migratory Mo-DCs required for
forinnateantifungaldefense.InresponsetoA.fumigatusinfection, fungus-specific CD4 T cell priming is not clear at this time. It is
CCR2+ Mo-DCs produced TNF and iNOS and are likely possible that a subset of Tip-DCs migrate to the lung-draining
comparable to Tip-DCs induced by L. monocytogenes infection. lymphnodeforantigentransportandCD4Tcellprimingorthat
Given the unique composition of fungal pathogens it will be a subset of Mo-DCs that do not produce TNF and iNOS are
important to examine how the recruitment and differentiation of responsible for fungal antigen trafficking. Further studies will be
CCR2+Mo into Tip-DCs is regulated by innate receptors required todissect these possibilities.
specialized infungal recognition. Although the current study addresses the role of inflammatory
During systemic listeriosis, Tip-DCs mediate protective effects monocytesinamurinemodel,itispossiblethathumanmonocytes
duetotheirroleasmajorproducersofTNFandnitricoxide[31]. similarly carry out an important role in defense against IA. The
The association of defects in TNF signaling with murine antifungal capacity of human monocytes against A.fumigatus has
susceptibility to IA and of TNF inhibitors with human suscepti- longbeenrecognized[46]andexogenouscytokines,includingM-
bility to IA indicates that TNF plays a critical role in antifungal CSF,IFN-candIL-12,enhanceantifungaleffectsofthesecellsin
immunityinthelung.AlthoughCCR2+Moareamajorsourceof vitro [46,61,62]. More detailed analysis of human monocyte
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VitalRoleforMonocytesinAntifungalImmunity
subsets showed that CD14+CD162 monocytes could prevent Dr.MichelleMomany.ForlungELISAstudies,weusedAspergillus
conidial germination [45]. In contrast, CD14+CD16+ monocytes fumigatus strain Af293. A. fumigatus was cultured on Sabouraud
mounted robust inflammatory responses to conidia but did not dextrose agar (SDA) for 7–10 days prior to infection. Mice were
preventgerminationinvitro[45],suggestingdistinctcontributionsof challenged with 4–86107 live conidia per mouse using a non-
human monocyte subsets to antifungal defense. Interestingly, invasive intratracheal (i.t.) infection procedure as previously
CD14+CD162 monocytesexpressCCR2andhavebeenproposed described [51]. The viability of A. fumigatus conidia in the
to be analogous to murine CCR2+ Mo [63]. Thus, the direct inoculum was confirmed by plating serial dilutions on SDA. For
conidiacidal activity observed in murine CCR2+ Mo and their assessment of fungal burden in infected mice lung single-cell
derivatives in the lung is likely functionally conserved in human suspensions were serially diluted and plated on SDA at various
CD14+CD162 monocytes andtheir derivatives.In human neutro- times after infection. For histological examination, lungs were
penicpulmonaryaspergillosisthereissignificantpulmonaryrecruit- perfused with 10ml of PBS to remove blood and fixed in 10%
mentofCD1a+DCs,whichrepresentmonocyte-derivedcells[25]. buffered formalin. Fixed lung tissue was paraffin embedded and
Patients with autosomal dominant or sporadic deficiency in stained with modified GMS stain at the Histology Core Facility
monocytes, DCs, and NK cells (termed MonoMAC syndrome) (Rutgers-NJMS).
duetomutationsinthetranscriptionfactorGATA2areproneto
disseminated nontuberculous mycobacterial infections (incidence Cell depletion strategies
,80%), invasive fungal infections (incidence ,30%), primarily For the selective removal of neutrophils, mice were injected
histoplasmosis but also aspergillosis, and to viral infections (e.g. daily with 1A8 monoclonal antibodies (anti-Ly6G). Mice were
human papilloma virus; incidence ,80%). The clinical manifes-
injectedwith500mgi.ptogetherwithanotherdoseof100mgi.tof
tations of patients with MonoMAC syndrome support the notion 1A8antibodiesinordertoachievesignificantdepletionofLy6G+
that circulating myeloid cells, independent of neutrophils and
neutrophils in the lung as previously reported [71]. Highly
tissue-resident macrophages, play an essential role in antifungal
concentrated, purified 1A8 antibodies were isolated from ascites
defense [64,65,66]. The ablation of circulating monocytes and
fluid following IACUC approved protocols (Rutgers-RWJMS).
monocyte-derived DCs as well as the partial loss of NK cells in For depletion of CCR2+ cells, CCR2-DTR mice and control
CCR2 depleter mice is similar to the quantitative defects in
CCR2-DTR negative littermates received 250ng of diphtheria
circulatingmonocytes,DCs,andNKcellsobservedinMonoMAC
toxini.p.onedaypriortoinfectionandeveryotherdaythereafter
patients and in both instances, hosts are vulnerable to invasive
in order to maintain depletion. Diphtheria Toxin was purchased
fungaldisease(thisworkand[21]).Althoughneutropeniahaslong
from List Biological Laboratories (Campbell, CA), and reconsti-
beenconsideredthemostimportantriskfactorforIAdevelopment
tuted at 1 mg/ml in PBS. Aliquots were stored in 280uC. The
inpatientswithhematologicmalignanciesandinallogeneicHCT
specificityandefficiencyofdepletioninthelungwasconfirmedby
patients, there is clinical evidence that monocytopenia represents
flowcytometric analysis.
anadditionalriskfactorforIAdevelopment[67,68].Inaggregate,
these linesof evidencesuggest that theimportance of CCR2+Mo
Lung cell isolation and flow cytometry
inantifungaldefenseislikelynotexclusivetomurinemodelsofIA,
Lung samples were minced in PBS with 3mg/ml collagenase
but reflective of a conserved essential function of these cells in
typeIV(Worthington),andwereincubatedat37uCfor45minto
antifungal defense.
obtain single cells suspensions. After digestion, lung suspensions
underwent RBC lysis. All antibodies were purchased from BD
Materials and Methods
Biosciences. The staining protocols included combinations of the
Mice following antibodies: Gr-1 (RB6-8C5 FITC), Ly6C (AL-21 PE),
Ly6G(1A8APC),CD11b(M1/70,PerCPCy5.5),CD11c(N418
TheCCR2depleter(CCR2-DTR)andCCR2reporter(CCR2-
Pacific Blue), MHC Class II I-A/I-E (M5/11.415.2, Alexa Fluor
GFP) strains were generated on the C57BL/6 background as
previously described [38,69]. Control animals for CCR2+Mo- 700),andCD45(30-F11APC-Cy7).Sampleswerecollectedzona
BDLSRIIFlow Cytometerand analyzedusing FlowJosoftware.
depletionexperiments weresexandage-matched,non-transgenic
littermates. For antibody depletion experiments, sex and age-
matched C57BL/6 mice were purchased from Jackson Labora- Analysis of cytokines and RNA expression in lung tissue
tories. RAG2/2cC2/2 (RAG-22/2IL2rg2/2) lymphopenic mice Total RNA from lungs was extracted with Trizol (Invitrogen).
were purchased from Taconic. All strains were maintained and Relative mRNA levels were determined by qRT-PCR. One
bred in the Rutgers-NJMS Cancer Center Research Animal microgram of total RNA was reverse transcribed using High
Facility or in the Fred Hutchinson Cancer Research Center Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
Animal Health Resources Facility under specific pathogen-free Taq Man Fast Universal PCR Master Mix (26) No Amp and
conditions.Mixedbonemarrowchimericmiceweregeneratedas TaqMan probes (Applied Biosystems) for each gene were used,
described in (Jhingran et al., 2012) [27] by transferring an equal andnormalizedtoGAPDH.Geneexpressionwascalculatedusing
mixture of CD45.1+ p47phox(+/+) and CD45.2+ p47phox(2/2) DDCT method relative to na¨ıve sample. For cytokine and
bone marrow cells using lethally irradiated CD45.1+CD45.2+ chemokinemeasurementsweperformedELISAsonlunghomog-
recipients. Recipient mice were rested for 6 weeks prior to enates according to the manufacturer’s instructions. Mouse
experimental infection. Animal studies were performed following CXCL1 and CXCL2 ELISA kits were purchased from R&D
biosafety level 2 (BSL-2) protocols approved by the Institutional systems. IL-12p70 and TNF ELISAs were obtained from BD
AnimalCareandUseCommittee(IACUC)ofRutgersUniversity Bioscience.
and ofFredHutchinson Cancer ResearchCenter.
Cell Sorting, RNA sequencing and analysis
Infection, culture, and histology CCR2GFP+CD45+CD11b+NK1.12CD11c2 (CCR2+Mo) and
For these studies, we employed an Aspergillus fumigatus-DsRed CCR2+CD45+CD11b+NK1.12CD11c+ (Mo-DC) populations
expressing strain (Af293.1RFP) [70], a generous gift from were isolated to more than 97% purity using a BD FACS ARIA
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