nomenclature

ImmunityPerspective

MacrophageActivationandPolarization:

NomenclatureandExperimentalGuidelines

,1,*

,,,,5SergijGoerdt,6

SiamonGordon,on,iv,9TobyLawrence,10MassimoLocati,11AlbertoMantovani,11

ez,12Jean-LouisMege,,14GioacchinoNatoli,,16

ze,17KariAnnShirey,18AntonioSica,19,20JillSuttles,21IrinaUdalova,derachter,23,24

,25

1DepartmentsofInfectiousDiasandImmunology,ildren’sRearchHospital,Memphis,TN38105,USA

2CentreforImmunity,Infection,andEvolution,SchoolofBiologicalSciences,UniversityofEdinburgh,EdinburghEH93JR,UK

3SingaporeImmunologyNetwork,A

*

STAR,8ABiomedicalGrove,ImmunosBuilding,Level4,Singapore138648,Singapore

4CenterforthePreventionofCardiovascularDia,NewYorkUniversitySchoolofMedicine,Smilow7,522FirstAvenue,NewYork,

NY,USA

5DivisionofMedicine,RayneInstitute,UniversityCollegeLondon,5UniversityStreet,LondonWC16JJ,UK

6DepartmentDermatology,UniversityMedicalCenterMannheim,UniversityofHeidelberg,68167Mannheim,Germany

7SirWilliamDunnSchoolofPathology,UniversityofOxford,Headington,Oxford,OX13RE,UK

8DepartmentofMedicine,UniversityofMelbourneandRoyalMelbourneHospital,Parkville,VIC3050,Australia

9HospitalforSpecialSurgeryandWeillMedicalCollege,CornellUniversity,535East70thStreet,NewYork,NY10021,USA

10Centred’ImmunologiedeMarille-Luminy,13009Marille,France

11UniversityofMilanSchoolofMedicine,IstitutoClinicoHumanitas,ViaManzoni56,20089Rozzano,Milan,Italy

12BotnarRearchCentre,NuffieldDepartmentofOrthopaedics,Rheumatology,andMusculoskeletalSciences,UniversityofOxford,

Headington,OxfordOX37LD,UK

13InfectiousDias,AixMarilleUniversity,27BoulevardJeanMoulin,13285Marille,France

14DepartmentofCellBiology,UniversityofMaryland,CollegePark,MD20742,USA

15DepartmentofExperimentalOncology,EuropeanInstituteofOncology,ViaAdamello16,20146Milan,Italy

16DepartmentofBiology,MassachuttsInstituteofTechnology,Cambridge,MA02139,USA

17Genomics&Immunoregulation,LIMES-Institute,UniversityofBonn,32115Bonn,Germany

18DepartmentofMicrobiologyandImmunology,UniversityofMarylandSchoolofMedicine,Baltimore,MD21201,USA

19IstitutoClinicoHumanitas,ViaManzoni56,20089Rozzano,Milan,Italy

20DepartmentofPharmaceuticalSciences,Universita

`

degliStudidelPiemonteOrientale‘‘AmedeoAvogadro,’’

ViaBovio6,28100Novara,Italy

21Microbiology&Immunology,UniversityofLouisvilleSchoolofMedicine,319AbrahamFlexnerWay,Louisville,KY40292,USA

22KennedyInstituteofRheumatology,UniversityofOxford,Headington,Oxford,OX37FY,UK

23LaboratoryofCellularandMolecularImmunology,VrijeUniversiteitBrusl,Pleinlaan2,1050Brusls,Belgium

24LaboratoryofMyeloidCellImmunology,VIB,Pleinlaan2,1050Brusls,Belgium

25LaboratoryofParasiticDias,NationalInstituteofAllergyandInfectiousDias,NationalInstitutesofHealth,Bethesda,

MD20892,USA

*Correspondence:@

/10.1016/.2014.06.008

DescebiblicalTowerof

Babel,macrophakof

connsusonhowtodefinemacrophageactivationinexperimentsinvitroandinvivoimpedesprogress

inmultipleways,includingthefactthatmanyrearchersstillconsidertheretobeonlytwotypesofactivated

macrophages,,wedescribeatofstandardncompassingthreeprinci-

ples—thesourceofmacrophages,definitionoftheactivators,andaconnsuscollectionofmarkersto

describemacrophageactivation—withthegoalofunifyingexperimentalstandardsfordiverexperimental

tively,wepropoacommonframeworkformacrophage-activationnomenclature.

Overview

Activationofmacrophageshamergedasakeyareaofimmu-

nology,tissuehomeostasis,diapathogenesis,andresolving

andnonresolvinginflammation(BiswasandMantovani,2010;

GordonandMartinez,2010;LawrenceandNatoli,2011;Manto-

vanietal.,2008;Mantovanietal.,2005;Martinezetal.,2008;

MurrayandWynn,2011b;NathanandDing,2010;Wynnetal.,

2013).Overthelastveralyears,divertermshavebeen

appliedtomacrophageactivationand‘‘polarization,’’wherea

stimulussuchasacytokineortoll-likereceptor(TLR)agonistpro-

,we

utheterm‘‘activation’’tomeantheperturbationofmacro-

phageswithexogenousagentsinthesameveinasmanyu

‘‘polarization.’’Wealsonotetheabilityofmacrophagesto

changetheiractivationstatesinrespontogrowthfactors

(e.g.,CSF-1andGM-CSF)andexternalcues,suchascytokines,

microbes,microbialproducts,andothermodulators,including

nucleotidederivatives,antibody-Fcreceptorstimulation,gluco-

corticoids,infection,phagocytosis,andpotentiallyanyotheren-

e

macrophageactivationisinvolvedintheoutcomeofmanydis-

eas,includingmetabolicdias,allergicdisorders(suchas

14Immunity41,July17,2014ª2014ElvierInc.

airwayhyperreactivity),autoimmunedias,cancer,andbac-

terial,parasitic,fungal,andviralinfections,weneedtoestablisha

commonlanguagefordescribingthepropertiesofthemacro-

phagesunderinvestigation.

BackgroundtotheProblem

Wenotewidespreaduofatleastfourdefinitionsofmacro-

phageactivation,includingtermssuchasM1andM2,alternative

andclassicalactivation,‘‘regulatory’’macrophages,andsubdi-

ginsofthe

termsoriginatedintheearly1990swhendifferentialeffectsof

interleukin-4(IL-4)incomparisontothoofinterferong(IFN-

g)and/orlipopolysaccharide(LPS)onmacrophagegeneexpres-

sionweredescribed(MartinezandGordon,2014;Steinetal.,

1992).ComparedtoIFN-g,IL-4wasdescribedtoinduce‘‘alter-

nativeactivation.’’Itshouldbenotedthattheterm‘‘classical’’

activation,whichoriginallyreferredtomacrophagesstimulated

withIFN-g,isnowinterchangeablyudwithIFN-gandTLR

stimulation(MartinezandGordon,2014).Theconddefinition

cameveralyearslaterwhenMillspropodtheM1-M2

terminology(Milltal.,2000).Mills’sideaoriginatedfromthe

differentialmetabolismofargininebetweenmacrophagesfrom

C57BL/6andmacrophagesfromBalb/cmice,aneffecthecorre-

latedwithdifferencesbetweenThelper1(Th1)andTh2cellre-

ndcolleagueswentfurther

andpropodthattheM1-M2dichotomywasanintrinsicprop-

ertyofmacrophagesassociatedwithtransitionsfrominflam-

mationtohealing,wouldoccurintheabnceofanadaptive

immunerespon,andaroearlyinevolution(Mills,2012).

Severallinesofevidencesuggestthatthistheoryrequires

,C57BL/6micebearadeletioninthepromoter

ofSlc7a2,encodingthekeyargininetransporterinmacro-

phages,causinglargedifferencesinarginineutilizationbetween

C57BL/6andBALB/neticdifferencebetween

thestrainswasnotknownatthetimethatMills’shypothesis

waspublishedandwasthereforenottakenintoaccount

(Sans-Fontal.,2013).Second,althoughMills’snotionon

‘‘innate’’shiftsinmacrophageactivationmightbetrue,most

immunologistsareconcernedwithimmunityintheprenceof

lymphocytes,whichprofoundlyaffecttheactivationstateof

,nomolecular

definitionhasyetaccountedforan‘‘innate’’M1-to-M2transition,

althoughnewinformationfromepigeneticsandmetabolism

(ebelow)mightprovideameansofdisctingintrinsicmacro-

phageactivationstates.

Thethirdtofnomenclature(M2a,M2b,etc.)expandedthe

M1-M2definitionstoaccountfordifferentactivationscenarios

andwasbalancedbytheideathatactivationexistsonaspec-

trumandcannotbeeasilybinnedintodefinedgroups(Biswas

andMantovani,2010;Edwardtal.,2006;Mantovanietal.,

2005;MartinezandGordon,2014;Stoutetal.,2005;Stoutand

Suttles,2004).Thefourthdefinitionreferstomacrophages

growninGM-CSF-1asM1andmacrophagesgrowninCSF-1

asM2(Joshietal.,2014).Notably,significantdifferenceshave

beendocumentedinthetranscriptomesofmacrophagepopu-

lationsprimarilygeneratedwiththeuofCSF-1orGM-CSF,

withandwithoutexogenousperturbation(Fleetwoodetal.,

2009),butthereisnocompellingevidencetoassignCSF-1-or

GM-CSF-derivedmacrophagesasM1orM2.

Thediversityofterminologyandinconsistentuofmarkers

todescribemacrophageactivationimpedesrearchinveral

,rearchernteringthefieldencounterconfusion

aboutwhichtermstouandwhichmarkersarereprentative

oftheirexperimentalorhuman-badsystem;manyrearchers

mighterroneouslyconsidertheretobeonly‘‘twotypesofmac-

rophages.’’Second,establishedrearchersinthefieldhaveyet

toagreeonnomenclatureorstandardsfordescribingactivation.

Third,grantandmanuscriptwritersandtheirreviewers,funding

andregulatoryagencies,andjournaleditorscanbeexasper-

,thelackof

experimentalstandardsimpedesstudieswherecomparisons

arerequired(e.g.,microarrayandproteomicdatats).Fifth,

deploymentoftherapeuticmacrophagemodulatorsrequires

thatstandardsbetranslatableacrossdisciplinessothatphar-

maceuticalandregulatorybodiescandrawmeaningfulcompar-

isonsintermsofdiagnosticoreffiandfinal

issueisthediversityinmacrophageactivationacrossspecies

(discusdbrieflybelow).

Toaddresstheobstaclesandpitfallsindescribingmacro-

phageactivationandinachievingexperimentalstandards,a

smallgroupofmacrophagebiologistsmetinformallyattheInter-

discusdtheissuessurroundingterminologyandtouttopro-

videaninitialtofnomenclatureandexperimentalguidelines.A

draftletterwasthencirculatedtoabroadergroupofrearchers

perspective,wedonotattempttocap-

tureeveryonewhohaspublishedonmacrophageactivationand

polarization;rather,weaimtoattainconnsusabouttheprob-

lemswithinthefi,discus-

sionandrevisionwillbeesntialforrefiningthepropertiesand

mechanismsofmacrophagepolarization.

Recommendations

AReproducibleExperimentalStandard

Weconcludedthatastartingpointwastoframeanomencla-

turesystemwithinareproducibleinvitroexperimentalstandard.

CSF-1-culturedmacrophagesfrommurinebonemarrowand

humanperipheral-bloodmonocytesremainthepredominant

invitrosystemsudforgeneratingmacrophagesandtherefore

willbeudasreferences(Figure1A).Othercommonlyud

macrophagesourcesareperitonealmacrophages(residentor

elicited)frommiceandGM-CSF-culturedmacrophagesfrom

murinebonemarrow(Figure1A),andeachofthecanbeper-

turbedtogenerateactivatedpopulationsofmacrophageswith

gene-expression

profilesoverlappingthoofCSF-1-generated

basis,thecultureconditionsforgeneratingthe

twoparadigmaticinvitroM1andM2populationsarestraightfor-

ward,i.e.,-4

andIFN-goftenexertclear-cutantagonisticeffectsonmacro-

phagepolarizationmediatedbySTAT6andSTAT1,respectively.

Furthermore,IL-4andIFN-ginducedefinedandcomprehen-

sivelyinvestigatedmacrophagesubpopulations(Lawrenceand

Natoli,2011;Mills,2012;Rutschmanetal.,2001;Tauband

Cox,1995;Wynnetal.,2013).

RecommendationforMinimalReportingStandards

Incompletedescriptionsofhowmacrophagesareisolated,stim-

ulated,andanalyzedarecontrarytothevalueofreplicationand

end,macrophages

Immunity41,July17,2014ª2014ElvierInc.15

ImmunityPerspective

A

B

C

(legendonnextpage)

16Immunity41,July17,2014ª2014ElvierInc.

ImmunityPerspective

isolatedfrominvitroandinvivosystemsrequire,ataminimum,

estan-

dardsasaguide,invitroexperimentsfromdifferentlabora-

y,wefavortheuof

purifiedendotoxin-freerecombinantCSF-1ratherthanL-cell-

conditionedmediumasthesourceofCSF-1togenerate

bone-marrow-derivedmacrophagesbecauthelatterisnot

readilydefimple,

L-cell-conditionedmediumcontainsvariableamountsoftypeI

interferonsthatcouldcauconfoundingeffectsinsubquent

activationexperiments(WarrenandVogel,1985).

DefinetheActivator

Ingeneral,giventhatdivermediatorshavebeenudalone

orinvariouscombinationsforthegenerationofpolarized

macrophagepopulations,wepropothatrearchersdescribe

stimulationscenariosandadoptanomenclaturelinkedtothe

activationstandards,i.e.,M(IL-4),M(Ig),M(IL-10),M(GC),

M(IFN-g),M(LPS),andsoforth(Figure1B).Suchasystemavoids

thecomplexityofM2a,M2b,etc.,whereonelaboratorymight

experimentallydefineactivationdifferentlythananother,and

allowsnewactivationconditionstobecomparedandcontrasted

1alsodepictstheconcept

ofa‘‘spectrum’’ofactivationtodenoteactivation‘‘states’’

commonlyobrved(MosrandEdwards,2008;Stoutetal.,

2005;StoutandSuttles,2004;Xueetal.,2014).Theemployment

ofthespectrumconceptisufulwhereambiguityexistsor

whenrearchersareoperatingoutsidetheinvitroCSF-1

ary,wenotethatstandards

needtobesimpleforadoptionbutatthesametimenotcau

ore,rearchersshould

considerharnessingtheterminologyandmarkersforCSF-1-

grownmacrophagesactivatedunderdefinedconditionsasa

referencestandard(Xueetal.,2014).Whereambiguityexists—

forexample,inamacrophagepopulationisolatedfromaninvivo

system—rearchersshouldemphasizethemarkercombina-

tionsudandstatetheclostrelative(s)alongthespectrum

showninFigure1(discusdbelow).

TermstoAvoid

Wepropothattheterm‘‘regulatory’’macrophagesshouldbe

avoidedbecauallmacrophagesareregulatoryinsomecapac-

ofmacrophagesderivedfrommicewithspecific

targetedmutationsthatpreventdevelopmentofanM(IL-4)pro-

file(e.g.,throughtheuofIL-4Ra-orSTAT6-deficientmacro-

phages)isrecommendedtoconfirmaspecificphenotype.

SomerearchersoftenascribethesubtterminologyM1

andM2toGM-CSF-andCSF-1-generatedmacrophages,

respectively;

CSF-1orGM-CSFisudforgeneratingactivatedmacrophage

populations,ercomplication

isthatGM-CSFculturescontainsubstantialnumbersofCD11c+

cellswithdistinctantigen-prentingactivitiesthatneedtobe

accountedforingeneprofilingorfunctionalanalys.

MarkersofActivation

CD4definesCD4+CD4+cells,Foxp3defines

rejusttwoexamplesofmarkers

defirast,macrophageactivationis

orkforDescribingActivatedMacrophages

(A)-1-grownmouadherentmacrophagesfrombonemarrow(BM)orCD14+monocytesareudas

thehagescanalsobegeneratedwithGM-CSF,whereaCD11c+dendriticcell

(DC),thioglycollateinjectionfollowedbyperitoneallavagesisudforgenerating

macrophagepopulationswithdifferingyieldsandproperties,whereasmanyorgansystemsinmiceandhumansaresourcesoftissue-infiltratingmacrophages.

(B)refunctionalsubdivisionsaccordingtostimulationofmouCSF-1macrophagesorhumanmonocyte-

derivedCSF-1macrophageswiththeexistingM1-M2spectrumconcept(MartinezandGordon,2014;MosrandEdwards,2008;StoutandSuttles,2004).

StimulationconditionsareIL-4,immunecomplexes(Ic),IL-10,glucocorticoids(GC)withtransforminggrowthfactorb(TGF-b),glucocorticoidsalone,LPS,LPS

andIFN-g,dataweredrawnfromawiderangeofpublishedandunpublisheddatafromtheauthors’laboratoriesandreprentastarting

connsus(Edwardtal.,2006;Fleetwoodetal.,2009;Gratchevetal.,2008;Gundraetal.,2014;Krausgruberetal.,2011;Langetal.,2002;Shireyetal.,2008;

Shireyetal.,2014;Shireyetal.,2010;Xueetal.,2014).AnasteriskindicatescorroborationofhumanIL-4genesbydeepquencing(.N.V.,datanot

shown).

(C)onsinAkt1andKlf4caua‘‘switch’’toM(LPS)-andM(IFN-g)-associatedgeneexpression,

onsinStat6,Ppard,Pparg,andIrf4andIRF5depletionareinvolvedinthemaintenance

and/oramplitudeofactivation.

ingStandardsforInVitroExperiments

ParameterNotes

Moustrainhowthebonemarrowisisolatedandprocesd

Startingcellnumber,media,andsupplementsmedia(DMEMversusRPMI)havesubstantialeffectsofgrowthrate,development,

andactivationstatus

Tissue-cultureconditionsdifferenttypesofplasticaffectmacrophagegrowthandactivation;tissue-culture

conditionsshouldbedocumentedforreproducibility

Timeofculturethepreciconditionsudandwhethercytokinesand/ormediaaresupplemented

duringthecultureperiod

SourceandconcentrationofdifferentiationcytokinesthesourceandconcentrationofCSF-1

Macrophageyieldtheyieldrelativetothestartingnumbershouldberecorded

Activationconditionsvariablesincludewhethermacrophagesarerestedpriortoactivationandhow,

whetherCSF-1isprentintheactivationcultures,thesourceandconcentrations

oftheactivatingagents,andthetimetoassay

Processingandanalysishowthecellsareprocesdandwhatmarkerreadoutsareud

Immunity41,July17,2014ª2014ElvierInc.17

ImmunityPerspective

associatedwithsubstantialshiftsingeneexpression(hundreds

ofgenes)dependingonthespecificstimuli,butnonedefine

rearcheroutsidethemacrophagesphere,markeruprob-

ablyappearsconfusingbecauimmunologistsareaccustomed

pleofproblematic

markeruixpressionofArgina-1(Arg1)asa‘‘marker’’

forM2orM(IL-4)spectrummacrophages,whichhasledtointer-

pretiveproblemsbecauArg1isalsoinducedinM1spectrum

macrophages,expresdinsomeresidentmacrophagepopu-

lations,andhighlyinducedinmycobacteria-infectedmacro-

phages,furtheremphasizingtheneedforcriteriaencompassing

multiplemarkers(ElKasmietal.,2008).Accordingly,wefavor

anapproachusingcombinationsofmarkers(oralackof

markerexpression)toascribeactivationoutcomesasoutlined

y,thereissignificantscopetoexpandupon

markerassignmentsuchastranscriptionfactorandcell-surface

markercombinationswithinthestandardizedexperimental

frameworkpropodhere,andthisshouldrveasastarting

cartographyforthefield.

TranslationtoInVivoExperiments

Whenisolatingmacrophagesfromtissueandanalyzingtheir

activationstate,eachlaboratorywillconfrontafamiliarproblem:

whatdowecallthem?Whatiftherearedifferentpopulations

prent?Ourrecommendationistoacquiresufficientevidence

toplaceagivenpopulationwithintheframeworkshownin

sunlikelythataparticularinvivoscenariowill

r,asmore

macrophagepopulationsaredisctedexvivo,moreinforma-

tionwillaccumulatetohelpusunderstandthegeneralandspe-

cificnatureofinvivomacrophageactivation.

ExVivoCharacterizationofMacrophageActivation

Eachlaboratoryhasindividualizedmacrophageisolationproce-

eofthebreadthofconditionsud,wefavor

describingindetailhowmacrophagesareisolated,whichtissue

andpathologicalorhomeostaticconditiontheyarefrom,and

whichmarkercombinationsareudforascertainingmacro-

horsstresstheneedforrapidisolation

techniquestoprervetheunderlyingphenotypequicklyand

esintechnologiesfor

insitugeneexpressionwithinindividualtissuesandcellswill

mostlikelyadvancetheunderstandingofspatialmacrophage

lessofthetechnologyemployed,combina-

tionsofmarkersneedtobeappliedtothepopulationsbeing

analyzed,andafulldescriptionoftheisolationtechniquesneeds

mple,theImmgenConsortiumhasa

mandateforisolationandsortingconditionsforimmunecells,

andwefavortheirdegreeofdescriptiverigorforexvivomacro-

phages(Gautieretal.,2012).Anothercomplicationfromexvivo

analysisofmacrophageactivationisplasticityacrossdifferent

mple,inobesityrearch,macrophages

residinginadipotissuearethoughttobecomemoreproinflam-

matoryasfataccumulatesandthusfalltowardtheM1endofthe

activationspectrum(Wynnetal.,2013).Inatherosclerosis,

resolutionoflesionsisassociatedwiththerever:macrophage

populationsontheM1spectrumconverttotheM2partofthe

spectrumwithoutevidenceoflocalSTAT6activationbyIL-4or

IL-13(Mooreetal.,2013).Onesolutiontotheproblemof

describingmacrophageactivationinscenariosinvivoistobegin

withanexplicitdescriptionofthepopulationsunderinvestigation

andhowtheywereisolated(asImmgendefines,forexample).

Markerscanthenbeudtoreflecttheperturbationstheyhave

mple,Arg1hiRetnlahipSTAT6+pSTAT1

À

couldbeudtoenhancethedescriptionofaspecificlung

macrophagepopulationisolatedfromaTh2-cell-type-driven

diaandthusbereasonablyrelatedtotheM(IL-4)cells

(Figure1B).Reportingthetimepointsofexvivomacrophage

isolationandanalysisarethereforemandatoryinthedescription

oftissue-anddia-associatedmacrophagepopulations.

TranslationtoHumanMacrophages

Howcanwedefineandcategorizeactivatedhumanmacro-

phages?Thisquestioncontinuestoconfoundrearchersin

partbecauhumanmacrophagesaregenerallyisolatedfrom

bloodmonocytesasoppodtobonemarrowortissues

stinctionisparticularly

importantwiththenewknowledgethatmanytissue-resident

populationsarenotofbonemarroworigin(SiewekeandAllen,

2013).Manyofthemarkersudformurinemacrophages

blereasons

forthediscrepancieshavebeendiscusd(Murrayand

Wynn,2011a),butitisworthemphasizingthatnostudyhas

systematicallycomparedtheresponsofblood-monocyte-

derivedmacrophagesfrommiceandhumansinaside-by-side

ctarangeofinterspeciesvariabilityonmacro-

phageactivationtoreflectdifferentevolutionaryoutcomes

sculptedbydifferentpathogens,diets,longevity,e

thevariablesinvolved,experimentalrigorcanbeudinthe

archforinformationabouthuman(andanyotherspecies)

macrophagebiologyaccordingtotheprinciplesandpractices

ly,systematicstudieshavebegunto

exploretheconrvationbetweenmacrophagesfromdifferent

species,includingswine,wherelargenumbersofdifferenttissue

macrophagescanbeisolated(Fairbairnetal.,2011;Martinez

etal.,2013;Schroderetal.,2012;Xueetal.,2014).Therefore,

rearchersshoulddescribehowtheygeneratetheirmacro-

croarray,

deepquencing,andproteomicstudiesarecombinedtointer-

rogatehumanmacrophages,aconnsuswillemergeabout

whichpathwaysofhumanmacrophageactivationareamenable

tonewdrugdiscovery.

GeneticstoAlterActivationStates

Recentworkhasidentifiedgeneticmodificationsproducing

mple,deletionoftran-

scriptionfactorIRF4orKLF6failstomakeM(IL-4)macrophages,

whereasPPARgandPPARvarerequiredfortheamplitudeofthe

M(IL-4)state(Chawla,2010;Dateetal.,2014;Ivashkiv,2013).

Ablationofproteinsinvolvedinanabolicgrowth,suchAKT2

andPTEN,enhancesanactivationstatewheregeneexpression

islinkedtoM(IL-4)macrophages,whereasdeletionofTSC1,an

inhibitorofmTOR,caustheoppositeeffect(Arranzetal.,2012;

Byletal.,2013;Yueetal.,2014).OthermutationsinthemTOR

r,usingthe

principlesdescribedhereforsystematicinvestigationofmTOR

pathwaymutantswillmostlikelyresolvewhyrapamycin-treated

macrophagesandmacrophagesfromRaptor,Rictor,andTSC1

mutantshavediverphenotypes(Aietal.,2014;Byletal.,

2013;Festucciaetal.,2014;Weichhartetal.,2008).Someof

18Immunity41,July17,2014ª2014ElvierInc.

ImmunityPerspective

endthat

theandrelatedmutantswillbeincreasinglyufulfordefining

y,itisimportanttorecognizetheeffectof

lparameterscan

affectactivationstateacrosstime;theinclude(1)removalof

thestimulus,(2)enforcementoffeedbackandfeed-forward

signalingloops,includingautocrineproductionofcytokines,

and(3)epigeneticand/ordevelopmentaleffectsbuiltintothe

lifehistoryofamacrophage(Ivashkiv,2013;Lawrenceand

Natoli,2011;Portaetal.,2009).ThiswouldgobacktoMills’s

notionofanactivated-to-healingtransition.

PerspectivesandConclusions

Understandingmacrophagebehaviorisakeystoneofdecipher-

raightforwardtoisolateand

propagatemacrophages,

contrast,nomenclatureandstandardizationissuesarestunting

progressbecaualinguafrancahasyettobeestablished

ourattemptsareastartingpointto

asizethatour

goalistoinitiatedialogratherthanactasarbitersoflanguage

gso,wehopescientistsnewto

macrophagebiology,establishedrearchers,pharmaceutical

companies,andregulatoryagenciescanappreciatethehistory

ofourfieldandtheneedforacommonframeworkopento

frequentrevision.

ACKNOWLEDGMENTS

ThisworkwassupportedbyNIHgrantsAI062921(P.J.M.),AI080621(J.P.S.),

HL084312(E.A.F.),andAI18797(.N.V.),Alex’sLemonadeStand

Foundation(P.J.M.),theHartwellFoundation(P.J.M.),CancerCenterCore

grantP30CA21765(P.J.M.),theAmericanLebaneSyrianAssociatedChar-

ities(P.J.M.),andtheNIHIntramuralProgram(T.A.W.).

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