Neutrophils Aging: A Temporal Dimensionfor Review

Series: Neutrophils in Action

Review

Aging: A Temporal Dimension for Neutrophils

José M. Adrover,

José A. Nicolás-Ávila,

and Andrés Hidalgo

*

Neutrophils are first-responders, providing early protection against invading pathogens.Recentfindings haverevealedatemporaldimensiontoneutrophil function,associatedwiththeclearancecyclesforagingneutrophils,andalso withaprogramthatendowscirculatingneutrophilswithdistinctphenotypicand functionalpropertiesatdifferenttimesoftheday,beforetheyareclearedfrom blood. We review here the process of neutrophil aging and its impact on homeostasis and inflammation. We outline the features of aged neutrophils, examineproposedmechanismsthatdriveaging,anddiscusshowthese pro- cessesmaycontributetotissuehomeostasisandpathology.Inthiscontextwe proposethat neutrophilagingmayoptimizehostdefensebyallowingneutro- philstoanticipate infectionswhile avoiding permanentactivationandsubse- quentdamage.

Introduction

Neutrophilsareinnateimmunecellsspecializedinearlydefenseagainstpathogens[1].Within theperspectiveofthisspecialization,thephysiologyofneutrophilsmaybebestunderstoodin thecontextoftheexposureofananimaltoinfectiousmicroorganisms.Giventhegeophysical cyclesofourplanet,however,thelikelihoodofexposureisnotconstant,butdisplaysdiurnal rhythms[2].Inthiscontext,timebecomesanimportantparameterbecauseneutrophilsseekto synchronizetheiractivitywiththatofinvadingpathogens.Thetemporalregulationoftheiractivity isoffurtherclinicalimportancebecauseneutrophilsnotonlyeliminatepathogensbutcanalso damagesurroundingtissuesduringinfectiousorsterileinflammation[3].Thiscollateraleffectof neutrophilactivationunderlieshighlyprevalentformsofinflammatorydisease[4,5],whichare knowntodisplaydaily(circadian)oscillations.Epidemiologicalandexperimentalstudieshave demonstratedthatboththenumberofneutrophilsinthecirculationandthetimeofday(early morninghours)stronglycorrelatewiththeincidenceofcardiovascularevents[5,6].Temporal adaptationofneutrophilsforprotectionagainstmicroorganisms couldthereforeexplainwhy inflammatoryeventsarenotconstantovertime.

Theseconceptsimplythatan importantapproachtounderstanding diseaseliesindefining whetherandhowtheimmunesystemadaptstochangesintheenvironmentthatmaycompro- misehealth.Onewayisthroughregulationofthenumberofleukocyteswithinatissue,andthis hasbeenshown to relyonmultiple typesoftissue-residentcells[7,8].A differentbutless- understoodmechanismentailsthemodulationofleukocytefunctionovertime,aprocessthat appearstoinsteadrelyontheuseofcell-intrinsictranscriptionalprograms[9,10].Thestudyof thistypeofqualitativespecializationhasbeenlargelyneglectedforneutrophils,partlybecauseit appearstodemandanextendedcellularlifespanandtranscriptionalplasticitythatneutrophils were believed to lack. This view is now changing because recent studies suggest that

Trends

Acuteinflammatorysyndromes,aswell asothertypesofdisease,showcirca- dianpatternsofmanifestationthatpar- allelchangesinthenumberofcirculating leukocytes.

Neutrophils are the most abundant myeloidcellsinblood,andtheirnum- bersfollowcircadianpatternsofrelease andclearance.

Neutrophilsundergophenotypicchan- gesfromthetimetheyarereleasedinto blood(freshneutrophils)tothetimethey disappear from the circulation (aged neutrophils).Thisphenotypicdrift,which occurswithinasingleday,isreferredto asaging.

Freshandagedneutrophilsshowdis- tinct properties in the production of reactiveoxygenspecies(ROS),deform- ability,migration,and NETformation, suggestingashiftininflammatoryprop- ertiesovertime.

Becauseagedandactivatedneutrophils share phenotypicsimilaritiesbutonly agingisregulatedinacircadianfashion, metabolitesorcytokineswhoselevels followdiurnalchanges(e.g.,thosepro- duced by the microbiota)may drive aging.

1AreaofCellandDevelopmental Biology,FundaciónCentroNacional deInvestigacionesCardiovasculares (CNIC),Madrid28029,Spain

*Correspondence:[email protected] (A.Hidalgo).

neutrophilspossessamoreactivetranscriptionalprogram[11]andextendedlife[12]thanwas previouslythought,andthattheydisplaymultipleflavorsofphenotypesandfunctions[1,13].In addition,therecentdescriptionofanatural driftinthephenotypeandfunctionofcirculating neutrophilsoverthecourseofoneday(i.e.,diurnal)[14],aphenomenonreferredtoasaging, suggests that these cells can undergo qualitative alterations and adapt to the changing environment.

Wereviewhereexperimentalevidencesupportingthephenomenonofaging,themechanisms thatdriveit, andthefunctionalpropertiesthatagingimprints onneutrophils.From theper- spectivesofpathogen-drivenspecialization, optimizationofimmunedefense,andfluctuating susceptibilitytoinflammatorydisease,theconceptofagingmayprovideausefulframeworkto betterunderstandtheexpandingrepertoireoffunctionscarriedoutbyneutrophils.

NeutrophilAging

Theestimatedhalf-lifeofneutrophilsinthebloodstreamis6–12hformiceandhumans[15],and isfollowedbyrapiddisappearance,orclearance,fromblood.The shortlifeinthecirculation demandsconstantproductionandreleaseofneutrophilsfromthebonemarrow,estimatedto beintherangeof10¹⁰cellsperday[16].Therelease–clearancedynamicinturndemandsafine balancebetweenproductionandeliminationthatisregulatedatdifferentstagesinthelifeofthe neutrophil:differentiationfrommyeloidprogenitorsandstorageinthebonemarrow,releaseinto bloodandintravascularmargination(theretentionofdiscretepoolsofneutrophilsbyincreased transit times within the microvasculature of particular tissues), clearance from blood, and eliminationthroughphagocytosisinspecializedorgans[16,17].Fromateleologicalperspective, thecomplexregulationofneutrophilnumbersinthesteady-stateislikelydictatedbythebalance betweenthepermanentneedtoeliminatepathogensthatcanbreachmucosalsurfacesandthe potentialriskthatthepresenceofthesereactivecellsposestothevasculature[3].Toofewortoo manyneutrophilscanthereforecompromise thehealthoftheorganism.Undertheopposing physiologicalpressuresofdefenseversusinjury,evolutionhasdevelopedasystemwhereinthe numberofneutrophilschangesduringtheday,resultinginoscillationsthatfollowacircadian pattern(Figure1A).Thissystemmaybeoptimizedsuchthatelevationsinneutrophilnumbersare timedtomatchthedailyhikeinexposuretomicroorganisms[2].

Onemechanismbywhich neutrophilnumbersinbloodarecontrolled isthroughtemporally regulatedexpressionofadhesionmoleculesbytheendothelialcellsthatlinethevasculature[18].

Regulatedadhesivenessofthevascularwall,whichisdictatedbylong-rangesignalsdelivered bythesympatheticnervoussystem,allowsleukocytestobindtotheendotheliumandleavethe circulation[18].Thiselegantmodel,however,doesnotexplainadditionaltemporalalterationsin quality(i.e., beyond cell numbers) recently described incirculating neutrophils: analyses of neutrophilsinthebloodofhealthymiceevery4hoverthecourseofafulldayrevealedchangesin phenotype and morphology [14]. Starting from neutrophils freshly released from the bone marrow,thesequalitativechanges(describedindetailbelow)accumulatedovertimetoreach apeakshortlybeforethebulkofneutrophilsbegantodisappearfromblood.Therefore,although changesin cell number andphenotype coincide during the day, they likelyoccur through differentmechanisms.Weusetheterm‘aging’tospecificallydescribethephenotypicchanges undergonebyneutrophilsfromthetimetheyarereleasedintobloodtotheirdisappearancefrom thecirculationintheabsenceofinflammation.

Featuresof ExVivoAging

Thephenotypeofneutrophilschangesovertime(Figure1B).Studies overthepast decade focusedprimarilyontheidentificationofchangesthattakeplaceinneutrophilsmaintainedex vivoincultureforseveralhours.AprominentexampleistheincreaseinsurfaceCXCR4,the receptorforthechemokineCXCL12[19].Inthebonemarrow,CXCL12servesasaretention

signalforneutrophilsthatenhancesbindingtoVCAM-1onendothelialandstromalcells,and antagonizesCXCR2-mediatedsignalsthatpromotereleasefromthebonemarrow[8];conse- quently,CXCR4downregulationduringmedullarymaturationisbelievedtofacilitateneutrophil mobilization[20].Thespontaneous upregulationofsurfaceCXCR4 infreshlyisolated blood neutrophilswasapparent afteronly 4hinculture [21]–this wasproposed to promotethe migrationofthese‘senescent’neutrophilsbacktothebonemarrow,anorganwherethelevels ofCXCL12 areconstitutively high[19,22].Althoughthese studiesprovide an importantlink between (ex vivo) aging and clearance from blood, studies in mice with myeloid-specific deficiencyinCXCR4failedtodetectalterationsinthekineticsofclearancerelativetocontrol

Fresh Aged

(In vitro) migraon

Deformability/shape change Aged

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Microbiota–TLR signaling/internal clock?

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CD49d ICAM1 CD11c CD24 CD45 CXCR2

Ly6C/G CD62L

CD47?

Release

(A)

(B)

Clearance

Apoptosis?

ROS producon Intravascular NETosis Clearance from circulaon

Acve Resng

Total

Figure1.DynamicsandPhenotypeofAgedNeutrophils.(A)ThelevelsofCD62L^LOCXCR4^HIagedneutrophilsshow diurnaloscillations,withdistinctphasesofreleaseintoandclearancefromthebloodstream.Theirnumbersincreaseinthe circulationofmice(Release)uponegressfromthebonemarrowduringtherestingphaseofananimal,anddecrease thereafter(Clearance),reachinganadiraroundmidnightduringtheactivephaseoftheanimal.Thebulkchangesintotal neutrophilnumbersaremainlyduetotheoscillationsintheagedneutrophilpopulation,withlittlechangeinthelevelsof neutrophilsbearingaCD62L^HICXCR4^LO‘fresh’phenotype.Thesteadylevelsoffreshneutrophilsmaybeexplainedbya rapidtransitionfromthefreshtotheagedphenotypesoonafterthecellsenterthecirculation.Notethatthecurvefortotal neutrophilsisroughlyequivalenttotheadditionoffreshplusagedneutrophils[14].Theseoscillationsmayprovideapoolof protectiveneutrophilscoincidingwiththetimewhenexposuretopathogenicmicroorganismsismorelikely,whileatthe sametimeprotectingthecardiovascularsystemfromconstanthighlevelsofneutrophils.(B)Molecularandfunctional featuresofagedneutrophils.Neutrophilsthatfirstenterthecirculationfromthebonemarrow(‘fresh’)areexposedto externalcues,suchasToll-likereceptor(TLR)agonistsderivedfromthemicrobiota,thatdriveaging.Additionalsignals dictatedbyaninternalclockcouldalsoberequired.Thesesignalsoccurundersteady-stateconditionsandtriggeraging- associatedchanges,whichincludereducedcellsize,alterednuclearmorphology,andupregulationofcertainsurface markers.ThereisdiscrepancyintheliteratureregardingCD47,asurfacemoleculeknowntoprotectcellsfromphago- cytosis.Thesechangescoincidewiththeindicatedshiftintheirfunctionalproperties,butmuchremainstobecharacterized regardingthesephenotypicandfunctionaltransformations.

neutrophils[23],indicatingthatthebonemarrowmaybeonlyoneofseveralorgansofnatural clearance.OurownunpublisheddataonCXCR4-deficientneutrophilssupportsthenotionthat, whereastime-dependentupregulationofCXCR4iscrucialforneutrophilclearanceinthebone marrow,migrationtootherorgansmaycompensatefortheirremovalfromblood.

Anotherphenotypicchangeobservedinexvivoculturedneutrophilsisthedownregulationof CXCR2,thereceptorforCXCL1,apotentneutrophilchemoattractantshowntopromotethe release of neutrophils into the circulation and their migration into inflammatory sites [24].

Although downregulation ofthis receptor in the contextof in vivo aging has not yet been confirmed, this observation suggests that aged neutrophils may respond less efficiently to inflammatory signals.This is, however, incontrast with a recent report showing that aged neutrophilsarecompetentorevensuperiormediatorsofinflammation[25].Whilethereasonfor theseconflictingobservationsremainsunclear,onepossibleexplanationisthatdifferentche- motactic receptors guide the migration of each type of neutrophil to preferred tissues at particular times and in specific physiologic contexts. For example, the clearance of aged neutrophilsintissuesatthe endofthe resting phaseof ananimalis known to relyinpart onCXCR4[14,19],whereasfreshneutrophilsuseCXCR2toenterthecirculationattheendof theactivityphase[24,26].

Aremarkable aspectoftheex vivoexperiments isthatthe changesinphenotypeprecede apoptosis.After about1 day inculture a largefraction ofneutrophils enterapoptosis [27], although they preserve their cellular integrity longer than other cell types, a phenomenon proposed torelyonto the use ofglycolysisfor energyproduction[17].Anunderlyingidea promptedbythesestudiesisthatdelayeddeathwouldgiveneutrophilstimetoreachareasof phagocytosis,thusminimizingthepotentialreleaseoftheirtoxiccargo.Nonetheless,weraise cautionabouttheideathatanytypeofneutrophildeathisinitiatedwhileinthecirculationoreven asfreecellswithinnon-inflamedtissues.Thereis,toourknowledge,noexperimentalevidence supportingthiscontention,andmostformsofneutrophildeathhavebeenstudiedeitherinvitro (e.g.,apoptosisornecrosis)orduringinflammation(e.g.,NETosis,theformationofDNA-based neutrophilextracellulartraps,orNETs;reviewedin[28]).Themechanismofneutrophilapoptosis inthesteady-stateremainslargelyunknown,andmaybetheresultofchangesinthebalanceof short-livedanti-apoptoticMCL1proteinandlong-livedpro-apoptoticproteins,whereconstant denovosynthesisofMCL1isessentialforneutrophilsurvival[29].Notably,however,although deficiencyinMCL1resultsindecreasedsurvivalofneutrophilsinvivo[30],itslevelsarefoundto remainunchangedinbonafideagedneutrophilsrelativetothosefreshlyreleasedfromthebone marrow[14].Thesame wastrue forotherclassicalmarkers ofapoptosis,includingsurface exposureofphosphatidylserineandthepresenceofcleavedcaspase3[14].Theseobservations putintoquestionthepossibilitythatphysiologicalagingrepresentsanearlystageofdeath,rather thansimplybeingatransitiontoadifferentphenotypicandfunctionalstatus.Theyalsoraise cautionconcerningtheassumptionthatfindingsmadeinexvivoculturedneutrophilsreproduce physiologicalaging.

Featuresof InVivoAging

Incontrasttotheseexvivofindings,twostudieshaveapproachedtheprocessofneutrophil aginginvivoandhaveanalyzedneutrophilalterationsatdifferentcircadiantimes,neutrophilsat differenttimesaftertransferintohostmice,orneutrophilsthatwereforcedtoremainlongerinthe circulation by use of gene-deficient mice or treatment with antibodies that prevent their extravasation[14,25].Thestrengthoftheseapproachesisthattimewasacceptedasbeing atruephysiologicalparameter,andthattheytookintoaccounttheenvironmentinwhichaging naturally takes place. Following initial observations in rabbits [31], murine neutrophils that remainedlongerinthecirculationwereshowntopresentreducedlevelsofCD62L(alsoknown asL-selectin),whileexpressionofCXCR4increasedovertime[14].Importantly,thenumberof

CD62L^LOCXCR4^HIneutrophilsdisplayedcircadianoscillationsovertime,andtheseagedcells almostcompletelydisappearedintheevening,coincidingwiththebeginningoftheactivephase oftheanimal[14].

AgedneutrophilsalsoexpresshighlevelsoftheintegrinsubunitsCD11bandCD49d(the/ subunitsoftheintegrinsMAC1andVLA-4,respectively),andthissuggeststhattheymaybe moreefficientatadheringtotheinflamedendothelium.Bycontrast,expressionofthe‘don’teat me’moleculeCD47isslightlyreducedinagedneutrophils,achangethatwespeculatetagsthe cellsforenhancedrecognitionandphagocytosisbymacrophages,similarlytowhathasbeen describedforagederythrocytesorleukemiccells[32,33].Thesechangesinsurfacereceptors areparalleledbymorphologicalalterations:agedneutrophilsaresmallerandlessgranular,and arecharacterizedbythepresenceofamultilobullarnucleus[14].Thephysiologicalsignificance ofthesechangesispresentlyunknown,althoughitdoessuggestthatneutrophilsspontaneously degranulateovertime,suchthatnewlyacquiredsurfacereceptorsmayfacilitateextravasation.

Someoftheseinitialobservations havebeenrecentlyconfirmed byZhangetal.whofurther reportedelevatedexpressionofTLR4,ICAM-1,CD11c,CD24,andCD45,aswellasareductionin surfaceLY6C/GbutnotinCD47[25].Interestingly,RNAsequencingofneutrophilscollectedat differenttimesaftertransferintohostmicesuggeststhatanumberofcellularprocessesarealtered atthetranscriptomiclevelduringaging.Pathwaysrelatedtocellactivation(NF-kB,MAPK,andp38 signaling),dangersensing(Toll-likeandNOD-likereceptors),adhesionandmigration(integrinsand the RAC1 pathway), protein degradation and cell death, all appear to change in the aged population[25].Becausemanyofthesereportedalterationsinagedneutrophilsaresimilarto thosethatappearinneutrophilsactivatedbyinflammatoryagonists,itislikelythataginginfact representsaformofcontrolledcellularactivation.Nevertheless,thesetranscriptomicanalyses demonstratedthatthegeneticprogramsactivatedbyagingandbyaprototypicalinflammatory cytokine,TNF/,arepartiallydifferent[25].Wenote,however,thatthesetranscriptomicstudiesdid notuseanunbiasedanalysisandthatsomeoftheidentifiedpathwaysdidnotreachsignificance, probablyowing tothelownumberofcellsthatcould berecoveredin vivo. Futureunbiased analyseswillbenecessarytobetterunderstandthebreadthofmolecularandfunctionalchangesof invivoagedneutrophils,andtoassesswhethersimilarchangesoccurinhumans.

WhatDrivesAging?

Thusfarwehavedescribedthephenotypicchangesthatneutrophilsundergoduringthetime theyspendinthecirculationofunperturbedmice.Thesechangesstartearlyafterthereleaseof neutrophilsfromthebonemarrowandaccumulateovertime,whichsuggeststhatoneormore factorsinplasmamaydrive aging.Thenatureofthefactorsthatdrive thisprocessremains poorlyunderstood(Figure1B).Becausetheconceptofagingwascoinedaftertheobserved circadianclearanceofneutrophils,onesimplisticexplanationforthesephenotypicalterationsis thattheyarecausedbyastochasticprocessinwhichcellsthatundergoactivationareeliminated fromblood.Inthismodel,thecircadianoscillationsinnumberandphenotypecouldbeexplained bychangesincomponentsofthevascularenvironmentratherthanbyacell-intrinsicprocess.

Thispossibilityissupportedbytheobservationthattheexpressionofadhesiveandchemotactic molecules(includingP-andE-selectins,ICAM-1,VCAM-1,CXCL12,andCCL2)istemporally regulated atthe RNAandprotein levelson endothelialcells from varioustissues, including skeletalmuscle,liver,andthebonemarrow[18,34].Regulationofvascularexpressionofthese molecules,whichisdependentonsympatheticsignalsdeliveredbylocalnerves,couldtherefore explaintheegressfrombloodandaccumulationofneutrophilsandotherleukocytesintissues duringtheactivephaseofananimal.

Althoughthismechanismismostlikelyamajorregulatorofcellnumbersinblood[8],itfallsshort ofexplainingthecell-intrinsicalterationsfoundinneutrophilsdescribedintheprevioussection.

OnerelevantexampleistheupregulationofCXCR4onagedneutrophils,whichcontributesto theirclearanceinthebonemarrow[19,35],andappearstocoordinatewiththeexpressionofits ligandCXCL12 inthe marrow tooptimize thisprocess ina time-dependentmanner [14].

Supportforacell-intrinsicmechanismofagingcanbefoundinstudiesinmonocytesandTcells showingthat generationofproinflammatoryLY6C^HI monocytes andT_H17cellsis autono- mouslyregulatedbymembersofthemolecularclock(BMAL1andREV-ERB/,respectively) [9,36]. Interestingly, transcriptional regulation by BMAL1 of CCR2, the receptor for the chemokineCCL2,wasrequired for diurnaloscillations inLY6C^HI monocytenumbers that resemblethoseofneutrophils[9].Variationsinclockgeneexpressionhavebeenfoundacross otherhematopoieticlineages[37],suggestingthatthismaybeageneralmechanismforthe time-dependentregulationofcellfate. Althoughsimilartemporalregulators havenotbeen reportedinneutrophils,thesestudiessupporttheexistenceofintrinsicregulatoryprogramsof leukocyteactivationordifferentiation.

Microbiota-DrivenAging

Only recently has a mechanism of aging been reported. The resemblance between aged neutrophilsandthose treated with inflammatory agonists,as wellas the reportedability of metabolitesproducedbythemicrobiotatoenterthecirculationandregulatehostimmunity[38], promptedtheFrenettegrouptoexaminethephenotypeandfunctionofneutrophilsinantibiotic- treatedoringerm-freemice.Theyfoundthatthenumberofaged(CD62L^LO)neutrophilswas markedly reduced inthe absence of a normal microbiota.Treatment with bacteria-derived agonistsforpattern-recognitionreceptorsorfecaltransferpartlyrestoredtheagingphenotype.

Thisprocessrequiredcell-intrinsicsignalingthroughTLR4,TLR2andthetransducingmolecule MYD88,andrevealedthatagingisregulatedatleastinpartbythemicrobiome[25].

Apotentialconcernwiththisstudy,however,isthat,becauseabsolutenumbersratherthan frequencyofagedcellsweremeasured,itisdifficulttointerpretwhethermicrobiome-derived signalsregulatetotalneutrophilnumbers(aprocessknowntorelyontheIL17/G-CSFaxis[38]) ratherthanbonafideaging.Wealsocautionthattheantibiotics usedforthesestudiesmay directly affect neutrophils [39–42], and may therefore confound some of the conclusions.

Competitivetransferexperiments,inwhichtheseexperimentalconcernsarebypassed,how- ever,supporttheinterpretationthatagingisdependentonTLRsignaling[25].Thesefindings underscorethenecessitytobetterdefinewhethertime-dependentchangesinthesesignaling pathwayscanexplainthecircadianoscillationsthatareatthecoreoftheagingphenomenon.

Together,thissetofstudiessuggeststhatsignalsderivedfromtheenvironment instructthe physiologicalagingofneutrophils,butdoesnotruleoutthepossibilitythatintrinsicregulationof cellularsensorsfortheseexternalsignals(e.g.,TLRreceptors,MYD88orchemokinereceptors) alsocontributestoaging.

A series of recent studies, although focused on ex vivo models or set in the context of inflammation, have shown that NADPH-derived ROS, activation ofthe ataxia-telangiectasia kinase,andreleaseofgranularproteasesallcompromiseneutrophilsurvivalandactivity[43,44].

Thesefindingsfurthersupportamodelinwhichcanonicalpathwaysofcellactivationtriggered byphysiological levels ofsignals derived from bacteriaorother sources may contributeto neutrophilaging.Conceptually,thesefindingsareimportantbecausetheyhighlightthecurrent viewthatinnateimmunityisanessentialmediatoroftheinteractionsbetweentheeukaryotichost anditsprokaryoticmicrobiome,andthatalterationsinthesereciprocalinteractionscancause pathology[45].

Consequencesof Aging

Thefullextentofthephysiologicalandfunctionalpropertiesthatagingendowsoncirculating neutrophilsiscurrentlyunclear.Thisuncertaintyislargelyduetothedifficultyofexperimental

purificationandmanipulationofneutrophilswithoutperturbation(e.g.,activationornon-specific retentionintotissuesaftercelltransfer),whereasphysiologicalanalysesarecomplicatedbythe shortlifeoftheneutrophil,whichhasthusfarprecludedgeneticandphysicaltracingofspecific cell populations.Nonetheless, early studiesshowed thatCD62L^LOneutrophilspurifiedfrom rabbitsdisplayimpairmentsinF-actinassembly,shapechange,deformability,andchemotactic migrationupon stimulationwithinflammatory agonists.Bycontrast, productionofH₂O₂and CD18surfaceexpressionwereincreasedintheagedsubsetcomparedtothetotalcirculating pool[46].Thesestudiessuggestedthat,atleastinvitro,agingbluntsthecapacityofneutrophils toextravasateduringinflammationbutdoesnotcompromisetheircytotoxicfunctions.Further- more,theparadoxicalobservationthatagedneutrophilsmigratepoorlytoinflammatorysignals, althoughtheyareefficientlyclearedintissues,suggeststhatdifferentrecruitmentmechanisms mediateeachprocess,butthisneedstobeformallydemonstrated.

Onlyrecently,invivostudieshaveshedlightonthe(patho)physiologicalconsequencesofaging usingmodelsofTNF/-inducedinflammationandvascularocclusioninamousemodelofsickle celldisease[25].Intravitalimagingofneutrophilsadheredtothevascularwalldemonstratedthat, withinallrecruitedneutrophils,thesubpopulation expressinglowerlevelsofCD62Ldisplays moreactiveb2-integrinsandproducesmoreROSorismorepronetoundergoNETosis[47].

Consequently,inhibitionofagingbytreatingmicewithantibiotics bluntedintegrin activation, improvedtissueperfusion, andextendedthesurvivalof‘sickle’micebypreventingvascular occlusion[25].Whilethesestudiesdidnotexplorethecontributionofagingtocelltraffickingand migration into tissues, they suggest that aging primes neutrophils for a more aggressive inflammatoryresponseinvivo.Theseobservationsarepuzzling,however,becausethenadir of aged neutrophil numbers inblood coincides with the reported peak of susceptibility to inflammatoryinjury[18].Clearly,amorethoroughstudyofthecellularconsequencesofaging willbenecessarytounderstandhowthisprocessinfluencesvascularhealth.

Ananticipatedconsequenceofagingistoprepareneutrophilsforeliminationfromthecircula- tion,aprocessthatcouldinprincipleconferprotectiontothecardiovascularsystembylimiting itsexposuretothesehighlyreactiveleukocytes.Thepeakoftotalneutrophilcountsinblood coincideswiththepeakofagedneutrophils,andthesubsequentdeclineinneutrophilnumbers canbefullyaccountedforbythedisappearanceoftheCD62L^LOagedsubset,whereasthe numberofCD62L^HIcellsremainsrelativelyconstantthroughouttheday[14](Figure1A).While theseobservations suggestthat agingpromotes neutrophil clearancefrom blood,a causal connectionbetweenthetwoprocessesisyettobeformallyestablishedandwillrequiretheuse ofgeneticmodelsofimpairedneutrophilagingthatarenotyetavailable.Thesestudieswillbe importanttodelineatewhetherthehighersusceptibilityofmammalstoinflammatorydiseaseat particulartimesoftheday[4,5],andtheepidemiologicalrelationshipbetweenelevatedneutro- philcountsand cardiovascular events[6,48],canbe taken advantageoftherapeutically by modulatingneutrophilaging.Importantly,thereisnowincreasingevidencethatneutrophilsthat leavethecirculationcanperformspecialized,non-inflammatoryfunctionsinthetissuesthatthey infiltrate.Below wediscuss someunanticipated functionsofneutrophilswithin tissues, and proposethatthesefunctionsmaybeinpartaffordedbynewlyacquiredpropertiesofneutrophils thathaveundergoneaging.

Functionsof NeutrophilsinTissues

Thefulllifecycleofneutrophilsundersteady-stateconditionsisgenerallyportrayedasproduc- tionin,andreleasefrom,thebonemarrow,immunesurveillanceinthecirculation,andsilent disposalinspecializedtissues(bonemarrow,spleen,andliver),alltakingplacewithinthespanof oneday[15,49–51].Severalrecentstudies,however,challengethissimplisticview,andinstead arguethatneutrophilsclearedfromthecirculationmaypersistlongerinspecifictissuesandcan haveafunctionalimpactuponthem(Figure2).Indeed,itisbecomingincreasinglyclearthat

neutrophilsareactivemodulatorsofimmunityandimmunetolerance,angiogenesis,andorgan metabolism(discussedinBox1).

Thefunctionalinfluenceofneutrophilsintissues,however,iscurrentlybestunderstoodinthe bonemarrow(Figure2).Thebonemarrowishometohematopoieticstemandprogenitorcells (HSPCs), the source of all blood elements, which are physically retained and functionally regulatedbystromalelementsgloballyreferredtoasthehematopoieticniche[22].Alterations inthecompositionorfunctionofthesenichesthereforeaffectkeyaspectsofhematopoiesis, includingmaintenance, proliferation,differentiation, andextramedullary trafficking ofHSPCs [52].Forexample,inadditiontoitspotentgranulopoieticproperties,thecytokinegranulocyte- colonystimulatingfactor(G-CSF)causespotentrepressionofnichecomponentsthatallowsits clinicaluseformobilizationofneutrophilsandHSPCsintothecirculation[53].In2005thegroup ofKlausLeyreportedthatendogenousG-CSFlevelsareregulatedbyhomeostaticphagocy- tosisofapoptoticneutrophils. Micedeficientingenesthatmediateneutrophilmigrationdis- playedmarkedelevationsinG-CSFlevelsinplasmaanddevelopedneutrophilia,bothofwhich couldbeameliorated bytransferofmigration-competent neutrophilsthathadbeenagedin culture.Theyfoundthatmigrationofagedneutrophilsandtheirphagocytosisbytissue-resident macrophagesanddendriticcellsinthebonemarrow,spleen,orlungrepressedthetranscription ofIL23,acytokinethatregulatesIL17productionbyTcellsandinducesG-CSFproduction[54].

Although it remains unclear to what extent ex vivo culture of neutrophils reproduces the phenomenon of physiological aging, this study provided the first link between neutrophil clearanceandfunctionalregulationofthebonemarrow.

Itisinterestingthatthe‘neutrostat’modelproposedbythesestudiesuncoveredafunctional association between neutrophil clearance and medullary granulopoiesis [48]. They further

Bone

HSPC niche CXCL12

CXCL1 CXCR4

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Bone marrow HSPC niche control Angen transport LungReservoir?

Spleen B-cell support Lymph nodes Angen transport T-cell regulaon

Containment of immune responses

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Non-vascularized ssue Promoon of angiogenesis Extramedullary

clearance MΦ

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lease

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Figure2.ADayinthe(Neutrophil's)Life.Neutrophilsareproducedinthebonemarrowandreleasedintothe circulation,wheretheyundergoagingbeforebeingclearedintotissues,includingthebonemarrowitself.Thereleaseinto bloodisregulatedbytheinterplaybetweenretentivesignalsdeliveredbyCXCL12throughCXCR4,andmobilizingsignals mediatedbyCXCL1signalingthroughCXCR2.Agedneutrophilsareultimatelyclearedfromthecirculationintothetissues, wheretheymayperformspecializedfunctions(indicatedatrightanddiscussedinBox1).Oneconsequenceofclearanceisthe negativeregulationofthehematopoieticnichesinthebonemarrowwhenneutrophilsenterthisorganandareengulfedby marrowmacrophages(redbranch),resultinginnicheinhibitionandreleaseofhematopoieticstemandprogenitorcells(HSPCs) intoblood.Bycontrast,neutrophilsclearedinextramedullarytissues(light-bluebranch)causereductionsinplasmaG-CSF,a granulopoieticandmobilizingcytokine,andconsequentlyinhibitgranulopoiesisinthebonemarrow.MF,macrophage.

revealed,surprisingly,anintimateconnectionwiththemetabolismofcholesterol:regulationof Il23geneexpressionuponefferocytosisofneutrophilsrequiresactivationofliverXreceptors (LXR), which are nuclear receptors that respond to intracellular oxysterol and are central regulators ofcholesterol homeostasis [55]. Consequently, mice lacking LXR/ and b or its downstreamtargetMerdisplayinefficientphagocytosisandremovalofneutrophils,anddevelop neutrophilia[56].Studiesinmicewithconditionaldeficienciesincholesteroltransporters(Abca1 andAbcg1) further demonstrated afunctional connection between cholesterol handling by phagocyticcellsintissues(macrophagesanddendriticcells),activationoftheIL17/IL23/G-CSF axis,andsuppressionofosteoblasts(bone-liningcellsthatareanimportantcomponentofthe hematopoieticniche),ultimatelyresultingintheabnormalreleaseofleukocytesintothecircula- tion[57].

Neutrophil-mediatedregulationofhematopoieticnichescanalsobeorchestratedlocally.Aged neutrophilsthatenterthebonemarrowareengulfedby localmacrophages,aprocess that triggersactivationofLXRreceptors inthesephagocytes. Throughasyetundefinedmecha- nisms,thesemedullarymacrophagesthenblunttheexpressionofCXCL12andtriggerreduc- tionsinthenumberandfunctionofhematopoieticnichecells,ultimatelycausingthereleaseof

Box1.AgedNeutrophilsinTissues?

Recentstudieshavefoundneutrophilsinmultipleorgansinthesteady-state[16](Figure2).Herewediscussevidence supportingthepossibilitythatneutrophilagingprecedesphysiologicalclearanceintotissuesinwhichtheymayacquire specializedfunctions.

(i) Relativelylargenumbersofneutrophilsarefoundintherestinglung,bothadheredtothevascularlumen(i.e., marginated)andintheinterstitialspace.ThispoolofneutrophilsisretainedthroughCXCR4andendothelial-borne CXCL12signaling[59]andispoorlymigratoryunlessaninflammatorystimulus[60]orepinephrine[61]isapplied.The dependenceonCXCR4supportsthepossibilitythatagedneutrophils,whichdisplayincreasedlevelsofthisreceptor [14],acquiretropismtothelungs.

(ii) Largenumbersofneutrophilsarealsofoundinthespleenofhealthymiceandhumans,wheretheylocalizearound themarginalzoneandproducecytokinesthatpromotesomatichypermutationandimmunoglobulinproductionby splenicBcells[62].Interestingly,theCD62L^LOCD11b^HIICAM1^HIphenotype,andthepropensitytoproduceNETsof theseBhelperneutrophils,resemblethepropertiesofagedneutrophilsinthecirculation[14,25].

(iii) FunctionalassociationsbetweenneutrophilsandTcellshavealsobeenreported.Inthecontextofinfectionor antigenicchallenge,neutrophilstransportantigenstothelymphnodes(LN)andbonemarrow,wheretheyelicitantigen- specificresponsesbyTcells[63,64]whilesimultaneouslylimitingthemagnitudeoftheresponseanditspathogenic spreadbeyondthedrainingLN[65].MigrationtotheLNisexplainedbythefindingthatasmallsubsetofneutrophils expressesCCR7,areceptorknowntomediaterecruitmentoflymphocytestotheselymphoidstructures[66].Because neutrophilentryintotheseorgansadditionallyreliesonreceptorsthatareupregulatedinagedneutrophils(CD11bor CXCR4[67]),itisthought-provokingtospeculatethatexpressionofCCR7andtheantigen-presentingmachinerymay beahallmarkofaging.NeutrophilsarealsoinvolvedinTcell-mediatedresponsesthroughcontact-dependentinhibition andlocalROSdelivery[68].Notably,thisTcell-suppressivepopulationofneutrophilsappearsinthehumancirculation aftertreatmentwithlowdosesofendotoxin,anddisplaysmorphologicalfeatures(nuclearhypersegmentation)and surfacemarkers(CD62L^LO,CD11b^HI,andCD11c^HI)[68]thatresemblethosedescribedinagedneutrophilsinthe mouse[14,25].

(iv) Recentreportslinkneutrophilswithcentralmetabolismintheliverandadiposetissue,whereneutrophilsarepresent atlowlevelsinsteady-state,andrapidlyincreaseduringexperimentalobesity[69].Neutrophil-derivedelastase,a proteasestoredinazurophilicgranules,candegradeInsulinreceptorsubstrate1(IRS1)inadipocytesandhepatocytes, inducinginsulinresistanceandpromotinglipogenesisandcholesterolsynthesis[69].Consequently,animbalanceinthe levelsofelastaseanditsinhibitor,/1-antitrypsin,isassociatedwiththeonsetofobesityandmetabolicsyndromeinmice andhuman[70].Althoughobesityrelatestoaninflammatorystate,theseobservationsraisethepossibilitythatbasal neutrophilinfiltrationintheliverandfatregulatesmultipleaspectsoftheirmetabolicfunctions,andsuggestsacausal connectionbetweenimmunerhythmsandthereportedcircadianpatternsoflipidmetabolism[71].

(v) Tissue-infiltratingneutrophilscanpromoteangiogenesis.AsubsetofneutrophilsexpressingVEGFR1,thereceptor forVEGF-A,isefficientlyrecruitedtonon-vascularizedtissuesandpromotesangiogenesisbyprovidingthemetal- loproteaseMMP9tofacilitatevesselpenetration[72,73].Thisproangiogenicsubsetdisplaysanaged-likeCD49d^HI CXCR4^HIprofileandhasbeenassociatedwithenhancedvascularizationandgrowthoftumors[74].

Thesestudieshighlightthephenotypicandfunctionalheterogeneityofneutrophils,andraisetheprovocativeideathatthis diversityoriginatesinpartthroughphysiologicalagingandsubsequentclearanceintotissues.Itisconceivablethateven inpathologicalscenariossimilarmechanismsbecomeactivetoprepareneutrophilsfortheparticulardemandsofeach tissue.

HSPCsintothebloodstream[14].Importantly,thiscircadianinfiltrationofthemarrowandniche suppression by neutrophils entrains the oscillations of HSPC levels in the circulation[34].

Together,thesestudiesrevealacomplexrelationshipbetweenneutrophilclearancefromblood andbonemarrowactivity,withpositiveornegativeeffectsdependingontheinfiltratedorgan (peripheraltissuesorthebonemarrowitself)(Figure2).Furthermore,becauseextramedullary HSPCsare nowalso recognized as mediatorsof inflammation[58],regulationof thebone marrowbyneutrophilscouldbepotentiallymanipulatedtoamplifyortoresolveinflammation.

ConcludingRemarks

Recentyearshaveseenarenewedinterestinneutrophilbiology.Perhapsmoststrikinghasbeen therealizationthatneutrophilsarenotphenotypicallyuniformandthattheyareinfactasdiverse asothermorepopulartypesofimmunecells.Thesenewfindingshaveledresearchersinthefield tochallengedogmassurroundingtheseleukocytesthatwereoftenbasedonoldorambiguous data.Howthisdiversityisachievedbycellswithauniquelyshortlifespanisstillunderdebate.

Theconceptofagingintroducesaparadigminwhichsubtlechangesthataccumulateovertime cangeneratediversityofbothphenotypeandfunction.Althoughweappreciatethatthisisonly onewaytointerpretthemultiplefindingsdescribedinthisreview,weexpectthatitwillprovidea usefulframeworktomoveresearchontheseleukocytesintoanewage.Tothisend,westillneed tobetterdefineagingatthecellularandmolecularlevels.Globalproteomicandtranscriptomic analysesofagedneutrophilswillbeessentialtoestablishtowhatextentthesecellsdifferfrom the total pool of circulating neutrophils. In addition, because (circadian) time is a defining parameterforagedneutrophils,itwillbeimportanttodeterminehowneutrophilssensetime –eitherbyintegratingsignalsfromtheenvironmentorthroughaninternalclocksimilartothat describedforsubsetsofmonocytesorTcells.

Onlyafterthebasicmechanismsandfeaturesofagingareunderstoodwewillbeabletoasknew andrelevantquestionsaboutthecontributionsofneutrophilstohomeostasisorpathology.For example,whetherthetemporalsusceptibilityofmammalstocardiovasculareventsandother inflammatorydisorderscanbeattributedtotheoscillatingpropertiesofthesecells.Likewise, understanding ifaged neutrophils feedsmall, persisting populations of these leukocytes in healthytissues,andwhethertheyacquirespecializedfunctions,mayhelptointegratethemany surprisingfindingsmadeinrecentyearsintoamoregeneralandproductiveframework.

Acknowledgments

WearegratefultoDrsMariaCasanova-AcebesandLinneaWeissforinsightfulcommentsandediting,andJuanManuel GarcíaCáceresforart.SomeimagesusedforourfigureswereobtainedunderpermissionfromtheSomersaultLibraryof ScienceandMedicalIllustrations, CreativeCommons(CCBY-NC-SA4.0).WeacknowledgefundingfromMINECO (SAF2012-31142andSAF2013-49662-EXPandPCIN-2014-103toA.H;SVP-2014-068595toJ.A.N-A.;andBES-2013–

065550toJ.M.A).TheCNICissupportedbytheMINECOandthePro-CNICFoundation,andisaSeveroOchoaCenterof Excellence(MINECOawardSEV-2015-0505).

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OutstandingQuestions

Currentapproachestoidentifyagingare limitedtoacombinationofpoorly-spe- cificmarkers,makingthedistinctionand quantificationofagedneutrophilsinthe mouseandotherspeciesambiguous.

Canbetterandmore-specificmarkers ofaginginmouseandhumanneutro- philsbeidentified?

The phenotype of aged neutrophils largely resemblesthat of neutrophils activated by inflammatory agonists, although recent work has demon- strateddistincttranscriptionalprofiles betweenaged andactivatedneutro- phils.Doesagingrepresentaformof homeostaticactivation and,if so,to whatextent?

Agingcorrelateswithchangesinneu- trophilnumbersinthecirculation,and thenumbersofagedandtotalneutro- philsfollowstrictcircadianpatternsof clearance from blood. Is neutrophil agingcausally connectedwith clear- ancefrombloodandentryintotissues?

Peak levels of aged neutrophils are immediatelyfollowed byrapid clear- anceofneutrophilsfromblood,sug- gestinganelevatedtropismoftheaged cellsfortissues.Whatisthenatureof thesignals(andoftheirreceptors)that guideagedneutrophilsfrombloodinto tissues?

Theobservationthatagedneutrophils displayreducedinvitrotropismtowards inflammatorymediatorsisatoddswith thenotionthatagingprecedesmigration from blood intotissues. Do different mechanisms govern inflammatory migrationandhomeostaticclearance?

Neutrophilagingfollowscircadianpat- terns, implying some type of time- sensingbythesecells.Howdoneu- trophils sensetime? Dothe mecha- nismsinvolveintegratingsignalsfrom theenvironment,ortheuseofintrinsic temporalclocks,orboth?

Several acute inflammatory diseases displaycircadianpatternsofincidence andseveritythatparallelfluctuationsof neutrophilsinblood.Canthesedisease parametersbeexplainedbythepre- dominanceofagedorfreshneutrophils atspecifictimesoftheday?Ifso,isit theirpresence in blood, orratherin tissues,thatexacerbatesthedisease?

Aretheredifferencesdependingonthe subsetsofneutrophilsinvolved?