动脉粥样硬化综述Atherosclerosis--A matter of unsolved inflammation

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SeminarsinImmunology

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alhomepage:http://wendang.chazidian.com/locate/ysmim

Review

Atherosclerosis–Amatterofunresolvedin?ammation

JoanaViolaa,?,OliverSoehnleina,b,c,?

a

InstituteforCardiovascularPrevention(IPEK),LMUMunich,Germany

DepartmentofPathology,AcademicMedicalCenter(AMC),Amsterdam,TheNetherlandsc

GermanCentreforCardiovascularResearch(DZHK),MunichHeartAlliance,Munich,Germany

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article

info

abstract

Articlehistory:

Received27January2015

Receivedinrevisedform19March2015Accepted27March2015

Keywords:

Atherosclerosis

In?ammationresolutionMacrophagepolarizationEfferocytosis

Atherosclerosisiscommonlylookeduponasachronicin?ammatorydiseaseofthearterialwallarisingfromanunbalancedlipidmetabolismandamaladaptivein?ammatoryresponse.However,atherosclero-sisisnotmerelyanin?ammationofthevesselwall.Infact,thecardinalsignsofunstableatheroscleroticlesionsareprimarilycharacteristicsoffailedresolutionofachronicin?ammation.Incontrasttoacutein?ammatoryeventswhicharetypicallyself-limiting,atherosclerosisisanunresolvedin?ammatorycondition,lackingtheswitchfromthepro-in?ammatorytothepro-resolvingphase,thelattercharac-terizedbyterminationofin?ammatorycellrecruitment,removalofin?ammatorycellsfromthesiteofin?ammationbyapoptosisanddeadcellclearance,reprogrammingofmacrophagestowardananti-in?ammatory,regenerativephenotype,and?nallyegressofeffectorcellsandtissueregeneration.Herewepresentanoverviewonmechanismsoffailedresolutioncontributingtoatheroprogressionanddeliverasummaryofnoveltherapeuticstrategiestorestoreresolutioninin?amedarteries.

©2015ElsevierLtd.Allrightsreserved.

1.Atherosclerosis–continuedin?ammationandfailedresolution

Atherosclerosisisacomplex,progressivedisorderaffectinglargeandmedium-sizedarteries.Therapeutically,majorconcernsarisefromthesilentprogressionofthisworldwidemalady,oftenwithnoclinicalevidenceuntiloccurrenceofischemicdamageduetothrombosisorseverestenosis.Intrinsicallyarterioscle-roticvasculardiseaseisanin?ammatoryconditioncharacterizedbyaberrantlipidmetabolismandamaladaptivein?ammatoryresponse.Classically,arterialin?ammationistriggeredbyaninsulttotheendothelium,oftenatarterialbranchpointsoratareasexperiencingdisturbed?ow,ultimatelyleadingtoendothelialcellactivationandrecruitmentofin?ammatorycellstothevesselwall.Atthesiteofendothelialactivationstructuralalterations,inparticulartheexposureofproteoglycans,facilitatethereten-tionoflow-densitylipoprotein(LDL)particlesintheintima[1,2],wheretheyaresusceptibletooxidativemodi?cationbyreactiveoxygenspecies(ROS)andenzymesreleasedfromin?ammatorycells.Asmacrophagesprogressivelytakeupmodi?edlipo-proteinstheygiverisetofoamcells.Thecontinuousintracellular

?Correspondingauthorsat:IPEK,LMUMunich,Pettenkoferstr.9,80336Munich,Germany.Tel.:+4989440054677;fax:+4989440054352.

E-mailaddresses:joana.viola@med.uni-muenchen.de(J.Viola),oliver.soehnlein@http://wendang.chazidian.com(O.Soehnlein).

accumulationoflipids(includingcholesterol,oxysterolsandotherfattyacids)inducesendoplasmicreticulumstresstriggeringfoamcellapoptosis[3].However,aberrationsinfoamcellshavebeendescribed,suchasthede?ciencyofpro-apoptoticfactors(e.g.Baxandp53),thatpreventcellapoptosiscontributingtoatheroscle-rosisprogression[4,5].Inadvancedatherosclerosisthesourcesofapoptoticcellssturdilyoverwhelmtheefferocyticprogram.Suchdefectiveefferocytosisallowsapoptoticcellstoundergosecondarynecrosis,therebyfeedingthenecroticcoreandaconstant?owofpro-in?ammatorymediatorsthatoverrideexistingpro-resolutionsignals.Thishighlyin?amedandnecroticcoreiscentraltotheatheroscleroticplaque–vulnerabletostructuraldisruptionandanimmediateprecursorofacutecardiovascularclinicalevents.

Incontrasttoacutein?ammatoryeventswhicharetypi-callyself-limiting,atherosclerosisisanunresolvedin?ammatorycondition,lackingtheswitchfrompro-in?ammatorytoanti-in?ammatorymediatorsthatcharacterizestheresolutionphase.Theresolutionphaseofin?ammationembracesterminationofin?ammatorycellrecruitment,removalofin?ammatorycellsfromthesiteofin?ammationbyapoptosisanddeadcellclearance,reprogrammingofmacrophagestowardananti-in?ammatory,regenerativephenotype,and?nallyegressofeffectorcellsandtissueregeneration[6].Understandingthedifferentaspectsoffailedresolutioninatherosclerosisprovidestheopportunitytoidentifyalternativetherapeutictargets,theoreticallywithminimalside-effects.Thus,thisreviewwillconcentrateonmechanismsoffailedresolutioninatherosclerosis,speci?callyon(1)continued

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Pleasecitethisarticleinpressas:J.Viola,O.Soehnlein,Atherosclerosis–Amatterofunresolvedin?ammation,SeminImmunol(2015),http://wendang.chazidian.com/10.1016/j.smim.2015.03.013

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leukocyteaccumulation(asresultofcontinuedrecruitment,pro-liferation,andfailedegress),(2)unbalancedM1/M2macrophagepolarization,and(3)impairedefferocytosis.Finally,wewilloutlinepossibletherapeuticideas,manyofwhichstemfrompreclinicalstudiesofacutein?ammatorymodels.

2.Leukocytesgraduallyaccumulateinatheroscleroticlesions

Monocyte-derivedcellsarethemostabundantleukocytesub-setintheatheroscleroticplaque.Thepertinentroleofmonocytesinatherosclerosiswasclearlyevidencedwhendepletionofthesecellsfromthecirculationwasshowntodrasticallyreduceplaqueforma-tion[7,8].However,depletionofmonocytesatlateratheroscleroticstagesdidnothaveanyeffectontheaccumulationofmacrophageswithinthelesionandalsonotinplaquecompositionornecroticcoreformation[9],underscoringtheimportanceofcontinuedrecruitmentofthesecellstotheinitiationofthedisease.Consistentwiththisnotion,continuedmonocyterecruitmentisahallmarkduringatherosclerosisprogressionandregressionofatheroscle-roticlesionsisprimarilydrivenbyhaltedrecruitmentofmonocytes[10].Besidesmonocytes,alsothepresenceofneutrophilsinthe

atheroscleroticplaquehasbeenreported,andacausalcontribu-tionofneutrophilsduringvariousstagesofatherosclerosishasbeenestablished[11,12].Continuedleukocyteaccumulationinthelesionsitefeedsanin?ammatorymilieuandpreventsaturnovertowardresolutionofin?ammation.

Severalprocessescontributetotheprogressiveaccumulationofleukocytesintheatheroscleroticplaque,themostobvious,andsupramentioned,beingleukocyterecruitment[7,10,13,14].Howevermacrophagesurvival[15]andproliferation[16,17]intheplaqueaswellasthelimitedabilityofleukocytestoleaveatheroscleroticlesions[18]representimportantprocessescriti-callycontrollingthenumberofmacrophageswithinatheroscleroticlesions(Fig.1a).

2.1.Mechanismsofcontinuedleukocyterecruitment

Theclassicalcascadeofleukocyterecruitmentincludesleuko-cyterolling,activation,arrestandmigration.CaptureandrollingaremediatedbyselectinsandtheP-selectinglycopro-teinligand-1(PSGL1)receptor,whereasleukocytearrestisledbychemokine-activatedintegrinsmainlylymphocytefunction-associatedantigene1,LFA1,andverylateantigene4,VLA4,

Fig.1.Mechanismsoffailedresolutioninatherosclerosis.(a)Perpetuatedleukocyterecruitmentisoneofthehallmarksofatherosclerosis,anditcontributestoadjourningresolutionofin?ammation.Inthevesselwallothermechanismstakeplacethataidtoputtingoffresolution,namely:proliferationofresidentmacrophages,macrophagesurvival,failedegressofabundantmonocyte-derivedcells,andM1favoredpolarization.Macrophagespartialescapeofapoptoticmechanismsissupportedbyaberrantgeneexpression,whereuponsurvivalandtumorsuppressorgenesareupanddown-regulated,respectively.Atlaterstagesofdisease,efferocytosis(theprocessbywhichapoptoticcellsarecleared)ishampered,andlateron,whenplaqueruptureoccurs,plateletaggregatesarefrequentandtheclosecontactwithoxLDLfromtheplaqueismorelikelytooccur,aggravatingthealreadyongoingleukocyterecruitment.(b)Underhypercholesterolemia,increasedLDLandoxLDLactivateplateletsuponcontactcontributingtoleukocyterecruitmentviaplateletchemokine(CCL5)depositionontheendotheliumandtheformationofaggregates(neutrophil–plateletorplatelet–plateletaggregates).PlateletderivedP-selectinalsoplaysamajorrole,mediatingthedeliveryofpro-in?ammatorymoleculestotheendotheliumaswellastocirculatingmonocytes.Furthermore,theenvironmentcharacterizedbylowavailabilityofnitricoxide(NOS)andbountifulreactiveoxygenspecies(ROS)favorsthesynthesisofleukotrieneB4(LTB4)andCCL2secretedbyendothelialcellsaswellasmacrophages.Contrarily,pro-resolutionmediators,suchaslipoxinA4(LXA4),resolvingD1(RvD1)orProtectinD1(PD)stopthereleaseofcytokinesandleukocyterecruitment.Alsopentraxin3(PTX3)isknownasanendogenousinhibitorofengagementofneutrophils,whereasAnnexinA1actsonmonocyte-derivedcellsviaformylpeptidereceptor2(FPR2).(c)Underin?ammatoryconditions,stimulisuchastumornecrosisfactor(TNF)andinterferongamma(INF?)predominate,fuelingaM1macrophagepolarization.M1macrophagesexpresshighlevelsofCD86receptoraswellasMHCII,http://wendang.chazidian.comparedtoM2macrophages,M1macrophagesalsopossessahighercapacitytoprocesslipids.Inapro-resolutionenvironment,cytokinessuchasIL-4,IL-13andIL-10favoranM2phenotype,wherehighPPAR?andSTAT6activityresultinincreasedcapacityofdeadcellclearance.(d)Clearanceofdeadcellsisrequiredformanyprocesses,includingresolutionofin?ammation.Apoptoticcellsrelease“?ndme”and“eatme”signalsinordertobecleared.“Findme”signalssuchassphingosine1phosphate(S1P),lysophosphatidylcholine8LPC),CX3CL1orthenucleotidesadenosineoruridine-5??-triphosphate(ATPandUTP)encouragemacrophagesmigration,whereas“eatme”signalsareresponsiblefortheengulfmentprocess.Atlaterstagesofatherosclerosisthisactionishampered.Possiblecontributionsareattributedtolysophosphatidylcholine(LPC)decoys(oxLDL-derivedLPC)thatdeviateefferocyticcellsfromtheirapoptotictargetsbykeepingthecellreceptorsoccupied,ortolowexpressionofbridgingmolecules(suchaslactadherinorGas6)aswellasreceptors(Mertyrosinekinase,MERTK,orCD36forexample)directlyenrolledinthephagocyticprocess.Alternativeexplanationsrelatetosheddingofreceptorsdirectlyenrolledinefferocytosisordecreasedavailabilityofbridgingmoleculesorothersimilarmediators,suchascomplement1q(C1q)orthrombospondin-1(THBS1),whichcanbindtoapoptoticcellsandincreasetheirclearance.Overall,thelesionmilieuismainlypro-in?ammatoryfavoringanM1polarizationphenotypeofmacrophagesandcreatinganimbalancedM1/M2ratiothatdelaysresolutionofin?ammation.

Pleasecitethisarticleinpressas:J.Viola,O.Soehnlein,Atherosclerosis–Amatterofunresolvedin?ammation,SeminImmunol(2015),http://wendang.chazidian.com/10.1016/j.smim.2015.03.013

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activatedbychemokinesviainside-outsignal.Thereforeanystim-ulusthat,directlyorindirectly,feedsthisprocessultimatelyresultsinleukocyterecruitment.Inthiscontext,itisimportanttoconsideridiosyncraticmarkersoftheatheroscleroticconditionsuchaslowbioavailabilityofnitricoxide(NO),highlevelsofreactiveoxygenspecies(ROS),andlowdensitylipoprotein(LDL),asthesearecon-stantfactorsthroughoutthediseaseandrepresentacontinuous,persistentstimulus(Fig.1b).

Indeed,LDLisanimportantmediatortotakeintoconsider-ation,asinbothitsoxidized(oxLDL)andnon-modi?edformithasbeenshowntoactivateplatelets,herebyhavinganindi-recteffectonarterialleukocyterecruitment[19,20]andfoamcellformation[21,22].Plateletshavebeenstronglyimplicatedinleukocyterecruitmentinatherosclerosis[23,24].Theiractionismultifaceted:plateletscandeliverpro-in?ammatoryfactorstoleukocytesuponup-regulationofP-selectin[23,25],induceleuko-cyterecruitmentviatheCX3CL1–CX3CR1axis[26]orbychemokineCCL5depositionontheendothelium[7,11,20],andenhanceneu-trophiltransmigrationinresponsetooxLDL[27,28].Incirculation,oxidizedphospholipidsexistaspartoflipoproteins[29],butitisatlaterstagesofthedisease,uponendothelialdamageorplaquerupture,thatthelikelihoodthatadheringplateletsareputinclosecontactwithoxLDLincreases[30].Attheselaterstagesplateletscanalsoin?ltratetheplaquevialeakageorruptureofnewlyformedmicrovesselscontributingtolipidaccumulationandnecroticcoreformation[31–33].Indeed,whenthe?brouscapisvulnerable,thrombosisaswellasneoangiogenesisoccur[34],andprovidenewsitesforleukocytestoenterthelesion.Interestingly,clinicalandexperimentalevidenceshavedemonstratedthatthrombiarepre-ferredareastorecruitcirculatingleukocytes[35,36]–aprocessalsomediatedbyplatelets[36,37].

InatherosclerosistheavailabilityofNO(nitricoxide)islowwhileROS(reactiveoxygenspecies)areknowntobeabun-dant.Interestingly,thesynthesisofleukotrienes,drivenbythe5-lipoxigenaseenzyme,dependsonROSanditisinhibitedbyNO[38].Leukotrienesarein?ammatorylipidmediatorsderivedfromthearachidonicacid(AA),whichmediateleukocyterecruit-mentaswellassurvival[39–42].MechanisticallyleukotrieneB4(LTB4)inparticularpotentiatesatherosclerosismainlybyincreasingtheexpressionoffattyacidtranslocase/CD36andofmonocytechemoattractantprotein-1(MCP-1/CCL2)inducingapositivefeedbacklooptorecruitleukocytes[43–45].Theoutputoftheleukotrienesyntheticpathwayisregulatedbyseveralfac-tors,forinstancetheamountoffreearachidonicacidreleasedfromcell-membranephospholipidsandtheavailabilityofNOandreactiveoxygenintermediates,thatmodulatetheactivityof5-lipoxygenase.ThesefactscallattentiontothedrivingforceofLTB4synthesisinatherosclerosis,especiallyas5-lipoxigenasehasalsobeenshowntocorrelatewiththeseverityoftheatheroscle-roticcondition[46–48].Itmaybepossiblethatthenumberofapoptoticcellsandthein?ammatoryenvironmentsupportthehighavailabilityofAAbyphospholipaseA2,andthatthelowbioavailabilityofNO,togetherwiththeincreasedROS,fuelthehighactivity,andpotentiatetheexpressionof5-lipoxigenase.Simulta-neously,thecontinuedin?ammatorysignalsinducetheexpressionofLTB4receptors[49],amplifyingthislipidmediator’sresponses.Theexcessiveproductionofleukotrienesinrelationtoprores-olutionlipidmediators,suchaslipoxinA4(LXA4),resolvinD1(RvD1)orprotectin1(PD1),shiftsthebalancetowardaproin-?ammatorymilieu.Accordingly,twocasecontrolstudies[50,51]onhumanvariantsof12–15lipoxygenase(12/15-LO),thekeyenzymeinvolvedinLXA4biosynthesis,supportaprotectiverolefortheenzymeagainstcoronaryarterydisease.12/15-LOoverexpressioninmurinemodelsofatherosclerosisunderscoresthese?ndings,anditfurthersuggeststhatade?ciencyintheenzyme’savail-abilitypredisposesthechronicin?ammation[52].Mechanistically,

LXA4leadstodownregulationofCCL5inmurinemacrophages,andtogetherwithRvD1andPD1(twootherdownstreamproductsof12/15LO),suppressesavastarrayofproin?ammatorycytokinesandstimulatesefferocytosis[52].

Continuedleukocyterecruitmentmightalsobecausedbymalfunctioningofrestrainingorhomeostaticmechanisms.Morespeci?cally,anumberofendogenousinhibitorsofleukocyterecruitmenthavebeendescribed[53],includingpentraxin3(PTX-3)[54],developmentalendotheliallocus-1(del-1)[55],galectin-1[56,57],andgrowthdifferentiationfactor15(GDF-15)[58].Itisthereforereasonabletoassumethataninhibitionordecreasedavailabilityofthesefactorscancontributetocontinuedleukocyterecruitment.Inatherosclerosishowever,uptodateonlyPTX-3hasbeenshowntobeprotective,whereastheeffectsofGDF-15arecontroversialandthoseofgalectin-1anddel-1remainunad-dressed.Morespeci?cally,PTX-3de?ciencyresultsinincreasedatherosclerosisaccompaniedbyincreasedbonemarrowmonocy-tosisandmacrophageaccumulationinthelesion[59].Inaddition,Apoe?/?PTX3?/?micehaveanincrementinexpressionofadhe-sionmolecules,cytokines,andchemokinesinthevascularwall,suggestingamodulatorfunctionofPTX-3invascular-associatedin?ammatoryresponses.TheatheroprotectiveeffectofPTX-3anditsincreasedexpressionfoundinatheroscleroticsamples[60]thussuggestanefforttorestrictexcessiveleukocyterecruitmentandmaintainacertainhomeostasis.Whetherthecontinuedleuko-cyteengagementisduetoinsuf?cientincreaseintheendogenousinhibitororasignaloverridingowingtomassivepro-in?ammatorymediators,isunclearbutlikelyacombinationofboth.OntheotherhandGDF-15de?ciencystudiesinatherosclerosisarenotconclu-sive:althoughlesseraccumulationofmacrophagesinthelesionhasbeenreported[61],itseemstovarydependingonthemousestrainandthedurationofthewesterndiet[62,63].Alsoitseffectsontheatheroscleroticlesionarecontradictory[62,64].Recently,annexinA1wasaddedtothispoolofendogenousbreaksduringatheroscle-rosis[65].AnnexinA1actsasaproresolvingligand,viaformylpeptidereceptor2(FPR2),andabolisheschemokine-mediatedacti-vationinhibitingleukocyterecruitment,herebyresultinginsmallerearlylesions.

2.2.Leukocytesurvivalandproliferation

Macrophageaccumulationobservedduringthedevelopmentofatherosclerosishasbeenperceivedforsometimeasaconse-quenceofcontinuedleukocyterecruitment.Neverthelessinthelasttwodecadesnumerousreportshaveconsistentlyshowncellproliferationwithintheplaque,includingofmacrophageorigin,suggestingacontributionofthisactiontothechronicin?ammation[66].Moreover,severalapproachestargetingcellcycleregulatorssupportaroleforproliferationinatherosclerosis[67–69].How-ever,therelativeimportanceofmacrophageproliferationtothismaladywasonlyrecentlyaddressed.Thestudycombiningcontin-uousbromodeoxyuridine(BrdU)deliveryandparabiosisrevealedthatthemacrophageturnoverinthelesionsisrapid,thussigni?-cantlycontributingtoaccumulation[16].Indeed,macrophages,inparticularresident,canbeself-maintainedlocally[70]andrespondtoin?ammationwithproliferation[71],possiblyinvolvingtype1scavengerreceptorclassA,SR-A,inthecaseofatherosclero-sis[16].Inthecontextofresolution,theanswertowhatspeci?csignalsputanendtotheproliferationstimulus,soasthefullcharacterizationofthesemacrophagesplasticity,isnaturallyofinterest.Onceaddressed,thesequestionscouldpotentiallyprovideclinicianswithnewtherapeutictargetstopossiblypushtheenvi-ronmenttowardtheresolutionphase.

Inadditiontocellproliferation,sustainedcellsurvivalinpar-ticularofmacrophageandatearlystagesofatherosclerosiswhenefferocytosisisfunctional–acceleratesdiseaseprogression[72].

Pleasecitethisarticleinpressas:J.Viola,O.Soehnlein,Atherosclerosis–Amatterofunresolvedin?ammation,SeminImmunol(2015),http://wendang.chazidian.com/10.1016/j.smim.2015.03.013

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Survivalgenes,suchasTosoorcIAP2,cellularinhibitorofapo-ptosisprotein2[73],areupregulatedduringatherosclerosiswhilefewtumor-supressorgenes(i.e.p27Kip1)aresilenced,leadingtofoamcellaberrationandsurvival[67].Thisprocesscontinuouslypostponesresolution,asclearanceofapoptoticcellsinducesthereleaseofanti-in?ammatorymediators.RecentlyMafB,atran-scriptionfactorthatinducedmyelomonocyticdifferentiation,wasaddedtothelistofapoptosisinhibitorsinthecontextofatheroscle-rosis.MafBexpressionisdominantinplaquefoamcells,whereitinhibitsapoptosisviadeexpressionofapoptosisinhibitorofmacrophages(AIM)mediatedbytheliverXreceptor/retinoidXreceptor(LXR/RXR)axis[74].Lipopolysacharidebindingprotein(LPB),anothertargetgeneofLXR,alikeMafBismacrophage-speci?candpromotescellsurvivalandatherogenesis[75].Inaddition,alsochemokinesandnon-codingRNAshavebeenreportedtopro-motecellsurvivalandtocontributetothischronicdisease.TheabsenceofCX3CR1oritsligandresultsindecreasedatheroscle-roticlesion,inanApoeKOmurinemodel.TheintroductionofBcl2gene(involvedincellsurvival)restoresthewildtypemousephe-notype,andtheadditionofCX3CL1toculturedhumanmonocytesrevertstheinducedcelldeathprocess,con?rmingtheenrolmentofCX3CR1–CX3CL1axisincellsurvival[15].Thelongintergenicnon-codingRNA(Linc)RNA-p21isadirecttranscriptionaltargetofp53.Italsofunctionsasacomponentofthep53pathwaybyinteractingwithp53repressivecomplextodownregulateseveralotherp53targets,itsuppressestranslationbyassociatingwithtargetmRNAs,anditwasrecentlyfounddownregulatedintheatheroscleroticplaqueofApoe?/?murinemodels[69].LincRNA-p21wasfoundtoregulatep53pathwaybyinteractingwithmousedoubleminute2(MDM2)resultingindecreasedcellproliferationandincreasedapo-ptosis.Interestingly,theexpressionofLincRNA-p21wasfoundtocorrelatewithcoronaryheartdisease,asitsexpressioninpatientswasfoundtobesigni?cantlylowerascomparedtocontroldonors.Naturally,these?ndingsopendoorstonewpossibletherapeutictargetstorevertorslowdownthedevelopmentofatherosclerosisaimingatincreasingmacrophageandfoamcellapoptosisatearlystages,andfuellingefferocytosisasaproresolutionprocess.

ofmacrophageegressduringplaqueregression?tswellwiththeconceptofatherosclerosisbeinganexampleoffailedresolu-tion.Mechanistically,macrophagesareretainedinatheroscleroticlesionsduetothepresenceoffactorsthatboycottmacrophageegress.Onerecentlyidenti?edfactorisnetrin-1,whichinhibitsthechemotacticresponsesofmacrophagestoseveralchemokines[84].Otherfactorspromotingtheretentionofmacrophagesincludeenhancedexpressionofadhesionmolecules.Thesearemorehighlyexpressedinmacrophagesinprogressingversusregressingplaques[85]andmaythuscontributetomacrophageenrichmentduringatheroprogression.

3.Unbalancedmacrophagepolarizationdelaysresolution

2.3.Failedmacrophageegressisacharacteristicofatheroprogression

Inacutein?ammatoryresponsesthereturntotissuehomeo-stasisandfunctionalityischaracterizedbyegressofin?ltratedleukocytes.Earlyworkhaspostulatedthatafterperformingtheircentraltasksinresolution,macrophagesemigratetothedraininglymphnode,wheretheymayplayanimportantroleinthepre-sentationofantigensfromthein?amedsite[76].Herein,matrixmetalloproteinase(MMP)-mediatedsheddingof?2integrinseemsimportantformacrophageegressfromtheinjuredtissue[77,78].Althoughtheimportanceofmacrophageegressduringin?amma-tionresolutionwasrecentlychallenged[79]itisstillbelievedthatinacutein?ammationmacrophagesdisappearfromthesiteofin?ammationbyegress.Incontrast,afailureinmacrophageegressresultsintheiraccumulationandmaypotentiallyleadtochronicin?ammatorydiseases,suchasatherosclerosis[80].Infact,earlyworkhasshownthatlesionalmacrophageshavetheabilitytoegressfromatheroscleroticplaques.Herein,studiesinatheroscle-roticpigs[81]andinmonkeys[82],inwhichelectronmicrographsshowedimagescompatiblewithmacrophagefoamcellsexitingbetweenendothelialcellsandthearteriallumen,supportthecon-ceptofmacrophageegress.Theavailabilityofatheroscleroticmicecoupledwithmethodstofollowcelltraf?cking,whicharerel-ativelyconvenientinmice,ledtoadirectdemonstrationafteraortictransplantationthatduringdiseaseprogression,emigrationwaslow,whereasplacementofplaquesintoaregressionenviron-mentreadilydemonstratedmacrophageexit[80,83].Theincrease

Uponin?ammationcirculatingmonocytesin?ltratethedam-agedtissue,wheretheydifferentiateintomacrophages,whosephenotypeisheavilyshapedbythesurroundingmilieu:growthfactors(macrophagecolonystimulatingfactor,M-CSF,versusgran-ulocytemacrophageCSF,GM-CSF),cytokines(interferongamma,INF?,IL-4andIL-13orIL-10),chemokines(CXCL4)[86],andotherplaquecomponentssuchasoxidizedproteins[87].Macrophageheterogeneityisoverwhelmingwithseveralsubpopulationsdescribedintheliterature[88],typicallywellcharacterizedinvitrobuttoalesserextentinvivo.However,theinitialandsimplisticviewofclassically(pro-in?ammatoryphenotype)andalterna-tively(anti-in?ammatoryphenotype)polarizedmacrophages,alsoreferredtoastypeM1andM2,respectively(re?ectingtheTh1andTh2nomenclatureinTcells),isfrequentlyemployed.Owingtothecomplexityofmacrophageheterogeneityandthediffer-encebetweenmurineandhumanmarkers,thissimplisticviewispreferredtoassessthemacrophages’phenotypethroughoutthedifferentstagesofin?ammation.Evidenceimpliesthatanimbalanceinmacrophagepolarizationsustainsthein?ammatoryenvironment:plaquemacrophagesexpressarginase-1(Arg1,amarkerforM2phenotype)atearlystagesofatherosclerosis,whenefferocytosisisfullyfunctional,whereasatlaterstagesofdisease,whenthein?ammationischronicallyperpetuated,theexpres-sionofarginase-2(typicalforM1)predominates[89].Accordingly,inductionofdiseaseregressionshowsaswitchofmacrophagemarkersfrompro-in?ammatory(monocytechemotacticprotein-1andtumornecrosisfactor)toanti-in?ammatory(Arg1,mannosereceptor,MR,andCD163)[90].However,translatingthese?nd-ingstohumanpathophysiologyisnotstraightforward,asinvitropolarizedhumanmacrophagesdonotexpressArg1aswellasothermarkerswell-describedformurinemacrophagesubtypes[91,92].Nevertheless,inepicardialadiposetissueofpatientswithcoronaryarterydisease(CAD)theM1/M2ratioischangedcom-paredtonon-CADpatients,withtheM1/M2macrophageratiopositivelycorrelatingtothediseaseseverity[93].Recently,inanattempttoaddresstheM1/M2ratioinhypercholesterolemicpatients,peripheralbloodsampleswereanalyzedforcirculatingCD68+CCR2+andCX3CR1+CD206+/CD163+,whichratherthanrep-resentingthetraditionalmonocytesubsetsaimedatre?ectinghumanmacrophagepolarization(M1andM2,respectively),whichtakesplaceaftertissuein?ltration[94].Despitethisstudy’slimi-tations,includingthemuchdebatedassumptionthatM1andM2macrophagesderivefromclassicalandnonclassicalmonocytes,respectively,itclearlyshowedanincreaseintheCD68+CCR2+pop-ulationinhypercholesterolemicsamplesascomparedtohealthycontrols.Moreover,the(CD68+CCR2+)/(CX3CR1+CD206+/CD163+)ratio(referredinthestudyasM1/M2ratio)wasincreasedinpatientswithplaquescomparedtothosewithout.Impor-tantly,traditionalgatingtoidentifymonocytesubsetsshowednodifferencesinthedistributionofclassical,intermediateandnon-classicalmonocytes,supportingthestudy’scorrelationbetween(CD68+CCR2+)/(CX3CR1+CD206+/CD163+)andM1/M2ratio.

Pleasecitethisarticleinpressas:J.Viola,O.Soehnlein,Atherosclerosis–Amatterofunresolvedin?ammation,SeminImmunol(2015),http://wendang.chazidian.com/10.1016/j.smim.2015.03.013

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Hence,accordingtothiscurrenthypothesisclassicallyactivatedmacrophagesfeedin?ammationandplaquevulnerability,whereasanalternativephenotypeislikelytosupportplaquestabilityorevenregression(Fig.1c).Inagreement,humanatheroscleroticplaqueM2macrophages,identi?edasCD68+/MR+,poorlyhandlecholesterolandhavestrongphagocyticproperties,attributedtohighPPAR?andlowLXR?activities[95].TheroleofCD68+/MR?andCD68+/MR+macrophagesisfurthercorroboratedbytheirlocal-izationinthelesion:the?rstcellmarkerscolocalizewithruptureproneplaqueareasandlipidcore,muchunlikethesecond[95,96].Nevertheless,aninconsistentM1/M2markersexpressionpro?leoflesionalfoamcellsisobserved[96],oncemoreunderscoringtheintricacyincharacterizationofmacrophagessubtypeinvivo.Theambivalentexpressionspectrumbyfoamcells,combiningfewM1andM2markers,islikelytostemfromthemyriadofstimulithatpopulatethelesionaswellastheensuingmicroenvironments.

Inconclusion,inatherosclerosistheshiftofmacrophagephe-notypefromM1toM2appearstobeputonhold,andshapingplaquemacrophagestowardanalternativelyactivatedphenotypemayfacilitateplaquestabilityorevenregression,viaresolutionofin?ammation.CytokinessuchasM-CSF,andinterleukins4,13and10(IL-4,IL-13,IL-10)activatespeci?ctranscriptionfactorsinmacrophages(i.e.,signaltransducerandactivatoroftranscription3and6,STATs3and6,interferonregulatoryfactor3,IRF3,PPARandLXR?)thatresultintheexpressionofproresolutionmediatorssuchasArg1(inmice),tumorgrowthfactor?(TGF?)andIL-10,herebyinducingananti-in?ammatoryphenotype[97].Therefore,modulationofsuchtranscriptionfactorscanbebene?cialtoinduceaproresolutionenvironment.

4.Defectiveefferocytosisacceleratesnecroticcoreformation

Clearanceofapoptoticcellsbyphagocytes,involvingaprocesstermedefferocytosis,impedestheaccumulationofnecroticcorpsesandtriggersphagocytere-programmingtowardanti-in?ammatoryphenotypesherebyboostingin?ammationresolution[6].Duringatherosclerosis,effectiveefferocytosisishamperedresultingintheaccumulationofapoptoticcells[98,99]andthedelayofresolutionofin?ammation[100](Fig.1d).Apoptoticcellsproduceaplethoraof“?nd-me”and“eat-me”signalsthatmediatephagocyteattrac-tion,interaction,and?nallyengulfment[101,102].Consequently,aninsuf?cientproductionofattractionandrecognitionmoleculesand/oranalteredinteractionandphagocytosismayexplaindefectiveefferocytosisduringatherosclerosis.Several“?nd-me”signalsreleasedbyapoptoticcellshavebeenidenti?edincludinglysophosphatidylcholine(LPC),fractalkine(CX3CL1),sphingosine-1-phosphate(S1P)andthenucleotidesadenosine-5??-triphosphateanduridine-5??-triphosphate[101,102].Duringcellapoptosis,acaspase-3dependentactivationofthephospholipaseA2(PLA2)resultsintheproductionandreleaseofLPCtriggeringphagocytemigrationandengulfment[103].Duringhypercholesterolemia,oxLDL[98]orLPCgeneratedbylipoproteinassociated-PLA2(Lp-PLA2)-dependentoxLDLshydrolization[104]reducesapoptoticcellclearance.Bycompetingwiththesamereceptors,enhancedlevelsofoxLDL-derivedLPCmayimpairapoptoticcellelimination.Inadditiontoinhibitingefferocytosis,otherpro-in?ammatoryactionsareassociatedwithLPC[105]andmayexplainthepositivecorrelationofbothLPCandLp-PLA2withsymptomaticplaques,macrophagecontentandin?ammatorycytokineproduction[105].Ontheotherhand,S1Pactsas“?nd-me”and“eat-me”signalthathasbeenassociatedwithananti-in?ammatoryandatheroprotec-tivefunction,sinceadministrationofasynthetichomologinducesplaquestabilitybyreducingplaquein?ammationandnecroticcoreformation[106].Ofthevariousreceptorsmediatingresponses

toS1P,speci?cactivationofS1Preceptor1wasshowntoreduceplaquein?ammation[107].

Oncephagocytesareadjacenttotheapoptoticcell,“eat-me”moleculesexpressedintheapoptoticmembranesenabletheirrecognition[101,102].Leukocyte-bornepentraxin3isexposeduponneutrophilapoptosispromotingclearancebymacrophages[108].Interestingly,pentraxin3accumulatesinthelesionofagingmiceandgeneticdepletionofpentraxin3resultsinenhancedplaquesizecharacterizedbyahyperin?ammatoryphenotype[59].Othermoleculessuchastrombospondin-1orcomplementC1qbindtoapoptoticcellsactingas“eat-me”signalsanditsdepletionresultsinenhancedplaquevulnerabilityduetodefectiveapoptoticcellclearance[109,110].Phosphatidylserine(PS)isthemostcommoneat-mesignals.ThesolublemoleculeMFG-E8(lactadherin)actsasabridgemoleculelinkingPSonapoptoticcellswithvitronectinreceptoronmacrophages.GeneticdeletionofMFG-E8resultsinaccumulationofapoptoticdebrisandpromotesadvancedplaques[111,112].Interestingly,theauthorsfoundadecreaseofMFG-E8expressioninadvancedatheroscleroticlesionsothatimpairmentinbridgingmoleculeexpressionoracceleratedturn-overduetofacilitateddegradationmayprovideanexplanationforfailedeffe-rocytosisinatherosclerosis.

Ontheotherhand,severalphagocyticreceptorsareimplicatedinapoptoticcellclearanceanditsmodulationhasrelevanteffectsonapoptoticcorpsaccumulationduringadvancedatherosclero-sis.TheMertyrosinekinase(MERTK)receptorwhichbindsthemoleculeGas6iscruciallyimplicatedinlesionalefferocytosissinceitsdepletioninducesapoptoticcellaccumulationandexpansionofnecroticcoreandplaquesizes[113,114].Whilemechanismsofdefectiveefferocytosisatlatestageatherosclerosisdespitetheabundanceoflesionalmacrophagesarenotentirelyclear,shed-dingofreceptorsinvolvedindeadcellclearancemayprovideoneexplanation.Sheddingofengulfmentreceptorsnotonlydepletesphagocyticcapacityofthehostcellbutalsogeneratesapotentialcompetitiveinhibitorthatcanblockefferocytosisonneighbor-ingefferocytes.NotableexamplesofsolublereceptorsinvolvedindeadcellclearanceincludeCD36,lectin-likeoxidizedlow-densitylipoproteinreceptor-1,low-densitylipoproteinreceptor-relatedprotein-1,andMERTK.EvidenceforsolubleMERTKisfoundinavarietyofchronicin?ammatorydisorders,includingsystemiclupuserythematosus(SLE),rheumatoidarthritis,andcardiovascu-lardiseases.Interestingly,themetalloproteinaseADAM17inducesMERTKreceptorshedding[115]releasingasolubleformwhichinhibitsefferocytosis[116].Inaddition,ADAM17mayshedCD36,ascavengerreceptornotjustinvolvedinphagocytosisofLDLbutalsointhrombospondin-mediatedefferocytosis,andthusreduceapoptoticcellclearance[117].Finally,theenzymetransglutamin-ase2(TG2),whichisinvolvedinMFG-E8-dependentapoptoticcellengulfment,isalsoimplicatedinlesionalefferocytosis.Bonemar-rowtransplantofTG2-de?cientcellsinLdlr?/?miceresultedinenhancedplaquesize,andnecroticcorewithreducedefferocytosiscapacity[118].

Withtheimportanceofbridgingmoleculesandtheirrespectivereceptorsindeadcellclearanceinatheroscleroticlesions,recentstudieshaveaimedatidentifyingtranscriptionalregulatorsforthesemolecules.Geneticvariationatthechromosome9p21risklocuspromotescardiovasculardisease.ArecentstudyhasrevealedthatlossofCdkn2b,onecandidategeneatthislocus,resultsindevelopmentofadvancedatheroscleroticlesionswithlargenecroticcores[119].Furthermore,humancarriersofthe9p21riskallelehadreducedexpressionofCDKN2Binatheroscleroticplaques,whichwasassociatedwithimpairedexpressionofcal-reticulin,aligandrequiredforactivationofengulfmentreceptorsonphagocyticcells[120].AnotherrecentstudyshedslightonERK5asmasterregulatorofmoleculesinvolvedinefferocytosis[121].ERK5isamemberofthemitogen-activatedproteinkinase

Pleasecitethisarticleinpressas:J.Viola,O.Soehnlein,Atherosclerosis–Amatterofunresolvedin?ammation,SeminImmunol(2015),http://wendang.chazidian.com/10.1016/j.smim.2015.03.013