The emerging role of ECM crosslinking in T cell mobility as a hallmark of immunosenescence in humans Authors' names and affiliations

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The emerging role of ECM crosslinking in T cell

mobility as a hallmark of immunosenescence in humans

Authors’ names and aﬀiliations

Jean-Francois Moreau, Thomas Pradeu, Andrea Grignolio, Christine Nardini,

Filippo Castiglione, Paolo Tieri, Miriam Capri, Stefano Salvioli, Jean-Luc

Taupin, Paolo Garagnani, et al.

To cite this version:

Jean-Francois Moreau, Thomas Pradeu, Andrea Grignolio, Christine Nardini, Filippo Castiglione, et

al.. The emerging role of ECM crosslinking in T cell mobility as a hallmark of immunosenescence in

humans Authors’ names and aﬀiliations. Ageing Research Reviews - ARR, Elsevier Masson, 2017, 35,

pp.322-335. �10.1016/j.arr.2016.11.005�. �hal-01403839v2�

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ContentslistsavailableatScienceDirect

Ageing

Research

Reviews

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a r r

Review

The

emerging

role

of

ECM

crosslinking

in

T

cell

mobility

as

a

hallmark

of

immunosenescence

in

humans

Jean-Francois

Moreau

a,g,∗

_,

_Thomas

_Pradeu

a

_,

_Andrea

_Grignolio

b

_,

_Christine

_Nardini

c

_,

Filippo

Castiglione

e

_,

_Paolo

_Tieri

e

_,

_Miriam

_Capri

d

_,

_Stefano

_Salvioli

d

_,

_Jean-Luc

_Taupin

f

_,

Paolo

Garagnani

d

_,

_Claudio

_Franceschi

d

a_University_of_Bordeaux,_{CNRS-UMR5164,}₁₄₆_rue_Léo_Saignat,₃₃₀₇₆_Bordeaux,_France b_University_of_Rome_“La_Sapienza”,_Rome,_Italy

c_{Personalgenomics,}_Strada_le_Grazie,_Verona,_Italy

d_Department_of_{Experimental,}_Diagnostic_and_Specialty_Medicine,_{Interdepartmental}_Centre_{“L.Galvani”}_for_{Bioinformatics,}_Biophysics_and_{Biocomplexity,}

ViaSanGiacomo,12,UniversityofBologna,40126Bologna,Italy

e_Consiglio_Nazionale_delle_Ricerche,_Istituto_per_le_Applicazioni_del_Calcolo,_Rome,_Italy f_Université_{Paris-Diderot,}_INSERM_U1160,_Paris,_France

g_CHU_Bordeaux,_Place_Amélie_Raba-Léon,_Bordeaux,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1June2016

Receivedinrevisedform26October2016 Accepted7November2016

Availableonline19November2016 Keywords: Aging Immunosenescence Extracellularmatrix Mobility Immunecells

a

b

s

t

r

a

c

t

Immunosenescenceisthoughttoresultfromcellularagingandtoreﬂectexposuretoenvironmental stressorsandantigens,includingcytomegalovirus(CMV).However,notallofthefeaturesof immunose-nescenceareconsistent withthisview,and thishasledtotheemergenceof thesistertheoryof “inﬂammaging”.TherecentlydiscovereddiffusetissuedistributionofresidentmemoryTcells(TRM)

whichdon’trecirculate,callsthesetheoriesintoquestion.ThesecellsaccountformostTcellsresidingin barrierepitheliawhichsitinandtravelthroughtheextracellularmatrix(ECM).Withalmostallstudiesto datecarriedoutonperipheralblood,theage-relatedchangesoftheECMandtheirconsequencesforTcell mobility,whichiscrucialforthefunctionofthesecells,havebeenlargelyignored.Weproposeanupdate ofthetheoreticalframeworkofimmunosenescence,basedonanovelhypothesis:theincreasingstiffness andcross-linkingofthesenescentECMleadtoaprogressiveimmunodeﬁciencyduetoanage-related decreaseinTcellmobilityandeventuallythedeathofthesecells.Akeyelementofthismechanismis themechanicalstresstowhichthecellcytoplasmandnucleusaresubjectedduringpassagethroughthe ECM.Thishypothesisisbasedonan“evo-devo”perspectivebringingtogethersomemajorcharacteristics ofaging,tocreateasingleinterpretiveframeworkforimmunosenescence.

Contents

1. Introduction...323

2. Agingandimmunosenescence:currentknowledgeandthebiasesofpreviousstudiesbasedonblood...324

3. ThetightconnectionbetweenimmunecellsandtheECM...325

3.1. Mechanicalstressonthenucleus,thelargestandmostrigidcellcomponent...325

3.2. Lessonslearnedfromvarioushereditaryimmunedeﬁcienciesinwhichcellmobilityisaltered...326

4. Immunosenescence,cellmobilityandage-relatedchangesintheECM:the“mesh”connection...327

4.1. ECMchangesovertime:how,whenandwhy?...327

4.2. ConsequencesofECMalterationswithage...327

4.3. ECMandtheC.elegansmodelofaging...328

∗ Correspondingauthor.Presentaddress:CNRS-UMR5164ImmunoConcept, Bor-deauxUniversity,146,rueLéoSaignat,33076BORDEAUXCedex,France.

E-mailaddress:jfmoreau@u-bordeaux.fr(J.-F.Moreau).

http://dx.doi.org/10.1016/j.arr.2016.11.005

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4.4. ECMandthenakedmoleratmodelofaging...328

4.5. Hyaluronanscanalsobeinﬂammatory ... 328

4.6. ECM,mechanotransductionandthemobilityofimmunecells...328

5. ConsequencesofthelowermobilityofT-lymphocytesandtheirhigherdeathrate...330

5.1. Necrosis,apoptosis,pyroptosisandinﬂammasomeactivation...330

5.2. Tlymphocytedepletionanditslinktohomeostaticproliferationandautoimmunity...330

6. Conclusion...331

Conﬂictsofinterest ... 331

Acknowledgments...331

References...331

1. Introduction

Immunosenescence is defined asage-related changes in the immunesystem.Itisassociatedwithaprogressivedeterioration oftheabilitytomountimmuneresponsesandwithahigher mor-talityrateintheelderly.Immunosenescenceiscurrentlythoughtto dependonlifelongantigenload,leadingtothesenescenceofcellsin theimmunecompartment,withaprominentroleattributedtothe chronicanti-cytomegalovirus(anti-CMV)response.Thereseemsto beanincreasinguseofimmuneresourcesallocatedtotheanti-CMV responsewithaging,a processthat ultimatelyleadsto exhaus-tion.Thecauseremainsunclearandinhumansthefewstudies examiningthepresenceofviralreactivationintheblood,found itnegative.Moredataarethereforeneededinthefieldofhuman aginginordertoconcludeonthispoint(McVoyandAdler,1989; Stoweetal.,2007;PawelecandDerhovanessian,2011;Parryetal., 2016).TheroleofCMVinimmunosenescenceisclearlyimportant, but,ratherthanbeingdirectlycausal,canalsobeinterpretedas aconsequenceofmoregeneralage-relatedchangesinthe three-dimensionalmicroenvironmentinwhichmostimmunecellsare mobileandoperate,theECM.Immunologistshaveneglectedthe implications ofsuchchanges, partly becausemost of the stud-iescarriedoutonimmunosenescence,atleastuntilveryrecently, focusedonbloodbecauseitisthemostaccessiblesourceofcellsand biologicalfluidinhumans.Althoughofvalue,thesedata,leadtoan overestimatedqualitativeandquantitativeimportanceofthis com-partmentintheunderstandingoftheimmunesystemphysiology. TherecentdiscoveryofresidentmemoryTcells,orTRM,showed

immunesurveillancetobelargelylocaland,therefore,not read-ilyaccessiblethroughstudiesonblood[seeforreview(Carbone, 2015)].

Here, we argue that efforts to decipher immunosenescence mustconsiderboth bloodand theECM.TheTRM arelocatedin

theECM,and theknownbiochemicalandbiophysical modiﬁca-tionstothismediumassociatedwithagingconsequentlyhampers localimmunesurveillancebythesecells.ECMproteinsand proteo-glycanshavewell-documentedrolesinscaffolding,buttheyalso haveaprofoundeffectoncellbehavior,throughinteractionswith secreted ligands or cell-transmembranereceptors, in particular integrins.We suggestthattheprogressiveand irreversible age-relatedchangesintheextracellularmatrixmayactuallyprovide aunifyingframeworkexplainingallthemolecularandcellular fea-turesofimmunosenescence.Thekeypointisthatfortheimmune cellstobefunctional,theymust befree torecirculate,navigate and restwithin theextracellular matrix, in tissues and organs. Thispoint is instrumental in tissue surveillance andprotection (Ariotti etal.,2012)evenin theabsenceofperipheral lympho-cytes(Steinbachetal.,2016).Wewillconsiderimmunosenescence withinthisframework,focusingontheadaptiveimmunesystem andTcellsinparticular,eventhoughthesecellsareneithertheonly onestobeaffectedduringagingnortheonlyonesconcernedwith mobility.

Wearguethatthemobilityofimmunecellsinnon-lymphoid tissuesis anecessary elementforeffective immunity.Alackof immune cell mobility, either intrinsic, as in hereditary defects affecting actin remodelingfor example as wewill seelater, or extrinsic,asinaging,resultsinanimpairmentofimmuneresponses. Nothree-dimensional(3D)modelofderegulatedcellmobilityhas everbeenproposedorexploredinthecontextof immunosenes-cence.Weshowherethatourhypothesisismoreconsistentwith theavailabledatathancurrentalternativetheories.Wehopethat thishypothesiswhichisbasedonreviewsoffieldsthathavenot hithertobeconnectedtogetherwillpromotefuturestudies,insilico andinvitro,tovalidatethistheoryexperimentally.The3Dmodel canreconcilemanyfeaturesofaging,suchasthealteredresponses tovaccination,whichisinessencebothamemoryandalocal pro-cess,anddysfunctionsofperipheraltolerance(autoimmunity).The chronicprocessofTcelldeathduetomechanicalstresswithinthe cross-linkedmeshoftheagedECMmayalsoaccountforactivation oftheinflammasome(IL1,IL18,NF␬B),leadingtoinflammaging, andtoastateofimmunedeficiencytypicalofagedsubjects.These twoelementstogetherunderliethephenomenonofviral reactiva-tion(atthebeginninglocalandultimatelysystemic)leadingtothe clonalamplificationofCD8+_T_cells_and_an_increase_in_the

propor-tionofmemoryTcellsfoundintheblood(Sylwesteretal.,2005; Nikolich-Zugich,2008;Fulopetal.,2013;Fulopetal.,2015).

AlargeamountofTcellsinthebodyaretissue-residentmemory Tcellsthatdon’trecirculate,asdemonstratedbythemostrecent studies(ThomeandFarber,2015;ParkandKupper,2015;Carbone, 2015;Steinertetal.,2015;FanandRudensky,2016).Physiological mobilityinECManditsimpactonTcellsurvivalanddifferentiation arethereforeoftheutmostimportance,includingforlocal anti-CMVdefense(ThomandOxenius,2016).Tcellsurvivalisimpaired in veryconstrained environments, astheforced passageof the cellsinsuchconstrainedconditionsleadstomultipledamageto theplasmamembraneandnucleus,potentiallyculminatingincell death(Denaisetal.,2016;Raabetal.,2016).Inflammasomesare activatedinresponsetoincrementalproductionofdangersignals comingeitherfrominsideoroutsidethecells(Ostanetal.,2015) andleadingtoproductionofIL1,IL18aswellastheactivationofthe NF␬Bpathwaytypicalofinflammaging(FranceschiandCampisi, 2014).Furthermore,limited mobility decreasesthe numbersof themostneededTcellslocallypresentintissues,leadingto:(i) viralreactivationnotnecessarilydetectedinblood,duetoalackof properlocalimmunosurveillanceasshowninhereditaryimmune deficienciesresultinginseverelyimpairedlymphocytemobility; (ii)clonalexpansionofa verylimitedrangeofTcellsfollowing antiviralresponses;(iii)repertoirereductionduetohomeostatic forcesintheabsenceofthethymus,ashomeostasisispurelyabout maintainingcellnumbers,nottheirdiversity.Allthesefactorsare additionalfeaturestypicalofaging,mutuallyenhancedinavicious circlethat,wesuggest,ismediatedbyage-relatedECMdegradation andadirectconsequenceofimpairedlymphocytemobility.

Wewilldevelopthisideaanditsconsequencesthroughaseries ofsteps.Wewillﬁrst(Section2)discussagingbyfocusing,in

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par-ticular,ontheagingoftheimmunesystem(immunosenescence). Wewillrelatetheimportanceofimmunecellmobilitytothe mech-anismsunderlyingECMagingandcross-linking,whichincreasethe constraintsoncellmobility.

WewillhighlightthefunctionalconsequencesoflowerTcell mobilityandTcelldeath,throughwell-knownhereditaryimmune deﬁcienciesresultinginimpairedTcellmobility,suchasDOCK8, Coronin-1,CDC42orPGM3deﬁciencies(Section3).

Wewillthen(Section4)associatetheimpairedmobilityofT cellswithECMaging.Finally(Section5),wewilldiscussthelikely speciﬁcconsequencesofthislackofmotilityandinducedcelldeath forestablishmentoftheimmunosenescencephenotype.

2. Agingandimmunosenescence:currentknowledgeand thebiasesofpreviousstudiesbasedonblood

Overthelast30years,considerableeffortshavebeenmadeto understandtherelationshipbetweenagingandthedeclineofthe immunesystemandthecontributionofimmunosenescencetothe phenotypesobservedinagingindividuals(Franceschietal.,2000; Franceschietal.,2000a;Salviolietal.,2006).Thesephenotypes includetheaccumulationofCD8+_CD28−_cells,_CMV_{seropositivity,}

andaninversionoftheCD4/CD8ratio,partoftheimmune risk proﬁle(IRP)thatseemstopredictmortalityinpeopleovertheage of65(Hadrupetal.,2006).

Onekeyquestionconcernstheextenttowhichthymicmature lymphocyteoutputcontributestoTcellhomeostasis,and there-fore,theextenttowhich age-relatedchanges inthis organcan beconsideredtodriveTcellaging.Maintenance ofthenaiveT cellpoolishighlydependentonthymicoutputinagingmice.In humansitseemstobebasedmainlyontheperipheraldivisionof pre-existingTcells,inaphenomenonknownashomeostatic prolif-eration,asdemonstratedincasesofneonatalthymectomy(Johnson etal.,2012;Sauceetal.,2012;denBraberetal.,2012;Thomeand Farber,2015;vandenBroeketal.,2016).Theglobalrepertoireof naiveCD4+_T_cells_remains_diverse_until_ninth_decade_of_life,_when

thereseemstobeanincreaseincellturnover,rapidlyfollowed byrepertoirecontraction.AlossofthymicTcelloutputcan, there-fore,bequantitativelycompensatedbyhomeostaticproliferationin ordinaryconditions,withoutfurtherconsequencesduetothewide diversityoftherepertoire.However,homeostaticproliferation can-notcompensateforalossofTcelldiversity.Inelderlyindividuals witha continual, progressive,stochastic loss of T lymphocytes duetoanexternalcause,andcharacterizedbyacumulativeeffect overtime,homeostaticproliferationoftheremainingcells accel-eratesthelossofTcelldiversity,bydilutingoutexistingminority clones(GoronzyandWeyand,2005).Regardlessoftheactualage ofthepatient,advancedHIVinfection,characterizedbyamassive andcontinuouslossofTcells,seemstoreproducesomefeatures ofaging,withunderlyingimmunosenescenceandinﬂammaging (NixonandLanday,2010;ZapataandShaw,2014).Therefore,both in aging subjectsand in patientswith advanced HIV infection, immunosenescenceoftheadaptiveimmunesystemisnota sim-pledeteriorationoftheimmunesystem.Instead,itresultsfrom adynamicdriftunderthepressureofcontinuousexposuretoan antigenicloadand an increasingly limited capacity togenerate newTCR-bearingcells,leadingtotheaccumulationofmemoryT cellsandanage-associated declineinTcellrepertoirediversity (Yageretal.,2008).Notably,decreaseinnaiveTcelllevels, lead-ingtorepertoireshrinkage,hasalsobeenreportedinagingapes (Cicin-Sainetal.,2007).

Thecausalmechanismsunderlyingtheseadaptationshaveyet tobeidentiﬁed,butarealmostcertainlydiverse.However, home-ostatic proliferation to correct imbalances in the number of T cellsinvolvestherecognitionofself–determinantsbynaiveTcells

(Richards et al.,2016), whichmayconstitute animportantlink betweenagingandautoimmunity(Khiongetal.,2007).

Theenvironmentalcontextinwhichthecellsarefoundmust alsobeconsidered,inadditiontothereportedcell-autonomous defectsandstem-cellaging[seeforreview(Montecino-Rodriguez etal.,2013).Theimportanceofcellenvironment ishighlighted bytworemarkableexamples.Firstly,mouseCD4+_T_cells

gener-ated fromhematopoietic stem cells (HSC) from old donorsare functionalinyoungbutnotinoldrecipients(Eatonetal.,2008). Secondly,changesintheepithelialcomponentofthethymus,the lymphopoieticorgan,haveshowntobecrucialfortheearly pro-gressivedecreaseinthymicoutputwithage(Hamazakietal.,2016; Youmetal.,2016).

Immunosenescenceisalsoinfluenced bythegeneral mecha-nismsofagingoccurringinthebody,thoughthesemechanisms remain elusive (Grimm, 2015; Cohen, 2015). Severalhallmarks ofaginghavebeenidentified,allofwhichhaveprofounddirect or indirect effects on the immune system (Lopez-Otin et al., 2013;Kennedyetal.,2014).Thefirstmechanismofagingtobe identifiedwascellularsenescence,inwhichtelomereshortening limitsthenumberofreplicationcycles(Hayflickand Moorhead, 1961;Campisi,2013).SenescentcellsthathaveaccumulatedDNA damagehaveasenescence-associatedsecretoryphenotype (SASP), characterizedbytheproductionandsecretionoflargeamountsof proinflammatorycytokines,matrixmetalloproteinases(MMP)and othersolublemediators(vanDeursen,2014).Senescentcells accu-mulateinolderindividuals,andthisisthebasisof“inflammaging”, aconceptputforwardbyoneofus(CF)tostressthecloselinks betweenagingandchronicinflammation(Franceschietal.,2000b; Salviolietal.,2006;Franceschietal.,2007).Thestateofchronic inflammationthatisahallmarkofaginginhumansaccountsforthe comorbidities(Fig.1)andmortalityassociatedwithagingamong which atherosclerosis,osteoporosis,osteoarthritis, diabetes[see forreview(FranceschiandCampisi,2014;Kennedyetal.,2014)]. However,cellularsenescencealonecannotaccountfor immunose-nescence.

ThedecreaseinnaiveTcellsandtheincreaseinmemoryTcells canbothbeexplainedbyasustainedlossofcellsinacontextof chronicimmuneresponsesassociatedwithadecreaseinthymic output(Nikolich-Zugich,2008;Fulopetal.,2013).This immunod-eficiencywouldaccountforthestrongassociationbetweenCMV seropositivityandmortalityduetocardiovascularcausesobserved intheelderly(Savvaetal.,2013).ChronicCMVreplicationmaybe seenasanindirectconsequenceoftheslowdevelopmentofthis immunodeficiency,aslatentvirusesarereactivatedonceacertain thresholdofimmunodeficiencyisreachedasshownalsoinmouse models(Policetal.,2001).Fromthisstandpoint,CMVshouldnot beseenasthecausalagentofimmunosenescence,althoughwe acknowledgethisvirusandtheimmuneresponsetoit,contribute torepertoireshrinkageandinflammation(Fulopetal.,2013).

Crucially,thecurrentoverallviewofimmunosenescenceis par-tialbecausemost,ifnotall,studiesonaginginhumansarebasedon bloodsamples,forpracticalreasons.However,Tcellsintheblood aresubjectedtostrongselectionthroughtrafﬁckingregulation[see forreview(ThomeandFarber,2015)].Thetissue-residentmemory Tcells(TRM)(Sathaliyawalaetal.,2013),whichhaveyettobe

stud-iedinagingresearch,areofparticularimportancehere(Gebhardt etal.,2011).Intissue,TRMaremorenumerousthanTcells

recircu-latingfromtheblood(Steinertetal.,2015),andtheymayremain withintissuesfortheentirelifetimeoftheindividual.Thistissue retentioniscontrolledbyCD69expressionandthedownregulation offactorspromotingtissueegress.Itisdevelopmentallyregulated throughexpressionofthespeciﬁctranscriptionalregulatorsHobit andBlimp1(Mackayetal.,2016).Thelymphocytesresidentin tis-suesincludenotonlyTcells,butalsoNKT,orevenNKcellsinthe liver,reproducingthediversityofthesubpopulationsknowntobe

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ECM

-cross-linking -glycation

-growth factor storage - MMP activation -LMW hyaluronans -granzyme B Diabetes Cancer Sarcopenia Viral reactiva -tions Frail

ty

CV diseases Neurode-generation

AGING

Altered cell communicat

ions _instabGenomic_ili_ty

Telomere shrinkage

Epignetic alterations Loss

proteostasis Deregulated nutrient sensing Mitochondrial dysfunction Stem cell exhaustion Cellular senescence

IMMUN

OSENES

CENC

E

-inflammaging

-inflammasome activation - decreased naive T-cells -inflation of memory T-cells (T_EMRA) - decreased TCR diversity -CD4/CD8 ratio inversion

Fig.1.Relationshipsofimmunosenescencewithagingmechanismsandcomorbidities.AgingmechanismsfollowtheninehallmarksofagingestablishedbyLopez-Otin (Lopez-Otinetal.,2013).Externalcirclesdepicttheco-morbiditiesassociatedwithagingwhichalsocooperatetomodulateagingphenotype.TheECManditsalterations linkedtoagingwillconstraintimmunecellmobilitywhileinducingcelldeath.ECMalterationisahallmarkofagingandthereforeacrucialprocesstobetterunderstandthe phenomenonofaging.FundamentalmechanismsassociatedwithECMagingarerepresentedintheexpandedbox,inrelationwiththealteredcellularcommunications.ECM servesnotonlyforthecellstomigratewithin,butalsoforgrowthfactorsstorageandreceptoranchorageasitisthecaseforintegrinsandCD44.

presentinbloodandbarriertissues(Gasteigeretal.,2015;Fanand Rudensky,2016).Tissue-residentlymphocyteshavebeenfoundin thegastrointestinaltract,lungs,skinandreproductivetract(Farber etal.,2013)[seeforreview (SchenkelandMasopust,2014)and (Clark,2015)]butalsointhebrain(Steinbachetal.,2016).Inmouse, theyhavebeenshowntobeakeyelementinimmunedefenses againstall microbes,includingCMV (Smith etal., 2015) (Thom etal.,2015).Inbothmiceandhumanscytomegalovirusinduces TRMparticularlyinmucosaltissueswhichareimportantviral

sanc-tuariesandentrysites(ThomandOxenius,2016).Theyseemto functionasanorgan-autonomousfirstlineofdefenseeveninthe absenceofcirculatingCD8+memoryTcells(Steinbachetal.,2016) showingthatrecirculationofthesecellsbetweenthebloodand thesetissue-compartmentsaredispensableforefficientorgan pro-tection.KnowninmousemodelofCMVinfection,virallatencyof CMV,canpromotesthecontinuous,low-levelrecruitmentof cir-culatingCMV-specificTcellstotheTRMpopulationofthesalivary

glandmaintainingapoolofTRMatthesiteofviralreplication(Smith etal.,2014),apointwhichremainstobeformallyproveninhuman aging.

Given the crucial importance of TRM, any exploration of

immunosenescenceshouldtakeintoaccounttheECM,the envi-ronmentinwhichthesecellsarefound.Thisplacingofimmune systemphysiologyintocontextisofvitalimportance,butstillrarely (ifever),doneinstudiesonimmunosenescence.

3. ThetightconnectionbetweenimmunecellsandtheECM

3.1. Mechanicalstressonthenucleus,thelargestandmostrigid cellcomponent

Cellsmaybecarriedalonginamobilemedium,suchaslymph orblood,butherewewillexcludesuchpassivemobility,tofocus insteadontherequirementsfor theintrinsic motilityofTcells, particularlywithintheECM.The trans-endothelialmigration of lymphocytesisrelevantinthiscontext,becauseofthe

biochem-icalandbiophysicalnatureoftheECMinthevesselwall(Kohn etal.,2015).

We willdeal hereexclusivelywithT cells, butmany differ-ent cell types from both the adaptive and innate arms of the immunesystemaremotilewithintheECM.Neutrophils,for exam-ple,areprobablythemostmobileofallimmunecells,recirculating frequently and rapidly between the bone marrow, blood and then tissues. These cells display an age-related loss of migra-torycapacity,withpredictableconsequencesaging(Sapeyetal., 2014).Neutrophilsarehighlydeformableandcancrossporesonly microns in diameter (Rowat et al., 2013), due tothe flexibility oftheirnuclearmembrane,whichlackslamin-A,amoleculethat restrictsnucleardeformability,therebylimitingmigrationthrough constrictionsandtherateof3Dmigration(Haradaetal.,2014). Thereisadelicatebalancebetweenthemechanicalprotectionfrom ruptureaffordedbythepresenceoflamininthelamina,limitingcell motilityandnuclearplasticityallowingmovementsofcellsthrough mesh(GerlitzandBustin,2011).Unsurprisingly,laminopathies,an heterogeneousgroupofhereditarydiseasescausedbymutations ofthelamin-Agene,areoftencharacterizedbybothaccelerated agingandhighlevelsofinflammation(BurtnerandKennedy,2010). Tlymphocytesdisplaylamin-Aexpressionwhenactivated,butnot whenresting,possiblyreflectingdifferencesinmotilitybeforeand afteractivationdependingoncelllocationandfunction.Thefew studiesfocusingonthelymphocytecompartmentinlaminopathies have reportedmajor changesin T cellbehavior, due toaltered synapseformationandactivationprocesses,consistentwiththe hypothesisthatlamin-Aisrequiredforactivation(Rocha-Perugini andGonzález-Granado,2014).Lymphocytedevelopmental abnor-malitieshavealsobeenreportedinthelaminKOmodel(Haleetal., 2010),butarelationshipbetweenthequalityofimmuneresponses andthemobility ofimmunesystemcellshasyettobe demon-stratedinaffectedpatients.Matrixstiffness,lamin-Aproteinlevels inthenucleusandcellmobilityareknowntoberelated(Swiftetal., 2013),butthepotentialconsequencesoftheserelationshipsforthe immunesystemduringaginghavenotbeenexplored.

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Ithasrecentlybeenshownthatmigratingmammaliancellsare susceptibletoruptureofthenuclearmembranewhensubjected tostrongmechanicalconstraints,suchaspassagethroughsmall pores(3␮mindiameter).Suchruptureswouldresultinamixing ofthenuclearandcytoplasmcontents.Majoreventsofthistype arefrequent(90%ofthecellsinvitro,accordingtoarecentstudy (Denaisetal.,2016)),butseemtoberapidlyrepaired(alongwith theDNAdouble-strandbreakstheycreate)byspecificmechanisms (Raabetal.,2016)(Denaisetal.,2016),andamongwhichautophagy orproteasomerolescouldbehypothesized.However,therepair mechanismsmaynotbealwayscompletelysuccessful,potentially leadingtocelldeath, or canceroustransformation (Hniszet al., 2016;Zhangetal.,2015).Thestresstowhichthenucleusis sub-jected,inadditiontocausingDNAstrandbreaks,alsoinducesmajor inflammatorypathways(IL6andNF␬B),potentiallyaccountingfor theinflammatorystatusassociatedwithagingandaddingto cur-rentknowledgeofcellsenescence(LeBerreetal.,2012;McGregor etal.,2016).Thenucleus appearstherefore astheplace where geneticinformationisstoredbutalsoasamechanicalsensor[see forreview(BustinandMisteli,2016).Asobservedforthenuclear envelope,thestressontheplasmamembraneanditsmaintenance probablyplayimportantrolesalsoinaging(Lauritzenetal.,2015). AsdiscussedinSection4below,themobilecellsoftheimmune systemhaveparticularlyhighlevelsofexposuretotheserisks. 3.2. Lessonslearnedfromvarioushereditaryimmunedeficiencies inwhichcellmobilityisaltered

DOCK8isaguaninenucleotideexchangefactor(GEF)that acti-vatessmallGTPases(Cotéand Vuori,2007),and alsoactsasan adaptorintheTLR9-MYD88signalingpathway(Jabaraetal.,2012). DOCK8controlscellcytoskeletalfunctions(secretion,cell interac-tions)andmigration,andisexpressedonlyincellsoftheimmune system.DOCK8mutationsresultinacombinedimmunodeficiency syndrome.DOCK8-deficientpatientshaverecurrentotitis,sinusitis, andpneumonia,recurrentS.aureusskininfections,H.simplexorH. zosterinfections,andpersistenthumanpapillomavirusinfections. Mostpatientshave severe atopy withanaphylaxis, and several developsquamous-cellcarcinomas.Biologically,somehavehigh serumIgElevelsorhypereosinophilia,otherspresentlowcounts ofTcells andB-cells,andlowserumIgM levelswhiletheirIgG antibodyresponsesarevariable(Zhangetal.,2009).Tcell acti-vation,survival,proliferation andprimingbydendriticcells are affected.Other cells, including dendritic and NK cells, are also crippled,resultinginpoorcellcytotoxicityandlowlevelsof antivi-ralcytokineproduction.Notably,DOCK8-deficientdendriticcells migratepoorlytothelymphnodes(Lambeetal.,2011;Randall et al., 2011). Microscopy observations of T cells from patients, migrating withinthe three dimensionsof the dermis microen-vironment in human skin biopsy samples, showed that these cells had abnormalelongated shapesand long migration times withinthemesh,phenotypesobservedinnormalcellsafterDOCK8 silencingwithsiRNA.Remarkably,DOCK8-deficientcellssenseand migratetowardaSDF-1chemokine(CXCL12)gradientnormallyin two-dimensionalandliquidenvironments.Moreover,in3D envi-ronments,butnotinliquidmedium,Tcellsfromnormalindividuals inwhichDOCK8issilencedinduceaspecificformofdeathknownas “cytothripsis”(Zhangetal.,2014a).Thistypeofcelldeathresults fromtheexertionofmechanicalforcesontheplasmacell mem-braneandthemorerigidnucleus,leadingtotearingoftheplasma membrane.The elongatedcellphenotypeleading todeathalso occurswhenTcellsmigratethroughpores,agarose,ICAM-coated orcollagen-coatedsurfacestowhichtheyadhere,demonstrating aclearrelationshipbetweenshapeandlocalconstraintson mobil-ity.Thus,theabnormalshapeanddeathofcellslackingDOCK8 areassociatedwithmovementconstraintsduetoaconfinedspace,

observedinthedermis,accountingforthephenotypeofpatients, withtheirhighfrequencyofskindiseases(Mouwetal.,2014).

DOCK8activatesCDC42,whichregulateslymphocyteshapeand cytoskeletalstructuresduringcellmovements,includingdendritic cellmigration(Haradaetal.,2012).CDC42thenactivatesseveral effectors,includingP21-activatedkinase(PAK)andthe Wiskott-Aldrich Syndrome Protein (WASP). Knockout of the small Rho GTPaseCDC42 reproduces someofthefeatures ofDOCK8 defi-ciency,whereasWASPlossfromTcellsdoesnot(Humblet-Baron etal.,2007).However,WASPdeficiencyisassociatedwith abnor-malimmuneresponses,reflectingthecomplexinterplaybetween theseproteinsintheorchestrationofcellmobility.

Similarly,Coronin-1(Coro1)deficiencyleadstoapronounced immunodeficiencyphenotyperesemblingthatofDOCK8-deficient patients(Fögeretal.,2006;Shiowetal.,2008;Hogquist,2008). Coro1 regulatesactin polymerization. Mutation of the CORO1A genecausesprofoundperipheralTcelllymphopenia,thoughtto beduetoaninabilityofTcellstomigrateoutofthethymusandto enterandleavelymphnodes.However,thesecellswerealsoshown tobegenerallylessmobileinthepresenceofthismutation.

TheimmunedefectinDOCK8-deﬁcientindividualsprincipally concernsthemaintenanceoftheTRMcompartment,butinnormal

individuals,itcouldalsorelyonECMqualityandquantitywhichare speciﬁctoeachtissue(Bonnansetal.,2014).AlterationstotheECM wouldmodifythemobilityofcellsthroughthismatrix,inasimilar mannertoDOCK8mutation.Inaddition,themobilityofimmune cellsisrequiredforcorrectactivationofTcellsandisapreliminary stepforcontactbetweenTcellsorTregcellsandDCsinsecondary lymphoidorgans(Sixt,2011;Kastenmülleretal.,2012;Hondaetal., 2014;Liuetal.,2015).ECMalterationsmayalsoaffectdiverse pro-cesses,includingtheformationofthethymicepithelium,which playsakeyroleinTcellproduction(Shenetal.,1994;Mouwetal., 2014).

Thevariousdegreesoflymphopeniaobservedatdifferentsites inthebody(spleen,skin,etc.)inDOCK8-deficientpatients prob-ablyresultfromacombinationoffactorsdifferinginmagnitude betweenpatients.Lymphopeniainbloodandtissuesisassociated withpoorer controloverlatentviruses,in turntriggeringacute antigen-drivenclonalamplificationandinflationoftheTEMRA

com-partment.In thelong term,lymphopeniamaybe compensated by homeostatic proliferation and/or thymic output, depending on the age of the patient, but with a change in their respec-tivefrequencies.Indeed,CD8+Tcells thatareCD57+₍_Brenchley et al., 2003), CD57+_/CCR7−_/CD27− ₍_Papagno _et _al., ₂₀₀₄_), _or

CD45RA+_/CCR7−_/CD27−_/CD28−₍_Rufer_et_al.,_2003;_Romero_et_al., 2007)display the greatest expansion in vivo, as demonstrated byTCR excision circle(TREC) quantiﬁcation ortelomerelength measurement,but thesecells donot proliferate invitro follow-ingTCR-mediatedstimulation.DOCK8-deﬁcientCD8+_T_cell_subsets

havehigherproportionsofCD57+_CD27−_CD28− _cells_in_both_the

memoryandTEMRAcellsubsets,withnaivecellsdisplaying

unusu-allyhighlevelsofCD95expression(Randalletal.,2011).These features are similar to those observed in young HIV-infected patients(BoassoandShearer,2008;ZapataandShaw,2014)and intheelderly(Vescovinietal.,2014).

Actindynamicsandcelllongevityareknowntobelinkedin yeast,in aged miceand humans(Fögeret al.,2006; Brockand Chrest,1993).YeastswithslowactindynamicsaccumulateF-actin, releaseROSandhavehigherrateofcelldeath.Conversely, increas-ingactindynamicsinnormalcells canincreaselifespanby65% (Gourlayetal.,2004).Actindynamicsanditsregulationtherefore profoundlyaffectmanyaspectsoflymphocytelifeandsurvival,as notedsometimeagoforTlymphocytes(BrockandChrest,1993). Allthesestudiesconcentratedonintrinsicdefectsofcelldynamic butextrinsicfactorsshouldalsobeconsidered.

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Inthisview,abnormalitiesofcertaintypesofglycosylationdue toautosomalrecessivephosphoglucomutase3(PGM3)mutations (Zhangetal.,2014b)havealsorecentlybeendescribed.Affected patientspresentasyndromeresemblingDOCK8deﬁciency,with atopy, immune deﬁciency, autoimmunity and neurocognitive impairment,suggestingapossibledecreaseincellmobilityinthese patientstoo,potentiallyduetochangesintheextracellularmatrix witheffectsoncellmigration.

4. Immunosenescence,cellmobilityandage-related changesintheECM:the“mesh”connection

TheECMisanacellular3Dstructurecomposedoftissue-specific combinationsofalargenumberoffibrillarproteinssuchas col-lagens,proteoglycans,andglycoproteins(Hynes,2009).Collagen fibersmaintaintheshapeofthetissues,astheyareinextensible, butflexibleandstrong.Collagensarethemostabundantproteins intheECM(Bella,2016)Thereare28differentformsofcollagen, belongingtoeightclassesthatdifferbiochemicallyinthenatureof theiraggregatedformsandspeciescomposition.

Fibroblast-matrix interactions have long been known to be importantinaging(Baileyetal.,1998;Varanietal.,2006).These interactionsarecurrentlythefocusofintenseresearchin devel-opmentandcancerbiology.Inaging,stiffeningofthejointsand ofthevasculartreeinthekidney,retinaandheartareobserved, togetherwithchangesinbasalmembranepropertiesdueto pro-foundalterationstocollagenstructureandmetabolism,through thecross-linkingofﬁbers,inparticular.Moreover,therateof col-lagensynthesis isalsoaffected.It graduallyslowsdownduring childhood,reachingaplateauinadultsandthendecreaseinmost tissuesintheelderly.

Incancer,cross-linkingandsubsequentstiffeningoftheECM around the tumor seems to be a prerequisite for transformed cellinvasivenessandimportantlyfortheprotectionofthesecells againstimmunesystemcontrol(Leventaletal.,2009).ECM alter-ationsprobablyalsopromotecelltransformation(Seoetal.,2015). Cellularintegrins,whichbindtotheECM,providecancercellswith thepositivesignalsrequiredfortumorprogression(Chenetal., 2015).Thissituationresemblesthatdescribedforstemcells,the fateofwhichisalsolargelydeterminedbyECMinteractions(Guilak etal.,2009).

4.1. ECMchangesovertime:how,whenandwhy?

Thecross-linking theoryofaging datesfromthelate 1950s. According tothis theory, proteins, in particular collagens, lose theirfunctionsfollowingexcessivecross-linkingduetoreaction withaldehydemetabolites[see(Baileyetal.,1998)].Two differ-entmechanismsdrivethechangesinthemechanicalproperties of collagen with age. The first involves the specific enzymatic cross-linking of lysine or hydroxylysine, and is fundamental todevelopment.With age,a second,non-specific, cross-linking mechanismoccurs.Thismechanisminvolvesthenon-enzymatic chemicalreactionofprotein,peptides,aminoacids,nucleicacids, andlipidswithglucose,fructose,ascorbicacidorpentose(Selland Monnier,1989),inaprocessknownasglycation(Maillard reac-tion),togenerateadvancedglycationendproducts(AGEs)(Sjöberg and Bulterijs,2009).Glucosepane isthemostabundanttype of protein cross-link identified to date in vivo. It is found in the extracellularmatrix,whereitparticipatesincollagencross-linking. By increasing collagenstiffness and limiting porosity size, glu-cosepanecross-linksmayhavesignificantimplicationsforseveral age-related diseases, including cardiovascular disease,diabetes, andosteoporosis(Monnieretal.,2014;Boger,2015;Draghicietal., 2015).ProteinturnoverisanimportantdeterminantofAGE

accu-mulationinproteinsand,therefore,oftheirdegreeofcross-linking (GaggarandWeathington,2016).Collagenshaveaverylong half-life(117years forcartilage,15yearsfor skin),resultinginhigh andcumulativeratesofglycatedproductaccumulationintheECM (Verzijletal.,2000).Thisaccumulationisacceleratedby hyper-glycemiaindiabeticpatients,andthisisthoughttobethemajor causeof higher morbidityand mortalityin thesepatients. Dia-betic patientshave impairedtissue repair mechanismsand are knowntobepronetoskininfections.Theprevalenceofdiabetes increaseswithage,potentiallyworseningagingoutcomesoverall. Glycationisthoughttooccurmostlyintheextracellular environ-ment,butproteinswithincellsmayalsobespeciﬁcallyglycated. Thisisthecaseforvimentin,whichseemstobeahighlysensitive targetforchemicalglycation,butwithahighturnover,likely lim-itingtherelevanceofthisfactorinourdiscussion(Kueperetal., 2007).Thisobservationis,however,ofinterestwhenconsidered togetherwiththoseforlamins,asbothmoleculesplaykeyrolesin nuclearenvelopebiology.Inaddition,glycatedcollagenscan oxi-dizelipids,generatingmoleculessuchasmalondialdehyde,which hasa longhalf-lifeand diffusesaway toreactwithproteinsor nucleicacids,therebymodifyingtheirbiologicalproperties. Rele-vanttocellmobility,invitrotreatmentwithmethylglyoxal,another oxidizingagent,hasbeenreportedtodecrease celladhesionto matricesby70–90%(Bailey,2001).

Proteoglycansareanotherabundantcomponentofthe connec-tivematrix involved inthe age-relatedchanges tothephysical propertiesoftissues.Throughtheirelectriccharge,these compo-nentsof theECMare alsoimportantfor thebindingof growth factors,suchasIGF1,totheirscaffolds(Parkeretal.,1998)andfor thereleaseofIL1alphafollowingECMmodificationbygranzymeB (McElhaneyetal.,2012).Decorin,themainproteoglycaninskin, regulates collagen matrix assembly. This protein is distributed along collagen fibrils and the decorin glycoaminoglycan (GAG) chain controlsthedistance betweenthesefibrils. Reducing the lengthofdecorinGAGchainsreducesthedistancebetween colla-genfibrils,decreasingmeshporosity,asobservedinaging(Bailey, 2001).

4.2. ConsequencesofECMalterationswithage

Changesto“mesh”porosityduetocross-linkingoralterationsin relativecollagenspeciescompositionwouldbeexpectedto mod-ifycellmobilityprofoundlyintheECM.Thischangeinmobility wouldparticularlyaffecttheimmunecells,althoughmodiﬁcations areexpectedtobebothlocation-dependentduetovariable com-positionsofECMindistincttissues(Groulxetal.,2011;Soretetal., 2015;Hallmannetal.,2015)andcell-dependent,dueto variabil-ityintheadaptationofnucleusstiffnesstotheenvironment(Wolf etal.,2013)(Swiftetal.,2013).

Inmice,lowlevelsofgrowthhormoneproductionduetoan embryonicpituitaryglanddefectresultintheproductionofmice onethirdthesizeofnormalmice,butwitha40%higherlifespan (Flurkeyetal.,2001).Interestingly,collagencross-linkinglevelsin thetailwerefoundtobeonlyonethirdofthoseinnormalmice, whichsuggeststhatacomplexinterplaybetweenpituitarygland andECMexists.Asshownbythenakedmoleratmodelofagingseen below,onelinkisembodiedbyCD44signaling(Tianetal.,2013). Inthisregard,ageddwarfmicehaveCD4+ _and_CD8+ _memory_T

celllevels(CD44+₎_similar_to_those_seen_in_young_control_animals,

andmuchlowerthanthoseinagedcontrolmice(Flurkeyetal., 2001).Furthermore,verysignificantdifferencesareobservedinfive othertestsprobingtheimmunestatusoftheseanimals, support-ingtheconclusionthatinthesemice,thehigherlifeexpectancy andthebetterimmunestatusthanwild-typemice,arecorrelated withdifferencesintheECM.However,norelationshiphasyetbeen experimentallyconfirmedinthisfield.

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InEcuador,agroupofhumanswithlifelongIGF-1deﬁciency causedbyaGHreceptor(GHR)mutation(Laronsyndrome)have beenshowntobemuchmoresensitivetoinsulinthanage-and BMI-matchedcontrolrelatives,despitehavingahighpercentage ofbodyfat.Noneoftheseindividualswerediabetic,whereas6%of theirunaffectedrelativeswerediabetic,andonlyoneofthe20 indi-vidualswithGHRdeﬁciencydiedfromcancer,whereas20%oftheir relativesdiedfromthisdisease(Guevara-AguirreandRosenbloom, 2015).Interestingly,theoffspringofonecentenarianwasfoundto havelowlevelsofcirculatingIGF1bioactivity,inverselycorrelated withinsulinresistance(Vitaleetal.,2012).

Takenaltogether,thesedataareconsistentwitharoleforthe IGF-1pathwayinaging,butthisrolemaybeatleastpartlyindirect, andshouldconsiderthepossibilityofECMalterations.

4.3. ECMandtheC.elegansmodelofaging

In nematodes, mutations preventing insulin/IGF1 signaling, suchasdaf-2mutations,doublelifespan.Removalofthegermline precursorcellsalsoextendswormlifespan60%,probablyby alter-ingendocrinesignaling.Thesetwoeffectsareadditive,resulting inaquadruplingoflifespan.Bymanipulatingtheexpressionofa fewgenesfromtheinsulin/IGF1axislifespancanbeincreasedby afactorofsix,withnoapparentlossofhealthoractivity( Arantes-Oliveira,2003).Aboutadozenpathwaysareknowntobeimportant inaging,butmatrixremodelinghasbeenidentiﬁedasanessential signatureoflongevityinallspeciestested,includingnematodes, leadingtotheconclusionthatthepromotionof ECM conserva-tionishighlybeneﬁcial(Ewaldetal.,2014)andcouldserveasan additionaltargetinthecontrolofaging.

ThemolecularroleofECMinthepreventionofagingremains tobeunderstood,butdiversemechanismsappeartobeinvolved. Thesemechanismsmayberelatedtoresistancetooxidativestress ormayoperateattheinterfacebetweenseveralsignaling path-ways,includingthoseinvolvingCD44(Tianetal.,2013;Pontaetal., 2003),TGFbeta,boundIGF1,andintegrins.Theymayalsorelateto themechanicalrelationshipsbetweenthenucleusandtheECMas pointedoutbefore.TheabilityoftheECMtobindgrowthfactors isanotherkeyaspectthatcouldbemodifiedforresearchpurposes (Martinoetal.,2014).Inthisregards,parabiosisexperimentshave shownthatthetransferofbloodfroma youngmousetoanold mouseincreasesbraincellgrowth,promotesbrainplasticity, mem-oryformationandtherepairofdamagedspinalcord,andreverses theage-relatedthickeningoftheheartwalls.Theserejuvenation processesmayreflectareversalofthedegradationofECM func-tioninagedindividuals,includingthequenchingofROSandAGE, decreasesinECMcross-linking,andthereplenishmentoftheECM withgrowthfactors,suchasIGF1(Conboyetal.,2005;Loffredo etal.,2013;Villedaetal.,2014;Elabdetal.,2014;Scudellari,2015). Theeffectsprobablydifferbetweentissues,reflectingdifferencesin ECMcompositionandinterestinglyalsolinkedtothedistinctrates ofagingnotedfordifferentorgans(Ceveninietal.,2008).

Suchtreatmentwouldalsoreversethedeclineinimmunestatus associatedwithaging,leadingtoadecreaseininﬂammaging,the replenishmentofnaivematureTcellsandhematopoieticstemcells, andanabolitionoflatentvirusreactivation,buttheseeffectshave yettobedemonstratedexperimentally(ConboyandRando,2012). 4.4. ECMandthenakedmoleratmodelofaging

Thenakedmolerathasanexceptionallylonglifespan,atover30 years,muchlongerthanthefouryearsforrelatedmousespeciesof similarsize.Furthermore,nocaseofcancerhaseverbeenreported inthisspecies,despitemanyyearsofobservationofnakedmole ratcolonies.Thisremarkableresistancetocancerseemstobedue tothesecretionbyﬁbroblastsoflargeamountsofanECM

com-ponent,thehigh-molecularmassmoleculehyaluronan(HMMH), duetohighlevelsofsynthesisandlowlevelsofcatabolism.The hyaluronansynthaseofthemoleratdiffersfromthoseof13other speciestestedbytwoaminoacidsinthecatalyticdomain(N178S andN301S).Oneofthesedifferencesconcernsanasparagine(N) residuetotallyconservedinallotherspeciestested.Thesefindings shouldledtoasearchforpolymorphismsofthehyaluronan syn-thasegeneinhumansthatmightbeassociatedwithcentenarians (Tianetal.,2013).Theskin,heart,brainandkidneyofnakedmole ratsarehighlyenrichedinHMMH.Thedisruptionofsignaling path-ways,inducingthemalignanttransformationofmousefibroblasts (H-RASandSV40),doesnotleadtothetransformationofnaked moleratfibroblasts.However,theeliminationofhyaluronan over-production,byknockingdownexpressionofagenerequiredforits synthesisoroverexpressinggenerequiredforitscatabolism, ren-derstheresistantcellssusceptibletomalignanttransformations andleadstotumorformationinmice.Thisremarkablephenotype seemstoinvolvesignalingthroughthehyaluronanreceptorCD44. TheintracytoplasmicpartofCD44interactswithNF2,which par-ticipatesinapathwaymediatingcontactinhibition.Inaddition,the affinityofCD44tohyaluronaninnakedmoleratcellsistwicethatin mouseorhumancells.TRMdoexpressCD44,whichisahallmarkof

memoryTlymphocytes,raisingthepossibilitythatthehyaluronan effectmayalsobemediatedpartlybyimmunecells.

However,tothebestofourknowledge,nostudieshaveyetbeen carriedoutonthenakedmolerateimmunesystem,with investi-gatorsinsteadfocusingincell-intrinsiccluestocancerresistance ratherthan onextrinsicfactors,suchas theimmunesystemin relationtoECM.

4.5. Hyaluronanscanalsobeinﬂammatory

Hyaluronandegradationproductsatinjurysitescanstimulate theexpression ofinflammatory genes byvarious immune cells (Jiangetal.,2007).CD44seemstoberequiredfortheclearance ofhyaluronandegradationproductsinlunginjuryand transplan-tation,in which hyaluronan clearance maybeimpaired bythe absenceofdraininglymphvesselsinthegraft,resultingin persis-tentinflammationandrejection(JiangandNicolls,2014;Maltzman etal.,2015).Intype1diabetes,autoimmuneinsulitisisassociated withtheislet-specificdepositionofhyaluronan,whereasthe inhi-bitionofhyaluronansynthesispreventsthediseaseinmice(Nagy etal.,2015).HyaluronanfragmentsusebothToll-likereceptor(TLR) 2 and TLR4 tostimulate theexpression of inflammatory genes in macrophages(Scheibneret al.,2006).Low-molecularweight hyaluronanfragmentsandingeneraldegradationsproductsofECM (matrikines)arethereforecandidatesforadirectrolein inflammag-ing,mediateddirectlyorindirectlyasDAMPsthroughtheimmune system(Evankoetal.,2012;GaggarandWeathington,2016).

ECMalterations mayhaveindirectpro-inﬂammatory effects, bydisrupting theinteractionwithcellintegrins responsiblefor connectingthecellsurfacetotheactinnetwork.Interestingly,in dendriticcells,theabsenceofbeta2-integrin-mediated cytoskele-talorganizationleadstomembranecompartmentalizationandan absenceofassociationoftheGM-CSFreceptorwithactin, result-inginhigherlevelsofsignalingviathis receptorandconferring amigratorymaturationphenotypeondendriticcells, leadingto theTh1primingofnaiveTcellsandanhigherneutrophilsurvival (Morrisonetal.,2014).

4.6. ECM,mechanotransductionandthemobilityofimmunecells Besidesthemechanicalstressofthenucleusmentionedbefore, cells can be sensitive toECM ageing through others pathways importantforcellmigration(Friedletal.,2011).Themechanism by which cells sense ECM stiffness is called

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mechanotransduc-Loss of T-cells/ Lymphopenia

Inflammasome activation

-Hereditary defects of cell mobility -HIV infections

- Immunosupressive regimens -Chemotherapeutics -Ageing Homeostatic proliferation of T-cells T_CM Decreased mobility in tissue

Viral reactivations CMV, EBV, etc.

Autoimmunity

-inflammaging

- decrease of naive T-cell compartment -inflation of memory T-cells T_EMRA - decreased TCR diversity -increase of T-cells T_CM

-CD4/CD8 ration inversion linked to antiviral responsesT_C_M

Immunosenesce

nce in blood

ECM al

terat

ions

Fig.2. Roleofextracellularmatrixalterationsinimmunosenescence.TheincreaseinECMcross-linkingwithagingplacesconstraintsonthemobilityofimmunecells, accountingforthephenotypeassociatedwithaging.Othersituationsoftenencounteredinclinicalpracticemayalsoleadtothisphenotype(hereditarydefectsofcell mobility,andTcelldepletionasinHIVinfection,immunosuppressivetreatmentsorchemotherapy).

tion(Iskratschetal.,2014).Mechanotransductionplaysakeyrole inadjustingECMmechanicstocellbehaviororfunction,mostly through integrins (Humphrey et al., 2014).For this reason, 2D invitroexperimentalsettingsarenotentirelyrepresentativeof3D situationsinvivo,asreportedinpreviousstudies(Harunagaand Yamada,2011;Hortonetal.,2016).Mechanotransductionisalsoa potenttriggerofepithelialmesenchymaltransition(EMT)(Nelson andBissell,2006;BissellandHines,2011;ArendtandKuperwasser, 2015).Italsoplaysawidelyacceptedandstudiedrolein develop-ment(EMTtype1)(Dupontetal.,2011;Halderetal.,2012),(Piccolo, 2012;HeisenbergandBellaiche,2013;Porazinskietal.,2015).It hasbeencloselylinkedtotheprogressionofcancerstometastasis (EMTtype3)andimplicatedincancerinitiation(Seoetal.,2015; ArendtandKuperwasser,2015;BissellandHines,2011),butrarely associatedwithwoundhealing(EMTtype2),andimmunology.

Immunecellshaveanumberofspeciﬁcfeaturesofimportance in this context, and theirintrinsic mobility is closely linkedto

surveillance,asillustratedbythedescriptionsof immunodeﬁcien-ciesprovidedabove.LifeonEarthbeganwithsinglecells,some of which much later,grouped togetherand evolvedinto meta-zoans(DaviesandLineweaver,2011).Inmulticellularity,thereis aneedforcellstoanchorthemselvestogethertoachieve mechan-icalcoherence.Wecanstillseeevidenceofthestepsleadingto thedevelopmentofcomplexmulticellularindividuals from sin-glecells, in intermediateforms, fromChlamydomonas toVolvox (Kirk,2005)(SheltonandMichod,2014).Fromthismodel, exper-imentaldatashowthatECMplaysastrikingroleinthisprocess (HallmannandKirk,2000).Noinformationabouttheroleofthe ECMinimmunesystembiologyisavailable,withtheexception ofsecondarylymphoidorganphysiology,whichisnotconsidered here(Kastenmülleretal.,2012).Mobilecellsmightthereforebe expected to have evolved speciﬁc mechanismsmodulating the consequencesofanchoragewithintheECM.Anunderstandingof thesemechanisms would greatlyimprove theway we seeand

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understandimmunityandthepathophysiologyofmanydiseases, includingautoimmune(Sofatetal.,2015)andinfectiousdiseases andofcourseaging.

Insummary,themechanismsofECMcross-linkinginagingare wellestablished,buttheireffectsonimmunecellmobilityinthe bodyremainlargelyunknown.Givenrecentﬁndingsformemory residentTcells andthefunctionalimportanceofcelltrafﬁcking betweenlymphnodes,bloodand,aboveall,withintissues,we sug-gestthatalinkbetweenthesetwoaspectscouldaccountforthe featuresassociatedwithimmunosenescence.

5. ConsequencesofthelowermobilityofT-lymphocytes andtheirhigherdeathrate

5.1. Necrosis,apoptosis,pyroptosisandinflammasomeactivation The preservation or loss of membrane integrity in dying cells determines whether cell death is inflammatory (Wallach etal.,2016).Someofcellcomponentsleakingoutofcells have beenidentifiedasdamage-associatedmolecularpatterns(DAMPs). These components,together withpathogen-associated molecu-larpatterns(PAMPs),constitutethegeneric“dangersignals”that, accordingtothedangertheory(Matzinger,2002),aresensedby dendriticcells, leadingtoanupregulationoftheirexpressionof costimulatorymolecules on theantigen-presenting cell surface (PradeuandCooper,2012;Konoetal.,2014)necessaryfornaive Tcellactivation.Conversely,membraneintegrityismaintainedfor awhileduringearlyapoptosis,beforecaspase-mediated fragmen-tation.Thisallowsthemacrophagestoengulfandclearthedanger signals,thus preventinginadequateactivation of Tcells (Green etal.,2009).However,iftoomanyapoptoticcellsareproduced, overwhelmingtheclearancecapacityofthemacrophages,orifthis capacityisdecreasedforsomereason,thenapoptoticcellsmight notbeclearedrapidlyenough,resultingintheleakingofapoptotic bodies.ThesebodieswouldreleaseDAMPs,resultinginahighly inflammatoryenvironment.Fromourhypothesis,wecanpredict thatthesetwosituationswouldoccurinsynergyoverlong peri-odsduringaging,asECMremodelingwouldseverelyimpairthe mobilityoflymphocytesandmacrophages,ultimatelyleadingto thedeathofthesecellsinsituinresponsetomechanicalstress. Othertriggersmayalsobeimportant.Inparticular,HighMobility GroupBox1(HMGB1)isanuclearproteinreleasedbynecroticcells thatpromotescytokinereleasebyinteractinginflammatorycell recruitmentviaTLR4andCXCL12cellmigration(Schiraldietal., 2012).Thisandotherexamples(Limetal.,2015;Vacchellietal., 2015)demonstratethatinflammationmaybecloselyconnected toimpairedmobility,potentiallyleadingtotheestablishmentofa viciouscircleinthecontextsofagingandECMmodifications.

Furthermore,thereleaseofIL1betainducesahighly inflamma-toryformofcelldeathknownaspyroptosis,whichhasrecently beenshowntoaccountforthemassiveTcelllossand inflamma-torystatusofHIVpatients(Doitshetal.,2014).Theinnatemolecular partoftheimmunesystemsensestheseotherwisehiddencell com-ponents(Davisetal.,2011;LamkanfiandDixit,2012), whereas cells,suchasmacrophages,drivea viciouscirclebyresponding toIL1andfurtherdegradingtheECMleadingto“macroph-aging” (Franceschietal.,2000b).IL1betaandIL18areprototypical inflam-matorycytokinessecretedfollowingcytoplasmiccleavageofthe correspondingproproteinsbycaspase-1activatedfollowing poly-merisationofinflammasome. Thetranscriptionalactivation and expressionoftheseproproteinsandofinflammasomecomponents followssignalingthroughTLRandcytokinesindiversecells[for detailssee(Martinonetal.,2009)].IL1isthensecreted,activating signalingviaitsreceptor,throughtheNF-␬Bpathway,and trig-geringtheinflammatoryprogramintargetcells(Mathewsetal.,

2008).Sixtypesofinflammasomeshavebeendescribedinhumans, eachessentiallyspecificforanarrayofPAMPsorDAMPs,mostof whichareabundantmoleculeswithimportantfunctions,enabling theinflammasometosensecellularinjuries.Forexample,ATP,RNA, DNA,cholesteroldepositionandcrystalsareknowntoactivatethe NLRP3inflammasome,whichplaysamajorrolein atherosclero-sis(Zhengetal.,2014).Interestingly,theinflammasomehasalso beenreportedtosenseactindynamics,whichisessentialforthe detectionofintracellularpathogens(Kimetal.,2015).Asdescribed above,thelow-molecularweightproductsofhyaluronan break-down,aresultofECMinjury,bindtoTLR2,inducingproductionof pro-IL1andpro-IL18(Scheibneretal.,2006).Inflammasomeshave alsobeenshowntoacceleratethedeclineofthymicfunction(Youm etal.,2012).Insummary,thisarrayofobservationsshowsthetight intricacywhichexistsbetweenECMandinflammation,pledging foritsconsiderationinimmunosenescenceandaging.

Withinthisinflammatoryframework,thehighlevelsofIL6 con-sistentlyobservedinthebloodoftheelderlymaydirectlyreflect thenuclearstressresultingfromtheECMremodeling.Indeed,the magnitudeofnucleardeformationisrelatedtoexpressionlevelsfor aspecificarrayofgenes,themosttranscribedofwhicharehistones H4(A-D)andH3F,butalsoIL6(LeBerreetal.,2012).Nuclear enve-loperupturehasbeenshowntocauseDNAbreakageandrepairthat mightcontributetotheDNAdamageresponse(Zhangetal.,2015; Raabetal.,2016),butexchangesofmaterialbetweenthecytoplasm andnucleusmightalsoprovideasourceofinternalDAMPsdirectly sensedalongthesevariouspathways.

Overall,theubiquitousECMmodificationsassociatedwith col-lagenglycationandcross-linkingareprobablydirectlyorindirectly followedbyaseriesofeventsleadingtothechronicproductionof highlypotentinflammatorycytokines,underlyingthe inflammag-inganditsconsequencesseenintheelderly.

5.2. Tlymphocytedepletionanditslinktohomeostatic proliferationandautoimmunity

ChronicTcelllossinducesthreehighlyregulatedprocessesof Tcellreplenishmentinmammalswhichare:1)thematureTcell egressfromthethymus,2)theclonalampliﬁcationofcellsengaged inanimmuneresponse,and3)thehomeostaticproliferationofT cells.

Intheelderly,asthymicfunctionisabsent,Tcellcompartment replenishmentisdependentexclusivelyonhomeostatic prolifer-ation.Inthisprocess,existingTcellsproliferateintheabsenceof exogenousantigen,duetotheirintrinsicself-recognition proper-tiesresultingfromtheirpreviouspositiveandnegativeselection in thethymus(Vrisekoopet al.,2008; den Braber etal., 2012; Johnson etal., 2012).Homeostatic proliferation may,therefore, alsobelinkedtothedevelopmentofauto-immunity(Goronzyand Weyand,2012)astheTcellrepertoireisbuiltonaprincipleof basicbutlimitedrecognitionofself(Mason,1998),knownas auto-reaction[see(Pradeu,2012)].

Innormaladults,thisbasalautoreactivestatedoesnotleadto auto-immunediseasesbecauseofseveralmechanisms,collectively called“peripheraltolerance”, butmostly involvingregulatoryT cells,whichinhibiteffectorTcellfunctionandwhichhavebeen showntoaccumulatewithage(Sharmaetal.,2007).

Most of our insight into T cell dynamics replenishment originatesfromanalysesofTcellreconstitutionintheblood fol-lowingperipherallymphopenia,asobservedduringHIVinfection, chemotherapy totreat cancer, transplantation and aging. Lym-phopenia is known to break tolerance (Jones et al., 2013), as highlightedbyreportsforhematopoieticstemcelltransplantation (Matsuokaetal.,2010).Intheseconditions,theTcellswiththe highestafﬁnityforMHCplusself-peptidesproliferatefasterthan

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thosewithalowerafﬁnityleadingtodysregulatedimmunesystem activation.

Inmousemodels,homeostaticproliferationafterlymphopenia alsoinducesthespontaneousproliferationofnaiveandmemory Tcells butwithlittleauto-immunity(LeCampionet al.,2009), becauseoftheconcomitantexpansionofTregulatorycells(Tregs) tocontrolthis phenomenon(Piccaetal.,2006).However,ifthe expansionsofthesetwopopulationsweretobedissociated,then transientauto-immunedisordersarise.Thisiswhatisobservedin theimmunereconstitutioninﬂammatorysyndrome(IRIS),found inHIV-infectedpatientswithlowCD4+Tcellcountsgivenhighly activeantiretroviraltherapy(Shelburneetal.,2005)orinNODmice (LeCampionetal.,2009).

Tregsinteractwithdendriticcellsinthelymphnodes,inwhich theysuppresseffectorTcellpriming,subsequentlymigratingto non-lymphoidtissues,inwhichtheysuppresseffectorTcell func-tionslocally.ThesuppressionexertedbyTregsisnotspecifictothe antigen;itis,instead,highlydependentoncolocalizationwiththe effectorTcellstobesuppressed(Antunesetal.,2008).Tregsmigrate rapidlyfromthebloodtositesofinflammation,highlightingtheir strongdependenceonanormalmigratorycapabilitytomediate theirsuppressivefunction.Changesin theirmigration capacity, duetotheECMalterationsknowntooccurinaging,crippletheir regulatoryfunctions,leadingtohigherlevelsof auto-immunity. Tregsuppressivefunctionis,thus,highlydependentoneffective migrationmechanisms,whichmaybedisruptedbyECMalteration, therebyexacerbatinginflammatoryprocessesandpartly account-ingforage-relatedauto-immunity.

6. Conclusion

Tcellsarehighlymobilecellswithfunctionsinimmunitythat arehighlydependentontheirabilitytomigrateparticularlyfor thoseresidingintissues.WeargueherethatchangestoTcell migra-tioncapacityduetowell-characterizedECMchangesduringaging mayplayakeyroleintheagingprocess,bycripplinginteractions betweenimmune cells and preventingtheirtrafficking (Fig.2). Studiesofhereditaryimmunodeficienciesinvolvingalackof effi-cientactinremodelinghaveshownthatTcelllossresultsfromthe deathofmigratorycells.InadditiontotheconsequencesofTcell deathforinflammation,theprogressivedepletionofTcellsleads toviralreactivation(herpesvirus)andtriggersmechanismsofT cellreplenishmentthatmayleadtosomedegreeofautoimmunity. Thesemechanismsprovideinformation abouttheconsequences ofECMremodelinginfundamentalimmunologyaswellassome explanationfor immunosenescence,but theymayalsoserve as appropriatetreatmenttargets.Earlyin2015,twostudies convinc-inglyshowedthatprovidingthehostwithTcellsagainsttumorsin thecontextofascaffoldmatrixcreatedafavorableenvironmentfor thegenerationofeffectivehumoralandcellularimmuneresponses totumorantigens(Stephanetal.,2015;WeberandMulé,2015). Thisobservationreflectsalsotheexistenceoftertiaryectopic lym-phoidorgans,insynovialtissuefromrheumatoidarthritispatients forexample(Weyandetal.,2003),demonstratinghereagain,the three-dimensionalnatureofimmunity.

Conﬂictsofinterest

Noneoftheauthorshaveanyconﬂictofinteresttodeclare.

Acknowledgments

We would like to thank Maria Mamani-Matsuda, Myriam Capone, JenniferHoward, LynnChiu and Maureen O’Malleyfor helpfuldiscussionsandrevisionofthemanuscript.

ThomasPradeureceivedfundingfromtheEuropeanResearch Council(ERC)undertheEuropeanUnion’sHorizon2020research andinnovationprogram−grantagreementno.637647-IDEM.

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