CellandTissueResearch(2018)371:473–
488https://doi.org/10.1007/s00441-017- 2751-4
REVIEW
Ageistheworkofart?
Impactofneutrophilandorganismageonneutrophile xtracellulart rapf ormation
WeronikaO rtmann1&ElzbietaK olaczkowska1
Received:28August2017/Accepted:21November2017/Publishedonline:18December2017
#TheAuthor(s)2017.Thisarticleisanopenaccesspublication
Abstract
NeutrophilextracellulartrapsorNETsarereleasedbyhighlyactivatedneutrophilsinresponsetoinfectiousagents,sterileinflam mation,autoimmunestimuliandcancer.Inthecells,thenuclearenvelopdisintegratesanddecondensationofchromatinoccursthatde pendsonpeptidylargininedeiminase4(PAD4)andneutrophilelastase(NE).Subsequently,proteinsfromneutro-
philgranules(e.g.,NE,lactoferrinandmyeloperoxidase)andthenucleus(histones)bindtodecondensedDNAandthewholestructur eisejectedfromthecell.TheDNAdecoratedwithpotentantimicrobialsandproteasescanacttocontaindisseminationofinfectionandi nsterileinflammationNETswereshowntodegradecytokinesandchemokinesviaserineproteases.Ontheotherhand,overproductio nofNETs,ortheirinadequateremovalandprolongedpresenceinvasculatureortissues,canleadtobystanderdamageoreveni nitiationofdiseases.ConsideringtheprosandconsofNETformation,itisofrelevanceifthestageofneutrophilmaturation(immature, matureandsenescentcells)affectsthecapacitytoproduceNETsasthecellsofdifferentage-
relatedphenotypesdominateingiven(pathological)conditions.Moreover,theimmunesystemofneonatesandelderlyindivid- ualsisweakerthaninadulthood.IsthesamepatternfollowedwhenitcomestoNETs?
TheoverallimportanceofindividualandneutrophilageonthecapacitytoreleaseNETsisreviewedindetailandthesignificanceofthes efactsisdiscussed.
KeywordsNeutrophilextracellulartraps. Immatureneutrophils. Matureneutrophils. Neonates. Elderly.Scenescent
Introduction
Neutrophils,polymorphonuclearcells(PMNs),arethefirstle ukocytestoreachthesiteofinflammationwheretheyper- formtheireffectorfunctions,phagocytosemicrobesandkillthe mintracellularly.Alternatively,neutrophilsfightpathogensex tracellularlyineitheroftwoways:upondischargeofpotentantim icrobialsandproteasesfromtheirgranulesoronreleaseofneutro philextracellulartraps(NETs;KolaczkowskaandKubes201 3).
ThefirstreportonNETsrevealedthatneutrophilsstimu- latedbyagentssuchaslipopolysaccharide(LPS),interleukin8(I L-8)orphorbol12-myristate13-
acetate(PMA)formandreleasestructuressimilartothenetwo rk,hencetheirname
*ElzbietaKolaczkowskaela.k olaczkowska@uj.edu.pl
1
DepartmentofEvolutionaryImmunology,InstituteofZoologyandBio medicalResearch,JagiellonianUniversity,ul.Gronostajowa9,30- 387Krakow,Poland
(Brinkmannetal.2004).DetailedstudiesofNETsbyelectrons canningandconfocalmicroscopyaswellasproteomicanal- ysesshowedthatNETsarecomposedofthinchromatinfiberst hataredecoratedwithsome30neutrophilproteins,includingn eutrophilelastase(NE),bactericidal/permeability-
increasingprotein(BPI),defensins,cathelicidin(LL- 37),proteinase3andcathepsinGofgranularoriginandnucle arhistones(Brinkmannetal.2004;Urbanetal.2009) (Fig.1).NETscantakedifferentforms,fromabandform,bya cloud-
likestructure,whentheNETisfullyhydrated,toanetwork- likeshape,exceeding10–
15timesthevolumeofthereleasingcell(Brinkmannetal.2004
;BrinkmannandZychlinsky2012).Morerecentstudies,appl yingatomicforcemicroscopytore-
vealtheirnanoscaleproperties,reportedthatNETsarebran chingfilamentnetworkswithasubstantiallyorganizedporo usstructureandwithopeningsinthesizerangeofsmallpathoge ns(Piresetal.2016).Importantly,proteasesattachedtoNETss ecureassemblyofthewholestructureanditsme-
chanicalproperties.Whilesuchastructureincreasestheeffi- ciencyofcatchingpathogens,itcanalsofavorcollateraldam- age(Piresetal.2016).Thelatterobservationdirectlyrelatestop rosandconsofNETformation.
474 CellTissueRes(2018)371:473–488
Fig.1 B a s icc h a racteristicso f n e u t rophile x t racellulart raps( N ETs):
I)inducingfactors,II)involvedpathways,III)compositionandIV)fateofN ET-
releasingneutrophils.TheimagecapturesNETsformeduponLPSstimul ationofmurineneutrophils(greenarrowsextracellularDNA,reda rrows citrullinatedh iston e H3),sca leb a r50μm.PAMP
pathogen-associatedmolecularp attern,D AMPd amage-associatedmo- lecularpattern,PMAphorbol12-myristate13-acetate,ROSreactiveoxy- genspecies,N O nitricoxide,PAD4peptidylargininedeiminase4,MPOmye loperoxidase,BPIbactericidal/permeability-increasingprotein,LL- 37c athelicidinsc athelicidin
YingandyangofNETs
TherearemultiplereportsonNETsbeingabletocapture ,immobilizeandneutralizepathogens.Themicrobescaughtb yNETincludebothGram-
positive(e.g.,Staphylococcusaureus)andGram-
negativebacteria(e.g.,SalmonellatyphimuriumandShigel laflexneri;Brinkmannetal.2004),fungi(e.g.,Candidaalbicans
;Urbanetal.2006)andviruses(Saitohetal.2012;Jenneetal.201 3).Morecontroversialistheircapacitytokilltrappedpathogens.
AsNETsaredecorat-
edwithantimicrobialproteinsandproteases,theirkillingpo- tentialseemedtobeunavoidableandinfactitwasrepeatedlyrepo rtedtooccur(Brinkmannetal.2004;Urbanetal.2006;Guimarãe s-
Costaetal.2009).However,somestudiesruleditout(Gabrieleta l.2010).ArecentpaperbyMenegazzietal.
(2012)challengedthetechnicalapproachappliedinthema- jorityofthestudies,mostofwhichwereperformedonisolatedneu trophilsandrevealedthattheresultsdependedonthecho- senstrategy;i.e.,incubationwithDNasepriororpost-
additionofbacteriatotheNETformingneutrophils.Overall,the studyconcludedthatNETsentrapbutdonotkillmicrobes(M enegazzietal.2012).Thisisinlinewithsomeinvivostudies showingthat,afterintravascularapplicationofDNase,colon y-formingunits(CFUs)ofS.aureusdonotin-
creasedespitestrongdepositionofNETsinthevasculatureofmic
ewithS.aureussepsis(Kolaczkowskaetal.2015).ButevenifNE Tsindeeddonotkillpathogens,theirrolein
CellTissueRes(2018)371:473–488 5 capturingandimmobilizingmicrobesshouldnotbeunde
restimatedasNETspreventmicrobialdisseminationthrough outthebody.Thiswas,forexample,showninthecourseof Escherichia colisepsis(McDonald etal.2012).Mor eover,onecanspeculatethatNETscanindirectlycontrib- utetopathogenkilling,asimmobilizedmicrobesareexposedto microenvironmentalimmunefactorspresentinserumortissue saswellascytotoxicleukocytes(macrophagesandNKcells).I naddition,bymeansofproteasesattachedtoNETs,virulencefact orsofpathogenscanbeshedfromtheirsurfacelimitingtheirvirul ency,e.g.,IpaBonS.flexneriisbeingremovedbyNEdecorating thetraps(Brinkmannetal.2004).Anotherim-portant,anti- inflammatoryfunctionofNETscomesfromstud-
iesonsterileinflammation,asduringgout,serineproteasesat tachedtoNETswereshowntodegradepro-
inflammatorycytokinesandchemokinescontributingtother esolutionoftheimmuneresponse(Schaueretal.2014).
TheimportanceofNETsisfurtherstrengthenedbyfourfa cts:(1)theirevolutionaryconservation,(2)releasebymul- tiplepopulationsofleukocytes,(3)releaseoftheNETback- bone(DNA)fromeithernucleusormitochondriaand(4)strat egiesofpathogensdevelopedtoescapefromNETs.Itturnso utthatDNAdecoratedwithantimicrobialsandprote-
asesispreservedinevolution;notonlydoallvertebrates(onlyda taonamphibiansaremissing)releaseextracellulartraps(E Ts;Brinkmannetal.2004;AlghamdiandFoster2005;Paliće tal.2007;Pijanowskietal.2013;Reicheletal.2015)
butalsoinvertebratespecies(Ng etal.2013;Homaetal.20 16)andevenplants( Wenetal.2009,2017)andsocial am oebae(Zhangetal.2016)doso.Moreover,althoughnotallcells releasingETsareleukocytesorleukocyte-
like,theyallseemtoperformakindofdefensefunction,includin grootbordercellsofplants(Hawesetal.2000)andsentinelcellso fthemulticellularslugstageofthesocialamoebafunctioningas aprimitiveinnateimmunesystem(Chenetal.2007).Thus,itisno tsurprisingthat,invertebrates,asdepictedindetailinmammals, ETformationisuniversalamonginnateimmuneleukocytesan dalsocharacterizesmonocytes(Grangeretal.2017),macropha ges(Chowetal.2010;Liuetal.2014),eo-
sinophils(Yousefietal.2008),basophils(Morshedetal.2014)a ndmastcells(vonKöckritz-
Blickwedeetal.2008).Furthermore,thesourceofDNAcanvar ysinceneutrophilsandeosinophilsnotonlyejectDNAofnuc learbutalsoofmitochondrialorigin(mNETs;Yousefietal.20 08,2009).ThestudiesonneutrophilsrevealedthatDNAofmNE Tsin-
deedcontainsmitochondrial(e.g.,Cyb)andnotnuclear(e.g.,Ga pdh)genes(Yousefietal.2008).Interestingly,mNETsarerelea sedbyvitalneutrophilsandtheyprolongsurvivalofthereleasing cells(Yousefietal.2009).Finally,differentstrategiesofpathoge nstoavoidtrappingbyNETs,ortoescapefromthereleasedchrom atinfibers,havebeendescribed.Streptococcuspneumoniaeand S.aureusaregoodexamplesofbacteriaarmedagainstNETsbutf ungi(Leeetal.2015;Rochaetal.2015;Johnsonetal.2016)and parasites(Guimarães-
Costaetal.2014)havealsodevelopedsuchmechanisms.S.pne umoniaepossessestheabilitytoformpolysaccharidecapsulespro tectingthemfrombindingtoNETs(Warthaetal.2007)andthe irendonucleasesdegradethenetwork(Beiteretal.2006).More over,S.pneumoniaecanchangetheelectricalchargeoftheirmem branetopositive,byincorporationofD-alanineres-
iduesintoLTAs(lipoteichoicacids).Thisstrategyprotectsthem againstpositively-
chargedresiduesonNETantimicrobialsandproteasespreventin gthetrapping(Beiteretal.2006).S.aureusalsoreleasesnucleas esbutnotonlytodesintegrateNETs(Berendsetal.2010),asthey alsodegradeNET-DNAtointer-
mediateproductsthatareconvertedto2′-
deoxyadenosine.Thelatterdeoxyribonucleosideinducesapopt osisofmacrophagesthatotherwisecouldphagocytosepathoge nsimmobilizedinNETs(Thammavongsaetal.2013).
Havingdescribedt h e a d ventageso f N E T r e l e ase,o n e musta lsoa cknowledget hes idee ffectso f t heirf ormationlea dingtoeitherinitiationofbystanderdamageorevendis- eases.N umerousstudieshaver eportedt hatu ncontrolledand/
orexcessivereleaseofNETs,aswellastheirinadequateremoval ,leads,oratleastcontributes,tovariouspathologi-
calconditions,includingrheumatoidarthritis(RA;SurChow dhuryet al.2014;Carmona-Riveraetal.2 017),s ys-
temiclupuserythematosus(SLE;Landeetal.2011;Villanue
vaetal.2011),atherosclerosis(Knightetal.2014;Wangetal.20 17),vasculitis(Kessenbrocketal.2009;
SöderbergandSegelmark2 016),t hrombosis( Goulde t a l.2 014;M artinoda ndWagner2 014),s epsis( Kolaczkowskaeta l.2015)andcancer(Berger-
Achituvetal.2013;Tohmee ta l.2 016).SLEa nds epsisa rer epresentativee x-
amplesofexcessive/inapropiateNETreleaseandinadequater emoval,respectively.S L E i s m a n ifestedbyb enigns kinle sionstolife-threateningsymptomsresultingfromoverpro- ductionofautoantibodiesandlossoftolerancetotheirownant igens(Crispínetal.2010;Dörneretal.2011).Theauto- antibodies,anti-
neutrophilcytoplasmicantibodies(ANCAs)ared i r e c t e d a g a i n s t P R 3 , M P O,N E a n d t h e a n t i -
n u c l e a r antibodies(ANAs)againstDNAandhistones,all ofwhicharec o mponentso f N ETs( Fauzie t a l .2 004;Yua n d S u2013;Gajic-
Veljicetal.2015).CharacteristicforSLENETsisthepresenceo fLL-37andhumanneutrophilpeptide(HNP).TheDNA/LL- 37/HNPcomplexesactivateplasmacytoidden-
driticcells(pDCs)resultinginincreasedproductionofIFN- α(Landeetal.2011),whichplaysacentralroleinthepathogen- esisofSLEbypromotingimmunesystemactivationthatcon- tributestotissueandorganinflammationanddamage(Crow201 4).Inaddition,NETsofSLEpatientsareinadequatelyde- gradedastheyareprotectedbyDNaseinhibitors(Hakkimetal.2 010)butalsocomplementC1qboundtoNET(Leffleretal.2012) ,whileLL-
37canprotectDNAfromdegradation(Landeetal.2011).Ofimp ortance,duringSLE,numbersofcirculatingimmatureneutroph ilsareelevated(Bennettetal.2003).
Correspondingly,duringsepsis,NETscontributetoby -
standerdamageofendotheliumduetoactivityofhistones (Xuetal.2009;Saffarzadehetal.2012;McDonaldetal.20 17)andNE(Kolaczkowskaetal.2015)ofNETsthatarenottim elyremoved.Also,sepsisischaracterizedbyarapidrecruitme nttobloodofimmatureneutrophils(Mareetal.2015)andn otfullymatureneutrophilsarealso presentintumors wheretheydisplaya pro-
tumorgenicphenotype(Sagivetal.2015).Thesedatasuggest thattheageofneutro-
philsmightnotonlyimpactthephenotypeofneutrophilsbutals otheircontributiontodiseasepathology.
OnhowNETsarecreated
ThirteenyearsintoNETresearchandstillweknowlittleaboutt hemechanismsofNETformation,althoughnumerousstud- ieshavebeenpublishedonthistopic.Nottounderestimatean yofthestudies,onemustkeepinmindthat,toourestima- tion,approximately90%ofstudiesonNETsareperformedonis olatedneutrophilsortissuescollectedpost-
mortem.Thisdoesnotreflectonacomplexinvivomilieuandb ehaviorofneutrophilsandotherleukocytesinsitu,inbloodort
issues.However,themainconcernisthatmostofwhatweknowo nthemechanismsofNETscomefromstudiesinwhichPMAwa suseda solestimulant.PMAisasynteticp horbol12- myristate13-acetate,arobustactivatoroftwoofthethree
familiesofproteinkinaseC(PKC;LiuandHeckman1998;Neel iandRadic2013)and,assuch,enforcesparticularsig-
nalingpathways.Arecentpaperre-examingkineticsandsig- nalingpathwaysofNETsinducedbyvariousagentsconclud- edthatBPMAstimulationshouldberegardedasmechanisti- callydistinctfromNETformationinducedbynaturaltrigger s^(vanderLindenetal.2017).
VeryearlyinNETresearch,dependenceonreactiveoxy- genspecies(ROS)generatedbytheNADPHoxidasepathwayw asreportedtobeaprerequisitefortheirformation(Fuchsetal.2 007).Thestudiesweresubsequentlystronglysupportedbyobse rvationthatpatientswithchronicgranulomatousdis-
ease(CGD),withimpairedNADPHoxidaseactivity,didnotrele aseNETsbutthatthiscouldberestoredbyatargetedgenetherapy (Bianchietal.2009).Subsequently,theRaf-MEK-
ERKpathwaywasidentifiedasbeinginvolvedinNETfor- mationthroughactivationofNADPHoxidase(Hakkimetal.20 11).ButthennumerousstudiesreportedROS-
independenceofNETformation,whichresultedfrombothinv itro(Gabrieletal.2010;Byrdetal.2013;Pijanowskieta l.2013;Mejíaetal.2015)andi nvivostudies( Chenetal.2012;
Kolaczkowskaetal.2015;Barthetal.2016a)utilizingNAD PHinhibitorsandknockoutmice.Thisdiscrep-
ancyinthedataonROSinvolvementinNETreleaseisdiffi- culttoexplainatthisstage.Itmightberesultingfromtheexp erimentalmilieuorthenatureofNET-
inducingfactorsasnotallagentsactivateNADPHoxidase(Farl eyetal.2012).Thelatterstudyreportsonaninterestingdiscrepa ncy:PMAbutnotplatelet-
activatingfactor(PAF),generatedROSbuttheNADPHoxidase inhibitor(DPI)reducedNETreleasebybothPMAandPAF.The sedataindicatethat,onceagain,resultsfromPMAstudiesshoul dbecarefullyreviewedunlesssup-
portedbydatafromconcominantstudiesapplyingpathogen- orimmuneresponse-
relatedagentstoinduceNETs.However,mostimportantly,thes tudysuggestsaninterestingexplana-
tionofROSinvolvementinNETformationasDPIalsoin- hibitsarangeofflavoenzymesincludingmitochondrialoxi- daseandnitricoxidesynthase(Stue hretal.1991;Liand Trush1998),whichcouldBsubstitute^forphagosomalROS.Th us,insomecircumstances,NETformationmightdependonpha gosomalROS(NADPH-
dependent;e.g.,Fuchsetal.2007)butalsoonmitochondrialRO S(asshowninLoodetal.2016)orNO(asreportedinPateletal.2010 )ornone.Itisalsoofnotethattheonlyfamilyofendogenousinhibit orsofNETsknowntodatedoesnotinhibitROSformationandi nsteadblocksPAD4-
dependentcitrullination(seeBNETformationinneonates^) (Yostetal.2016).
AnothermechanismputativelyinvolvedinNETformationis autophagy.Thisprocessiscriticalfortheturnoverofdam- agedorganellesandproteinsduringhomeostasisbut,duringinf ection,playsaroleinthekillingofphagocytosedpatho-
gensanddown-
regulationofinflammasomeactivation(Birminghametal.20 06;Jabiretal.2014).Themajorityof
studiesshowinginvolvementofautophagyinNETformationa ppliedpharmacologicalinhibitorsofkeypathwaysormole- culesinvolvedinthisprocessthathowever,werealsoinhibit ingROS(Remijsenetal.2011;McInturffetal.2012;Kennoeta l.2016;Ullahetal.2017).Recently,theinvolve-
mentofautophagyinNETreleasewasstudiedintransgenicmi cewithconditionallydeletedatg5(itsproductiscriticalforauto phagosomeformation)ineitherneutrophilsoreosino- phils(Germicetal.2017).Thestudyruledoutaroleofau- tophagyinNETformation.Asimilarcontroversyconcernsth einvolvementofnecroptosis(aprogrammednecrosis- likecelldeath),whichiswellillustratedbytwocontradictorypa -perspublishedrecentlyhead-to-
head(Aminietal.2016;Desaietal.2016).
However,therearetwoenzyme-
basedmechanismsofNETformationthatwereconfirmedtoop erateindependentlyoftheinvitroorinvivosettingsandthe inducingagents.TheseincludetheinvolvementofNEan dpeptidylargininedeiminase4(PAD4)
(Fig.1).PAD4belongstothegroupofCa2+-
dependentenzymesandislocatedinthenucleusandgranul esofneutrophils(Asagaetal.2001;Nakashimaetal.2002;Kea rneyetal.2005).Theenzymeisinvolvedincata-
lyzingthecitrullinationofhistonesH2/H3/H4,whichis apost-
translationalmodificationconvertingthemethylarginineresi duestocitrullinetoformacarbonylgroup(Hagiwaraetal.
2002;Aritaetal.2006;Györgyetal.2006).Theconversionofpo sitivelychargedmethylargininetoneutralsidechainsofcitr ullineaffectsprotein(histone)-
DNAstabilizationandleadstochromatindecondensationan dNETrelease(Neelietal.2008;Wangetal.2009).Studieso nPAD4knockoutmice(PAD4−/
−)showedimpairedabilitytoformNETsincompar-
isontoWTanimalsindependentlyofstimuli,beitLPSorion omycin(Martinodetal.2013).Similarly,thePAD4inhib- itor(Cl-amidine)alsodiminishesNETreleasebothinvitro(Li etal.2010;Kusunokietal.2016)andinvivo(Knightetal.2013 ,2014).However,recently,PMA-inducedNETforma- tionwasreportednottobeconnectedwithhistonedeamina- tion(nocitrullinatedH3histonesweredetectedinPMA- inducedNETs),whichwasexplainedbythefactthatPMAacti vatesthePKCαisoformthatinhibitsPAD4whileitisthePMA- irresponsivePKCζthatactivatesdeamination(Neeliand Radic2013).Nevertheless,therearealsostudiesreportingdep ositionofcitrullinatedhistonesinPMA-
stimulatedNETs,althoughtoa lowerdegreethanuponothe rinducers(Martinodetal.2016;vanderLindenetal.2017).
AnotherenzymerequiredtoformNETsisaserineprote- ase:neutrophilelastase.Theproposedmechanismofitsactioni sspecificdegradationofhistonesthatdestabilizeschromatin(
Papayannopoulosetal.2010).Inaddition,blockadeofNETfor mationwasalsodemonstratedinvivoonNEKOmiceinf ectedwithGram-
negativebacteria(Papayannopoulosetal.2010;Farleyetal.2
012)orGram-
positivebacteria(Kolaczkowskaetal.2015).Also,theuseo fNEinhibitor
resultedintheinhibitionofC.albicans-inducedNETforma- tion(Papayannopoulosetal.2010).However,Martinodetal.
(2016)showedthatnumerousneutrophilsderivedfromNE−/
−miceejectedNETsuponinvitroionomycinstimulation,while4 0%ofthemdidnot(Martinodetal.2016).Interstingly,dur- ingmousesterilethrombosis,only20%fewerNETswer eproducedbyNEKOneutrophils(Martinodetal.2016).Thisin dicatesthatbothPAD4andNEareinvolvedinNETformationb utmightbemoreorlessredundantdependingonthediseasestatea nd/orstimuli.Forexample,duringS.aureussepsis,NE−/
−neutrophilsdidnotproduceNETswhilesomePAD4−/
−P MNs(c. 20%)did (Kolaczk owskaet al.2015),whereas duringdeepveinthrombosis,80%ofNE−/−neutro-
philsreleasedNETs(Martinodetal.2016)butnosuchstruc- tureswerecastbythePAD4−/
−cells(Martinodetal.2013).Thesefindingsreflectwellonthedi versityofNETtypes.Thetrapsseemtovarynotonlyintheirappea rance,involvedmol-
eculesandpathwaysbutal sointheconsequencesfortheprod ucingcells.ThefirstreportontheexistenceofNETspres entedmanyargumentssupportingthatthetrap-
releasingcellsremainviable(Brinkmannetal.2004)butsubseq uentstudiesreportedontheprocessbeinglethal(Fuchsetal.2007 )andeventuallyatermNETosiswascoined(Steinbergan dGrinstein2007).However,Yippetal.
(2012)showedbymeansofintravitalmicroscopyofS.aureus- inflamedskinthatanuclearneutrophilsthatreleasedNETsrema inaliveandkeepmovingandphagocytosing(Yippetal.2012 ).Thisseemsmoreeconomicalandefficientthanthebeneficia lsuicideandwasdetectedinthemilieuoftheliveorganism.S uccessively,viableNET-formingneutrophilswerealsore- portedininvitrosettings(Yousefietal.2009;Pilsczeketal.2010 ).Mostprobably,thetwomodesrepresentanothersetofparallel mechanismsbywhichNETsarereleased,eitheruponcellruptur e(Fuchsetal.2007)orvesiculartransporttothecellsurface(Pilsc zeketal.2010).
Westilldonotknowhowtounderstandthisvarietyofin volvedmechanismsandwhetherreportedNETsarealwaysBN ETs^,asadequate,multicomponentdetectionisakeybutnotago ldenstandard.Thisissueisevenbecomingatopicofopendiscuss ionswithBhealthycritisism^suchastheoneofNauseefandKub es(2016).
NETsandageofneutrophils
Immatureneutrophilsversusmatureneutrophils Neutrophilsariseandmatureinthebonemarrow.Thematu- rationconsistsofthemitoticstage( myeloblasts,promy elocytesandmyelocytes)andpostmitoticstage(metamyelo cyte,neutrophilbandandmaturesegmentedneu-trophils)
(Borregaard2010;Amulicetal.2012;Lahoz-
Beneytezetal.2016).Neutrophilsecretionfromthebone
marrowintocirculationiscontrolledbycircadianoscillations(
Casanova-Acebesetal.2013)anddependsontheinterac- tionsbetweentheCXCL12chemokineanditsCXCR4recep- tor(retentionofneutrophilsinthebonemarrow)andtheCXCL 1ligandwiththeCXCR2receptor(releaseofneutro-
philsintoblood)
(Martinetal.2003;Eashetal.2010).Incirculation,neutrop hilageandhumanneutrophilhalf-
lifeislessthan1day,about19h(Lahoz-
Beneytezetal.2016)andabout12hinmice(Pillayetal.2010a).E xpressionofCXCR4increasesonagingcellsandcausesneutro philstoreturntothebonemarrow,wheretheyareremovedbyma crophages(FurzeandRankin2008;Casanova-
Acebesetal.2013)butthecellscanalsoberemovedinthesple enandtheliver(Shietal.2001;Surattetal.2001).Inturn,thisl eadstosecretionfromthebonemarrowofacorrespondinglys mallnumberofma-
turebutnotimmature(Bruegeletal.2004;Nierhausetal.20 13),neutrophilstothecirculation(Semeradetal.2002).Assh ownrecently,theprocessiscontrolledbygutmicrobiota(Zhan getal.2015)andmostprobablyalsobyexosomeswhosenu mbersandcontentchangeduringaging(Prattichizzoeta l.2017).Ifduringtheirlifeneutrophilsarerecruitedtothesite ofinflammation,theirlife-
spanisprolongedandtheirdeathbyapoptosisisdelayed (Simon2003;MilotandFilep2011).Duringinflammation,es peciallythesystemicone,bothmatureandimmatureneutroph ilsarerecruitedfromthebonemarrow(Drifteetal.2013).I nterestingly,arecentstudyshowedthatthefirstneutrophilstoa rriveatthesiteofinflammationareagedneutrophilsandtheyar efollowedbynonagedcells(Uhletal.2016).Thefactthatagedc ellsdisappearfromcirculation,neatlyexplainswhyfreshcells arerecruitedtothebloodfromthebonemarrowinthecourseofin flammation.
Immatureandmatureneutrophilsdifferintheirgeneex- pression,theformerhavinghigherexpressionofgenescon- trollingtheirdifferentiationandgranularproteinsynthesis ,includingNE,MPOandBPI,whereasgenescontrollingche- motaxisorapoptosisaredown-regulatedinimmatureneutro- phils(Martinellietal.2004).Comparisonofhumanimmature(
bonemarrow)andmature(blood)neutrophilsintheircapac- itytoproduceNE TsuponIFN-
α/γprimingandfollowingstimulationwithcomplementfa ctorC5,showedthatonlythematureneutrophilsreleasedthe traps(Martinellietal.2004).Otherstudiesrevealeddiminishe dyetdetectableNETreleasebyimmatureneutrophils.Inthestu dybyTanejaetal.
(2008),circulatingneutrophilsconsistedofc.35%ofimma- turecells(vs.5%inhealthyvolunteers)duringsepsis.Andsi milarresultswereobtainedbyPillayetal.
(2010b).Theimmatureneutrophilshadalowerratioofphago cytosisand
Ca2+signaling(Tanejaetal.2008),antimicrobialrecognition andkillingandROSgeneration(Pillayetal.2010b).Also,inpat ientswithsterileburninjury,immatureneutrophilswerenum
erouslypresentincirculationandthesepatientshadhigherleve lsofcirculatingfreeDNA(cfDNA)andcitH3,
clinicalmarkersofsystemicformationofNETs(Hampsoneta l.2017).Thiswasespeciallyapparentattimeswhennum- bersofimmatureneutrophilsdominatedincirculation.Howe ver,whenneutrophilswereisolatedfrombloodandexvivo- stimulatedwithPMA,thecells(amixtureofmatureandimmatur eneutrophils)ofpatientswiththermalinjuryreleasedfewerNE Ts(Hampsonetal.2017).
ThereisalsoareportonnormalproductionofNETsbyhum animmatureneutrophilspresentincirculationthatcomesfroms tudiesonbonemarrowtransplantation(Glennetal.2016).
Important,althoughnotdirect,informationonNETproductio nbyimmatureneutrophilscomesfromstudiesondiseasesduri ngwhichtheundevelopedcellsareeitherpresentinbloodortiss ues.OnesuchexampleisSLE,aslupuspa-
tientsdisplayavaryingdegreeofneutrophilmaturation(Denn yetal.2010;Villanuevaetal.2011).Inparticular,twoneutroph ilsubpopulations,low-
densitygranulocytes(LDGs)andhigh-
densityneutrophils,wereidentifiedinthecourseofthedisease.
TheLDGsdonotcarryanyspecificmarkersidentifiedtodate buttheirnuclearmorphology(c.40%cellshavelobular,bando rmyelocyte-
likenucleivs.c.60%withsegmentednuclei)suggeststhatmany ofthesecellsrepresenttheimmaturephenotypeofneutrophils(D ennyetal.2010).Thecellshavehigherexpressionofazurophilic granulegenes,includingthoseencodingNEandMPOande xhibitincreasedspontaneousNETproductionandoverallrel easemoretraps(Villanuevaetal.2011).Asimilarsubsetofneu- trophilswasalsodescribedinthecourseofpsoriasisandpso- riaticLDGsalsotendtoformNETswithoutanystimulation,inc ontrasttocontrolorpsoriasismatureneutrophils(Linetal.2011).
Low-
densityneutrophils,consistingofbothimmatureandmaturene utrophils,havealsobeendescribedincancer(Sagivetal.2015).
Unlikehigh-densityneutrophils,thelow-
densitycellshaveapro-tumorphenotype(i.e.,decreasedche- motaxis,phagocytosisandROSproduction).Thetwopheno- typesoftumor-associatedneutrophils(TANs),i.e.,high- densityandlow-
densityneutrophils,arealsotermedN1andN2,respectively(Fri dlenderetal.2009).TheN2phenotypedominatesintheprese nceofTGF-βbutisdiminishedby IFN-
β(Fridlenderetal.2009;Andzinskietal.2016).Itwasshownthat bloodneutrophilscollectedfrommicewithtumorsinwhichN2p henotypewassuggestedtodominate(IFN-
βKOs),producedfewerNETs,eitherspontaneouslyoruponP MAexvivostimulation(Andzinskietal.2016).These,how- ever,werenotTANsandtheexactphenotypeofcirculatingneut rophilswasnotexamined,neverthelessimmatureneutro- philspresentinacourseofdiseasemightnotalwaysreleasespon taneouslyhigheramountsofNETs.Inaddition,thetumorenviro nmentisuniqueandthuswecanspeculatethatNETreleaseincr easesanti-
tumoralresponseasNETcomponentsmightdamagetumorcell
s.ButNETscouldalsofunctionasscaffoldsoftumorantigens,faci litatingtheirtake-
upbyDCsandmacrophages.Ontheotherhand,NETscantrigge r
metastasis,e.g.,high-mobilitygroupbox1(HMGB1)re- leasedfromNETsactivatestheTLR9-
dependentpathwayincancercellspromotingtheiradhesion,p roliferation,migrationandinvasion(Berger-
Achituvetal.2013;Tohmeetal.2016).Similarresultscamefro mastudyonimmatureandmaturegranulocytespresentinle ukemicpatients(Lukášováetal.2013).Inthisstudy,onlyd ataonPMA-
inducedNETswerereportedandacutemyeloidleukemia(
ALM)granulocyteswereshownnottoproducethetraps asopposedtogranulocytesisolatedfromperipheralbloodofh ealthydonors(Lukášováetal.2013).Theimmaturecellsexpre ssedhetero-
chromatinprotein1γ(HP1γ)anddimethylatedhistoneH3atly sine9(H3K9me2).Thetwoproteinsinteracttopreservethespr eadingofheterochromatinandHP1γisabsentinmaturegranu locytes.Terminallydifferentiatedmatureneutrophilsarechar acterizedbyatightlycondensedchromatinandgenere- pression,whileimmaturecellsdonot(Lukášováetal.2013).L ukášováetal.
(2013)hypothesizedthatitmightbenecessaryforchromatinto becondensedtofacilitatePAD4actionandforthisNETformat iontobeweakerinimmaturecells.
OnehastobearinmindthatthemajorityofdataonNETform ationbyimmatureneutrophilscomefromillpatients(withse psis,SLE,psoriasisorcancer).Nevertheless,manyofthem,a lthoughnotall,reportonspontaneousreleaseofthetrapsbyi mmatureneutrophils(ifthisaspectwasstudied/reported)andd iminished,oratleastnotfutherincreased,pro-
ductionofNETsuponstimulation(mostlywithPMA) (Fig.2).Inaddition,atleastonestudyreportedonconcomitant lyelevatedmarkersofNETsincirculation.Consideringallthe abovedata,onemighthypothesizethatimmatureneutrophilsp resentinbloodtendtospontaneouslyreleaseNETs,hencethe presenceoftheirmarkersincirculationandthus,whenisolat edandexvivo-
stimulatedtoproducethetraps,failtoformthem.Thisiseither duetoanexhaustedphenotypeofthecellsorthefactthatallneutr ophilswithapotentialtoreleaseNETshavealreadydonesoonc einvasculature.Especially,itisonlyabout25%ofneutrophilst hatreleaseNETs(NauseefandKubes2016).
Agedorsenescentneutrophil s
NotmuchisknownaboutNETproductionbysenescentneu- trophils.Agingneutrophilsup-regulateCXCR4andprogres- sivelyloseCD62L(L-
selectin)expressionthatfacilitatestheirre-
directiontothebonemarrow(Zhangetal.2015).However,the yexhibitenhancedadhesionmolecules(e.g.,Mac-1,ICAM- 1)andTLR4expression(Zhangetal.2015),whichisinlinewi ththeirrapidrecruitmenttothesiteofinflamma-
tion,priortomaturebutnotaged,neutrophils(Uhletal.2016).
Thisagingphenotypeisregulatedbymicrobiotaandislostin
micetreatedwithbroad-rangeantibioticsorgerm- freeanimalsbutrestoredbyapplicationofLPSorfecaltrans- plantation(Zhangetal.2015).TheCD62LloCXCR4hiaged
neutrophilsaresignificantlynumerousinSelp−/−m i c e(P- selectinKOs)oranti-P-selectin-
treatedanimals(Zhangetal.2015;Uhletal.2016).WhenNETpr oductionwasstudiedinthelattermice,neutrophilsstimulatedex vivowithLPSdra-
maticallyincreasedtrapformation.Thiswasfurthercon- firmedinanendotoxemicmodelbyintravitalimagingofNETsi nlivervasculature(Zhangetal.2015).Therefore,inthecaseofsc enescentneutrophils,theexvivoandinvivodataclearlycorrelat ed,indicatingtheirenhancedcapacitytore-
leaseNETs,whichisinlinewithapro-inflammatorypheno- typeofthesecells(Fig.2).However,nodataonhumanscenesce ntneutrophilsareavailable.
NETsandageofindividuals
Immunesystemmaturesduringfetaldevelopmentandthendec linesasweage.Thesefactshaveimportantimpactsonsuscept ibilitytoinfectionandthechancesofsurvivingit.And,assuc h,itisalsoimportanthowNETreleasechangeswithage.Especi ally,theworldisundergoingashiftindemo-
graphicswithlowbirthratesandagingofpopulations(Bouleand Kovacs2017).Independentlyoftheageofmothers,notonlyfew erbabiesarebeingbornbutalsomanyofthemarebornpreterma ndthereforetheyaremorelikelytobecomeill
ordie,aspreterminfantsaremorevulnerabletoinfectio n(Urquhartetal.2017).Inlinewiththis,theriskofseveres epsisinneonatesincreasesdramaticallywithdecreasingges- tationalage(Sperandioetal.2013).Ontheotherhand,theglob alpopulationisagingandthenumberofindivudualsolderth an65yearswilldoubleby2050(BouleandKovacs2017).Elderl ypeoplearemoresusceptibletoinfectionduetoinflamm- agingorimmunosenescence,i.e.,theage-
relateddysfunctionoftheimmunesystembuttheyalsodevelop chronicinflammatorystates(Boeetal.2017).
NETformationinneonates
Theimmunesystemplaysaveryimportantroleduringpreg- nancy,withthepurposeofprotectingthemotherandthede- velopingfetus(Moretal.2011).Pregnancyisaperiodthatischar acterizedbymodulationoftheimmunesystemassociatedwithb oththecourseandstageofpregnancy,aswellastheexposureto pathogens.Moreover,thepregnancyischaracter-izedbyapro- inflammatoryphase(firsttrimester),theanti-
inflammatoryphase(secondtrimester)andbytheendofthepreg nancyreturnstothepro-
inflammatoryphase(MorandCardenas2010).Pregnantwom enhaveanincreaseinthetotalnumberofleukocytes,whichco rrelateswiththecourseofpregnancy(thehighestlevelisinth ethirdtrimester)ofwhich
Fig.2N europhilmaturation-andage-
dependentchangesinneutrophilextracellulartraps(NETs)formation.Tos trengthenthegraphicalvisual-
ization,potentialtoformNETsismarkedwith–and+,where+<++
<+++;−/
+indicatesthat,forimmatureneutrophilsstimulatedexvivo,somestudiesre portedalackofNETformation(−)whereasothers
reportedsomeNETreleasealthoughweak(+).Phenotypeofmatur eversusagedneutrophilsisdefinedbyhighorlowexpressionofCXCR 4andCD62L.Immatureneutrophilsweremostlydefinedbytheirnucleus morphology.Referencedataareincludedanddiscussedinthemaintext
CellTissueRes(2018)371:473–488 480
themostabundantcellsarecirculatingneutrophils(Crockereta l.2000).Theseneutrophilsdisplayadecreasedrespiratoryburst duringthesecondandthirdtrimesters;however,thisactivit yreturnstonormalwithin7 weekspost-
partum(Crockeretal.2000).WithrespecttoNETs,increasedle velsofcfDNA(nucleosome/MPOcomplexes)areobservedin pregnantwomen’sserum,comparedtononpregnantwomen(S urChowdhuryetal.2016).Interestingly,thetendencytoforms uchcomplexesincreasinglyrelatestothedurationofpregnanc y.Nevertheless,thehighestserumcfDNAlevelisobservedinp reeclampsiawomen,asopposedtowomenwithnormalpregnan cyandnonpregnantwomen(Loetal.1999;SurChowdhuryetal.
2016).Moreover,thelevelofbothfetalandmaternalcirculating plasmaDNAfrompreeclampsiawomencorrelateswiththedeg reeofdiseaseseverity(Zhongetal.2001).
Thefetus,whichislocatedintheuterus,developsitsownimm unesystem(Daubyetal.2012).Afterbirth,bothpreterm(<37we eks)andterm(37–42weeks)neonatesarecharacter-
izedbyatolerogenicimmuneresponseduetothereducednum berofimmunecells,includingneutrophilsorlympho- cytes,whichincreaseinthefirstweeksoflife(Walkeretal.2011;
Nguyenetal.2016).Inthedevelopinghumanfetus,asmallnumb erofneutrophilsbegintoappearintheclavicularmarrowafter11 –12weekspost-
conceptionwiththemajorityobservedafter13–
15weeks(Slaytonetal.1998a,b).However,neutropoiesissta rtspriortothisinthefetalliver(aroundweek5;Slaytonetal.19 98a)andyolksac(aroundweek3;Sperandioetal.2013).Neutro philsofamatureindi-
vidualdisplayacapacitytomigratetothesiteofinflammationand effectivelyfightpathogensthroughphagocytosisorde- granulation(KolaczkowskaandKubes2013).Intermneo- nates,thephagocytosisanddegranulationareequallyefficienta sinadultsbutnotinpretermneonates(Bektasetal.1990;Nuppo nenetal.2002).However,bothpretermandtermneo-
natesshowimpairedmigrationofneutrophilstotheinflam- matoryfocus(McEvoyetal.1996;Nussbaumetal.2013).Hen ce,theyoungorganismisnotabletodefenditselfasefficie ntlyastheadultandthereforeneonatesarehighlysus-
ceptibletoinfections,includingsepsis,whichdirectlyaffectinc reasedmorbidityandmortality(Gardner2009;Lawnetal.2010) .Makonietal.
(2016)suggestedthatimpairmentoftheneonatalneutrophilsm aybeduetotheincreasednumberofdevelopmentallyimmature neutrophilsatbirthratherthanoth-
erabnormalitiessuchastheexpressionofsurfaceadhesionmol ecules,whichislowatbirthbutincreasesovertime(Carretal.19 92;Makonietal.2016).Anotherreasoncouldbekeepingd ownimmunitytopreventsideeffectsthatmightresultfro mitsoveractivity.
Furthermore,theformationofNETsinpretermortermin fants/neonateshasbeenreportedtobeweaker(Fig.3).Neutrop hilsisolatedfrominfants/neonatesdisplayedimpairedNETpro ductionafterstimulationwithLPS,PAFandfMLP,
incontrasttoneutrophilscollectedfromadultindividuals(Yos tetal.2009;Lippetal.2017).Th iswasdespitethepresenceoff unctionalreceptorsthatrecognizethesemole-
culesanduncompromisedphagocytosis.Nevertheless,whenb acteria(E.coli,S.au reus)orPMAwereusedt o induceNETs,n eonatalneutrophilsdidnotformNETs(Yostetal.2009).Ont heotherhand,Lippetal.
(2017)reportedthatthecellsofterminfantsreleasesomeNETsi nresponsetoPMAandthoseofpretermbabiesreleasesignific antlyfewerofthesestructures.Importantly,thedefectofN ETformationbyneutrophilsofpretermandtermneonateswas notrescuedbytheROSdonor(glucoseoxidase)
(Yostetal.2009).Also,thestudybyByrdetal.
(2016)showedthatNETsinducedbyneonatesinresponsetoaco mbinationoffibronectin(Fn)withpurifiedfungalβ-
glucanorFnwithC.albicanshyphaeareROS-
independent,althoughinthiscase,NETswereformednormally .Thus,neonateneutrophilsseemtobesensitivetofungalstimu lationbutnotnecessarilythebacterialcompo-
nents(Byrdetal.2016).However,incontrasttoLippetal.
(2017),Marcosetal.
(2009)showedthatneonatalneutrophilscancastNETsuponL PS(aswellasothernumerousTLRagonists)althoughatfirstt hesignalisweaker(Marcosetal.2009).Directcomparisonof thetwostudiesindicatesthatneonateneutrophilsreleaseN ETsbuttheyrequirealongertimefortheirmaximalformation.I nfact,furtherstudiesre-
vealedthateventhemostprematurelyborninfantsgainthecapa citytoreleaseNETbyday3post-
birthandmaximalcapacitytocastNETsisachievedbetweend ay3and14oflife(Yostetal.2016).Thischaracteristicseems alsotobepresentinothermammals,asthesamephenomenonw asob-servedinpigs(Nguyenetal.2016).Also,neutrophilsof21- day-oldmiceproducedfewerNETsthanthecellsof60-day- oldanimals(Barthetal.2016b).
Inthesearchformechanismsofimpaired/delayedNETf ormationbyneonates,afamilyofendogenousinhibitorsofNET swasdiscovered(Yostetal.2016).Thefamily,callednNIF- relatedpeptides(NRPs),afterthefirstidentifiedpeptide(NET- inhibitoryfactor,nNIF),alsoconsistsofcancer-
associatedSCMrecognition,immunedefensesuppressionan dserineproteaseprotectionpeptide(CRISPP)anda44–
aminoacidcarboxyterminuscleavagefragmentofA1AT(α1- antitrypsin),A1ATm358(Yostetal.2016).Thelevelsofinhi bitorsrapidlydecreaseinthecirculationoftheinfantafterdeliv ery.Thismightexplainwhy,insomestudies,dif-
ferencesinNETformationwerereportedbetweenpreterman dterminfants.Theinhibitorsweredetectedindifferentti ssues/bodyfluids-
umbilicalblood(nNIF),placenta(A1ATm358),plasma(
CRISPP-relatedpeptides)-
underlingingtheirimportance.TheyalsoinhibitedNETfor- mationinducedbybacteria(S.aureus),damage-
associatedmolecularpattern(DAMP;heme)andPMA(Yoste
17
CellTissueRes(2018)371:473–488
tal.2016)butdidnotdestroythem.Amechanismoftheiraction isalsoveryintriguing,asNRPsdonotaffectROSproduction
Fig.3I mpactofindividual(human)ageonneutrophilextracellulartrap(NE T)release.GraphicalrepresentationofneutrophilcapacitytoproduceNETs uponstimulation.Inthecaseofneonates,neutrophilpotentialtoreleasethet rapschangesintime.Tostrengthenthegraphicalvisualiza-
tion,potentialtoformNETsismarkedwith–and+where+<++;−/+
indicatesthatinthecaseofnewborninfantssomestudiesreportedalackofN ETformation(−)whereasothersreportedsomeNETrelease(+).Thepresen ceofendogenousNETinhbitorsshortlyafterbirthisindicatedbycircleswit hadiagonalline.Referencedataareincludedanddiscussedinthemaintext
norNEactivity(although,afterenteringthecell,theylocalizeini tscloseproximity)butinhibitPAD4andhistonecitrullinati on.Importantlyfromatherapeuticalpointofview,theinjectiono feithernNIForCRISPPintoadultmiceinfect-
edwithE.coliorLPSpreventedformationofNETsanddecrea sedmortality(Yostetal.2016).
Whywouldsuchinhibitorsfunctiononlyinfetuses/neo- nates?nNIFlevelswerenegligibleinhealthyadultsandun- detectableintheplasmaofadultindividualswithchronicin- flammatorydisorders(Yostetal.2016).Itispossiblybecause,dur ingpregnancy,NET-
inducingstimuliarepresent/generatedatthematernalfetali nterface(Guptaetal.2005;Marderetal.2016;MizugishiandYa mashita2017)andthusexcessiveformatiomofthetrapscouldc auseinflammatorypathologyinthefetomaternalenvironment.
Butthen,shortlyafterbirth,theinhbitorsaredegradedorneutra lizedbyun-
knownmeans.Intriguingly,thelattercorrelateswiththetimewh enresidentmicrobiotainhabitsthehumaninfantandthemicrob iotawasindeedshowntoregulategranulocytosisandhostresist ancetosepsisintheneonate(Deshmukhetal.2014).Theimpact ofmicrobiotaonthelife-spanandfunc-
tioningofneutrophilsinadulthoodhasjustbeenestablished(see BNETsandageofneutrophils^).
NETreleasebyelderlyindividuals
Ana gingo r g a n ism,l i k e t hen ewborn,i s susceptiblet o a var ietyofinflammatorypathogenesis,leadingtoincreasedmorb idity,whichisduetoimpairedimmunefunction(Collertonet al.2012;Tsengetal.2012;Boeetal.2017).
Therefore,thetermimmunosenescencehasbeenintroduced.I mmunsenescence,orinflamm-aging,isassociatedwithlow- grade,chronic,pro-
inflammatorystatus,resultingfromanimbalancebetwee npro-inflammatoryagentsandanti-
inflammatoryfactors(Franceschietal.2007;Collertonetal . 20 1 2).It i s charact e ri zed b y el evat ed l e ve l s of pro- inflammatorycytokines,includingIL-6andTNF-α,inphys- iologicalconditions(Bruunsgaardetal.2000;Krabbeetal.200 4;Ferruccietal.2005).Onehypothesissaysitisbecauseoftheco nstantimmunechallengesoverthelifetimeleadingtoahigherba salactivationstateofcellsoftheinnateimmunesystem(Fulopeta l.2017).Inaddition,arecentstudyreportsthattheseage- associatedchangesdependonthemicrobiota(Thevaranjanetal .2017).Ontheotherhand,theelderlyhaveaweakerresponsetov accination(Goodwinetal.2006;Sasakiet al.2011),w h ichm i ghtre s ultf r oman i m pairedabilitytopresentantigenstoTcel ls,thelatterleadingtoadysfunctionalimmuner esponse(De Martinisetal.20 04;Plowdenetal.2004;WongandGoldstein2 013).
Neutrophilsofelderlyindividualsarecharacterizedbyim- pairedbactericidalactivity(Wenischetal.2000),chemotaxis(F ulopetal.2004),phagocytosis(Butcheretal.2001;Simelletal.2 011)anddecreasedabilitytoperformarespiratoryburst(Wenisc hetal.2000).However,someparametersareeitherpreserved(c hemokinesis)orup-regulated(degranulation)
(Sapeyetal.2014).Thechangesarebelievedtoreflectontheb ehaviorofthecellsinagedindividuals.Theyperformaberrant migration(alteredchemotaxis/chemokinesisratio)sotheycans preadmoreefficientlythanthosefromyoungerin-
dividualsand,becausetheyreleasemoreprotease(asshown
482 CellTissueRes(2018)371:473–488
forNE;Sapeyetal.2014),possiblytofacilitatemigrationthr oughtheECM,morecollateraldamagecanoccur.
Interestingly,highlevelsofNE,alongwitheleventate dpro-
inflammatorycytokinelevels,arealsocharacteristicforthelo w-
gradeinflammatorystateaccompanyingobesity(Talukdaret al.2012).Infact,itisrecognizednowthatsuchaninflammatorys tateconnectsaging,metabolicsyndromeandcardiovasculard isease(GuarnerandRubio-Ruiz2015).
Inphysiologicalconditions,numbersofneutrophilsinthebo nemarrowaresimilarbetweenoldandyoungmice.However, duringinflammatoryconditions,suchassepsisin-
ducedbycecalligationandpuncture(CLP),fewerneutrophilsar eobservedintheperitoneallavageinoldversusyoungmice(Xuet al.2017).Thus,whileinhealthyagedorganisms,thepro- inflammatorystateisapparent,inthecourseofinflamma- tion,theimmuneresponseseemstobedimmed.Althoughnotma nystudieshavebeenundertakenonNETformationbyel derlyindividuals,theyallconsistentlyreportedweakerpro- ductionofthetraps,inlinewiththedataonotherneutrophilactivi ties(Fig.3).Itwasobservedwhenthecellswerefirstprimedwit hTNF-αandthenactivatedtoformNETswithLPSorIL- 8(Hazeldineetal.2014),stimulatedwithPam3CSK4,a TL R2ligand(Xuetal.2017),S.aureus(Tsengetal.2012)ormitoch ondrialDNA,aDAMP(Itagakietal.2015).Notably,expression ofnucleasesbyS.aureus(vs.thenucleasenullstrains)ledtoincrea sedbacterialdissemina-
tioninyoungbutnotoldmice,suggestingthatdefectiveNETfor mationinelderlymicepermittedbothnucleaseandnon- nucleaseexpressingS.aureustodisseminate,altogetherlead- ingtomoreinvasiveS.aureusinfection(Tsengetal.2012).Inter estingly,neutrophilsisolatedfromelderlyperiodontitispatient salsoreleasedfewerNETsthantheyoungonesbutthiswasnot observedinthecaseofhealthyage-
matchedcontrols(Hazeldineetal.2014).Inthestudiesapplyin gTLR2andTLR4ligands,neutrophilscollectedfromelderlype oplehadnormalexpressionofrespectivereceptorsrequiredfort hecellactivationbutdimishedROSproduction(Hazeldine etal.2014;Xuetal.2017).Andthusthelatterwasproposedasa mechanismofthelowerNETrelease.However,Hazeldineetal .(2014)aswellasTortorellaetal.
(2004)showedthatthereisnoimpairmentinp38mitogen- activatedproteinkinase(p38MAPK)activity,thesignalingcas cadeactivatedbyROS,inTNF-α-
primedneutrophilsinboththeelderlyandyoungerindividuals.
Furthermore,PMA,astrongchemicalinducerofROS,induceds imilarquantitiesofNETsinbothagegroups(Tortorellaetal.200 4;Hazeldineetal.2014).
AnotherpossiblemechanismleadingtodimishedNETfor- mationinagedindividualsisimpairedautophagy.Althoughinv olvementofautophagyinNETformationiscontroversial(seeB OnhowNETsarecreated^),itsimpairmentwassug-
gestedtobeco-
responsible,alongwithROS,foraweakNETreleasebyneutrophi lsofelderlyindividuals(Xuetal.2017).
CellTissueRes(2018)371:473–488 3 Inparticular,adefectofAtg5,involvedinautphagosomefor-
mation,waspointedouttocontributetodimmedNETrelease.
AndinsteadofformingNETs,neutrophilswereundergoinga poptosis(Xuetal.2017).
Thereisonereportonanincreasedcapacityofneutro- philsfromagedindividualstoproduceNETs.Thisobser- vationcomesfromstudiesonaorticlesionsinatheroscle- roticmiceandisstrengthenedbydatafromisolatedneu- trophilsactivatedtoproduceNETswith7-
ketocholesterol,anathero-
relevantstimulus,themostabundantoxysterolinhuman(Wa ngetal.2017).Suchaneffectresultedfromincreasedmitocho ndrialoxidativestress,thusmitochon-
drial(mitoOS)andnotcytosolic,ROSgeneration.Theforme rbeingindeedassociatedwithatherosclerosisdur-
ingaging(Vendrovetal.2015).Consideringthatnumbersofi nflammatoryneutrophilswerethesameinagedandyoungmi ce,theyounganimalshadsmallerlesionsandtheirNETforma tionwasmitoOS-
dependent,indicatingintrinsicchangesinneutrophilsofage dsubjects.Thisexperimentalsettingdiffersfromtheotherst udiesonNETformationbyneutrophilsofelderlyindividuals inclearrequirementofmitochondrialROSandnotNA DPHoxidase-dependent.
Asforwhatweknowtodate,neutrophilsofelderlysub- jectsingeneralcastfewerNETs(Fig.3).Nodataindicatesofart hatthisisbecauseofactiveinhibitonoftheirformationasinneo natesbutratheritresultsfromdysregulatedactivityofneutroph ils.Itistemptingtospeculatethatoneofthemecha-
nismsinvolvedmightbeconnectedtotheincreasedreleaseofN Eviadegranulation,asthisenzymeiscriticalforNETfor- mation.FortheseNETsthatrequireNADPHoxidase- dependentROS,adiminishedrespiratoryburstbyneutrophils ofelderlysubjectscanprovideanadditionalexplanation.Ho wever,theobservationthatincreasedmitochondrialROScani nfactincreaseNETformationbyneutrophilsofagedindivi dualssuggeststhatthecellsdonotlosethecapacitytoreleaseN ETsperseandthatthisisratherduetoupstreamdysfunctiona lpathways.
Conclusion s
Thephenotypeofanygivencellreflectseitheritsmaturationsta teortheimpactofextrinsicfactorsandmanifestsitselfbychang esincellmorphology,expressionpatternofintracellularandex tracellularmoleculesbut,foremost,its(altered)func- tioning.Thisisalsotrueforneutrophilsandtheircapacitytoind uceNETs.As,nowadays,NETsarethefocusofbiomed- icalresearch,mostlyduetothesideeffectsoftheirformation,as earchfortheirinhibitorsorremovingagentsdominatesthefiel d.Owingtostudiesonneonateneutrophilsisthediscoveryofen dogenousNETinhibitors.Thisisespeciallypromisinginthelig htoffindingthatimmatureneutrophils,whicharemore
abundantinnumerousdiseasesinwhichNETsplayapivotalrole ,releasethetrapsspontaneously.Obviously,thecellsdonotbeh aveuniformlyinallconditionsandstudiesonNETsarealsotech nicallychallengingastheymostlyrelyoneitherdetectionofsing ularNETcomponentsinbodyfluidsorexvivostimulationofis olatedneutrophils.Although,inthecaseofmicestudies,theseli mitationscanbeovercomewithintravitalmicroscopy,detectin gthetrapsdirectlyinbloodvesselsortissuesofliveanimals, thistechniquecannotbeappliedtohumanstudies.AndNETi nhibitioncanalsobedetrimental.Forinstance,atearlystagesof sepsis,thestruc-
tureshelptocontaindisseminationofinfectionanditisatlatertime pointsthattheirpersistentpresencecausescollateraldamage.T hus,NETinhibitionorremovalshouldalsobetime-
lyadjusted,which,however,isdifficulttocontrol.Now,ane wfactorhastobetakenintoconsiderationwhenitcomestotheco ntrolofNETformationanditsconsequences,namelythepresen ceofneutrophilsofcertainages(immature–mature–
senescent)aswellastheageoftheindividuals.
FundingThisstudywasfundedbytheNationalScienceCentreofPoland(
grantnumber2014/15/B/NZ6/02519).
Compliancewithethicalstandards
Conflictof interestTheauthorsdeclaret hattheyh aven o conflictofinterest.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo mmo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecom mons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,a ndreproductioninanymedium,providedyougiveappro-
priatecredittotheoriginalauthor(s)andthesource,providealinktotheCreat iveCommonslicense,andindicateifchangesweremade.
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