CellandTissueResearch(2018)371:407–
414https://doi.org/10.1007/s00441-018- 2787-0
REVIEW
Earthwormcoelomocyteextracellulartraps:structuralandfunctionalsim ilaritieswithneutrophilNETs
JoannaHoma1
Received:6October2017/Accepted:4January2018/Publishedonline:5February2018
#TheAuthor(s)2018.Thisarticleisanopenaccesspublication
Abstract
Invertebrateimmunityisassociatedwithnaturalmechanismsthatincludecellularandhumoralelements,similartothosethatplayaro leinvertebrateinnateimmuneresponses.Formationofextracellulartraps(ETs)isanewlydiscoveredmechanismtocombatpathoge ns,operatingnotonlyinvertebrateleucocytesbutalsoininvertebrateimmunecells.TheETcomponentsincludeextracellularDNA(e xDNA),antimicrobialproteinsandhistones.FormationofmammalianETsdependsonenzymessuchasneutrophilelastase,myelop eroxidase,thecitrullinationofhistonesandproteaseactivity.Itwasconfirmedthatcoelomocytes—
immunocompetentcellsoftheearthwormEiseniaandrei—arealsoabletoreleaseETsinaprotease-dependentmanner,depen- dentorindependentoftheformationofreactiveoxygenspeciesandrearrangementofthecellcytoskeleton.Similartovertebrateleukoc ytes(e.g.,neutrophil),coelomocytesareresponsibleformanyimmunefunctionslikephagocytosis,cytotoxicityandsecretionofh umoralfactors.ETsformedbycoelomocyteanaloguestoneutrophilETsconsistofexDNA,histoneH3andattachedtothesestruc turesproteins,e.g.,heatshockproteinsHSP27.ThelatterfactconfirmsthatmechanismsofETreleaseareconservedinevolution.The studyonAnnelidaaddsthisanimalgrouptothelistofinvertebratescapableofETrelease,butmostimportantlyprovidesinsidesintoinn atemechanismsofETformationinloweranimaltaxa.
KeywordsCoelomocytes.Amoebocytes.Eleocytes.Extracellulartraps.Histones
Introduction
Theearthwormimmuneresponsedemonstratesanumberofstr ucturalandfunctionalsimilaritiestotheinnateimmunesyst emofvertebrates.Ininvertebrateswithasecondarybodycavity(
e.g.,Annelids),coelomicfluidisrichinmanyproteins(lysozym e,fetidins,lysineprotease)andspecificcells,i.e.coelomo cytes,whichcanbeclassifiedasamoebocytesandeleocytes(B ilejetal.2010).Ontheotherhand,ininverte-
bratesthathaveanopencirculatorysystem,suchasarthro- pods(insects,crustaceans)andmolluscs,hemocytesarere- sponsibleforphagocytosisandcytotoxicity.Hemocytescanbe furthersubdividedintohyalinehemocytesandgranuloc ytes.Thesecells,togetherwithnumeroushumoralcomponent s(e.g.,cecropins,defensins,proteases)arepresent
*JoannaHomajoanna.homa@uj.edu.p l
1
DepartmentofEvolutionaryImmunology,InstituteofZoologyandBio medicalResearch,JagiellonianUniversity,Gronostajowa9, 30-387Krakow,Poland
inthehemolymph(Söderhäll2010).Regardlessoftheadopt edcellnamesofcoelomocytesandhemocytes,theirkill- ingmechanismsaresimilartoeachotherandpathogende- structionisbasedonphagocytosis,enzymeactivation(e.g.,ly sozyme),andformationofreactiveoxygenspecies(ROS)and antimicrobialproteins(e.g.,defensins)
(Bilejetal.2010;Söderhäll2010).Recentpapersalsoconfirm thepossibilitythatinvertebratephagocytesarecapabletoprod uceextracel-lulartraps(ETs)
(e.g.,Homaetal.2016a;Robbetal.2014).
Anatomyoftheearthwormimmunesystema ndimmuneeffectormechanisms
Theearthwormsareprotostomiananimalspossessingtrueco elomcavityfilledwithcoelomicfluidthatnotonlyformsastabl ehydrostaticskeletonbutalsoincludesmanycellsoftheimmu nesystem,coelomocytesandhumoralfactors(Bilejetal.2010;
Cooperetal.2002).Thecoelomocytesoriginateinthemesench ymalliningofthecavity(Bilejetal.2010)andaretheprimaryim munecellsofearthworms.Insimplifiedno-
menclature,coelomocytesaredividedintoamoebocytes(hy- alineandgranular)andcellsderivedfromchloragogentissue
CellTissueRes(2018)371:407–414 3 surroundingthegut,calledeleocytes/chloragocytes(Kureke
tal.2007;Bilejetal.2010)
(Fig.1a,b).Takingintoaccountphysicalparametersmeasuredb yflowcytometry,smallandlargecoelomocyteswithdifferentf unctionalcharacteristicsmaybedistinguished(Cooperetal.1 995,2002;Cossarizzaetal.1996;Quaglinoetal.1996).Inturn ,Engelmannandcoworkersidentifiedusingflowcytometrythr eedifferent
populationsofcoelomocytes:(1)R1–granularcoelomocytes, (2)R2–hyalinecells,and(3)R3–
chloragocytes/eleocytes(Engelmannetal.2004,2005).Moreo ver,insomeolderclas-
sificationsbasedoncytomorphologyandcytochemistry,theco elomocytesoftheannelid(e.g.,Eiseniafetida)weredividedint ofourmajorcategories:acidophils,basophils,chlorago cytescells,andneutrophils(SteinandCooper
Fig.1A natomyoftheearthworm(Eiseniaandrei)immunesystemandim muneeffectormechanisms.aCross-
sectionofearthwormandtheirelementsofimmunesystem:surroundingt hegut(G),chloragogentissue(Ch)andfree-
floatingcoelomocytes;amoebocytesandfreeeleocytesderivedfromc hloragogentissue.Representativeimagesofcoelomocytes’basicim munereactions:bcross-
sectionwithvisiblechloragogentissue(Ch)andincoelomcavityfreecoelo
mocytes(C),cphagocytosis,d ROSproduction,cellcontainingdarkblue NBT
408 CellTissueRes(2018)371:407–414 formazandeposits(*),emovingcells–
chemotaxis,fencapsulation,gROSandproPOactivationintheformedka psuleandhmelaninsynthesis(darkdeposits)whichfinallyleadingtobro wnbodiesformation,e.g.,inematodesclosure,visibleinsidethecapsul e(arrow),j thelatestmechanismofcoelomocytesresponse,production ofextracellulartraps(ETs)andkjointactionofencapsulationandET sformationprocess(Sytoxorangestaining).Scalebar25μm
1978).Amoebocytesareinvolvedintheimmuneresponsein cludingphagocytosis(ValemboisandLassègues1995),ROSp roduction(Homaetal.2013,2016b),andcytotoxicity(NKcell- likeactivity)(Cossarizzaetal.1996).Theyalsoex-pressToll- likereceptors(TLRs)
(Škantaetal.2013;Fjøsneetal.2015).Itisknownthatantimicro bialAMP-
likeproteinoftheneutrophilgranulecontentinthefunctionaresi milartolipopolysaccharide-
bindingprotein(LBP)andbacterialpermeability- increasingprotein(BPI)
(WiesnerandVilcinskas2010).Similarlytoneutrophils,coel omocytesoftheearthwormEiseniaandreiexpressgenesunco dingforatleasttwoconserveddomains(Ealbp/bpiandccf)w iththeabilitytobindlipopolysaccharide(LPS).Theydifferinth eirtissueexpressionandsharehomologywithLBP/BPIfamily(
Škantaetal.2016).Accordingtotheauthors,theup- regulationofmRNAlevelofEalbp/bpiafterbacterialin- fectionsuggeststheirsignificantroleinearthwormimmunedef ense(Škantaetal.2016).
Ontheotherhand,eleocytessynthesizeandreleasehumor- alfactors,suchasagglutininsandopsonins(Bilejetal.2010).Imp ortantantimicrobialpeptides(AMPs),belongingtotwostructu rallydistinctclasses,knownasthedefensinsandthecathelicidi ns,aremainlyproducedbyvertebrateneutrophils(Wiesnerand Vilcinskas2010).Severalauthorshavedemon-
stratedthatearthworminnateimmunityalsodependsoncoelo mocytesthatsynthesizeandsecretehumoralantimicro- bialmolecules(e.g.,lysenin,fetidin,coelomiccytolyticfactor1, CCF-1)
(e.g.,Bilejetal.2000,2001,2010;Engelmannetal.2005).A mongsubpopulationsofcoelomocytes,lyseninismainlyprodu cedbychloragocytesanditsexpressioncanbemodulatedbyGra m-
positivebacterialexposure(Opperetal.2013).Inturn,CCF- 1islocalizedinthecellsofchloragogenoustissueadjuncttot hegutwallandinthetrans-
lucentfreelargecoelomocytes,i.e.incellswithmacroph age-likefunction(Bilejetal.1998).Amongothers,CCF- 1isinvolvedinpathogenrecognitionandleadstoitsimmobiliz ation(Bilejetal.2001).Inaddition,eleocytes,de-
rivedfromchloragogentissue,areresponsibleformaintainingt heconstantpHofcoelomicfluidandstorageofglycogenandlipid s(Affaretal.1998;FischerandMolnár1992).Moreover,eleocyt egranulesstoreriboflavin(B2vitamin)
(Plytyczetal.2006).Intheearthwormcoelomcavity,numerou senzymessuchasproteasesarealsopresent.Theproteasesexert antimi-
crobialeffectsandtakepartintheactivationofthepropheno loxidasesystem(pro-PO)
(Valemboisetal.1994).Thefinalstageofpro- POactivationismelanizationandelim-
inationofpathogens(e.g.,nematodes)(Fig.1g–i).
Earthworms,duringtheirdefenseagainstpathogens,usesev eralelementarymechanisms.Phagocytosisbycoelomoc ytes,similarlytothatofvertebrates,canbemodu-
latedbyhumoralcomponents,opsonins,whichcoatthepar- ticleandthuspromoteitsphagocytosis.Moreover,theyarecapa bleofROSandnitricoxide(NO)production(Homaetal.
2013;Bernardetal.2015;Homaetal.2016b;ValemboisandLa ssègues1995).Furthermore,coelomocyteshaveavarietyofd efensemechanismstoresisttheharmfulsideeffectsofROS.
Theyincludeexpressionofsuperoxidedismutase(SOD)wh ichcatalyzestheconversionofsuperoxideintohy-
drogenperoxideandoxygen,aswellasglutathioneperoxi- dasesandcatalases,whichthendegradehydrogenperoxide(
Homaetal.2016b;Saint-Denisetal.1998).
Theabove-mentionedmoleculesarekeyfactorsinthepro- cessofchemotaxis,phagocytosisandencapsulation,i.e.clos- ingthepathogensinsidestructurescalledBbrownbodies^(B ilejetal.2010;Valemboisetal.1992)(Fig.1c–
i).Encapsulationisacellularimmuneresponseusedagainstp athogensthataretoolargetobephagocytosed(Valemboisetal .1994).BBrownbodies^aregraduallypushedintotheposteri orpartsoftheearthwormbody,andfinallydisposedwithseg mentsthroughthenaturalamputationcalledautotomy(Bilejet al.2010).
Inmanygroupsofinvertebrates,thepro-
PO,anelementofthehumoralinnateimmunesystem,isthefirstl ineofdefenseinthefightagainstpathogens.Phenoloxidase(P O)isapartofacomplexsystemofpatternrecognition,madeofpr oteinasesandproteinaseinhibitors,constitutingtheso- calledprophenoloxidase-
activatingsystem(Söderhäll2010).Thisinnateimmunerea ctionprovidestoxicquinonesubstancesandothershort- livedreactionintermediatesinvolvedintheformationofmore long-
livedproducts,suchasmelanin,thatphysicallyencapsulatepa thogens(Valemboisetal.1992,1994).Recentevidencealsost ronglyimpliesthatthemelani-
zationcascadeprovides,orisintimatelyassociatedwith,theap pearanceoffactorsstimulatingcellulardefensebyaidingpha gocytosis.Inannelids,thepro-POsystemisstrictlyin- volvedinencapsulationandtheformationofbrownbodies,in whichmelaninandlipofuscinaresynthesized.Therefore,itisn otsurprisingthatseveralstudieshaveunequivocallyshownt heimportanceofthemelanizationreactionfortheoutc omeofseveralspecificpathogen–hostencounters,includ- ingbacterialinfections.
Extracellulartrapproductio n
SincethediscoveryofETs,theresultsofresearchconductedon vertebratecellshaveaddedmuchinformationonboththecomp onentsofETsandthemechanismsnecessarytoinitiatetheirfor mation(Brinkmannetal.2004;Neelietal.2009;Papay annopoulosetal.2010;Kolaczkowskaetal.2015).Thepheno menonofcreatingETswasfirstdescribedformam-
malianneutrophils(Brinkmannetal.2004).Theauthorscon- cludedthat,uponstimulationwithGram-
positive(Staphylococcusaureus)orGram-
negative(SalmonellatyphimuriumandShigellaflexneri)b acteria,aswellasundertheinfluenceofphorbol12-
myristate13-acetate(PMA),LPSandinterleukin-8(IL- 8)neutrophilsareabletoproduceETs,
7
CellTissueRes(2018)371:407–414
so-calledneutrophilETs(NETs),inwhichDNAandcytoplas- micgranulefactorsarecontained.Thefollowingyearsbroug htreportsontheabilitytoalsocreateETsbyotherpop-
ulationsofmammalianleukocytes,i.e.,monocytes/macro- phages,eosinophils,andmastcells(Chowetal.2010;Yousefi etal.2008)inmice(Kolaczkowskaetal.2015),sheepandcattle(
Yildizetal.2017),aswellasbyothernon-
mammalianvertebrateneutrophilsandmacrophages,e.g.,tel eostfish(Pijanowskietal.2013)andchicken(Chuammitrieta l.2017).TheproductionofETsisimportantinthedefenseagain stpathogens,butthereisstillnoclearevaluationofthewholer angeofconsequencesoftheiracti-
vation.Although13yearshaspassedbysincethediscoveryofETs tructures,thenumberofreportsonETsininvertebratesisstilllimi ted.Todate,ithasbeenfoundthatETsareproducedbythehemoc ytesofshrimps(Ngetal.2013,2015;Koiwaietal.2016),crab(C arcinusmaenas)
(Robbetal.2014),oyster(Crassostreagigas) (Poirieretal.2014),gastropodslugspe-
cies(ArionlusitanicusandLimaxmaximus),andsnail(Ac hatinafulica)(Langeetal.2017).Thelatestreportsindi- catethatthecellsofsimplerorganisms,e.g.,thesocialamoeba(D ictyosteliumdiscoideum),alsohaveanabilitytoreleaseextracellu larDNAwiththeformationofstructuressimilartoNETs(Zhan getal.2016;ZhangandSoldati2016).Earthwormcoelomoc ytesshowasimilarmechanism(Homaetal.2016a).
InsomestudiesofthestructureofETsreleasedfrominver- tebrateimmunocompetentcells,onlythepresenceofextracel- lularDNA(extDNA)wasfoundaftercellimmunologicalstim ulation(Koiwaietal.2016).Otherstudieshaverevealedthathist ones(Ngetal.2013;Robbetal.2014;Homaetal.2016a),hsp27 (Homaetal.2016a)andc-
typelysozyme(Koiwaietal.2016)arealsoattachedtoextDNA.
ThemostdetailedcharacteristicofETswasrevealedinshrimph emo-
cytes(Ngetal.2013,2015).TheydemonstratedthatE.colicanb ecapturedbyETsandthathistoneH1proteinscolocalizedwi thDNAfibers.AveryinterestingprocessofETformationwasa lsofoundinsocialamoeba(Zhangetal.2016;ZhangandSoldat i2016).Duringtheemergenceofmulticellularity,theseanima lsdevelopedaprimitiveimmunesystemintheformofadedicated setofspecializedphagocyticcellsincludingcells(Sentinelcel ls)whichreleaseETstructures.
Basedonknowledgegainedthroughresearchonvertebratece lls,itisknownthatthemechanismofETformationconsistsofsev eralbasicsteps,asfollows:(1)productionofROSand
(2)thetransportofproteases,includingneutrophilelastasere sponsibleforthechromatindecondensation,fromcytoplas- micgranulestothecellnucleus(PapayannopoulosandZychli nsky2009).ThenextstepoftheETformationisthecitrullinatio nofhistones,and,finally,generationofETs,whichmeansthro wingunfoldedDNAtogetherwithgranulecomponentsoutoft hecell(Brinkmannetal.2004;
Kolaczkowskaetal.2015).Ingeneral,theproteinsattachedtone utrophilETsincludehistones,proteases(e.g.,neutrophilelasta se,cathepsinG),oxidativeenzymes(e.g.,myeloperoxida se,MPO)andantimicrobialproteinssuchaslactoferrin(Gold mannandMedina2013;VorobjevaandPinegin2014).Itshoul dbeunderlinedthathistonesarethemainproteincomponents ofchromatinthatcompact,helpcondensateDNA,andposs essantimicrobialproperties(Brinkmannetal.2004).Moreov er,recentresearchsuggeststhattheunderlyingstructureofNE Tsisconsiderablyorga-
nizedandthatpartoftheirproteincontentplaysanimportantrolei nmaintainingtheirmesharchitecture(Piresetal.2016).
Instudiesonearthwormcoelomocytes,wedemonstratedth eappearanceofNET-
likestructures(Fig.1j,k)asaresultofcoelomocytestimulationwi thLPS,zymosan,PMA,aswellasMicrococcuslysodeikusand Xenorhabdusbovienii(symbioticbacteriainhabitingnematod es).Moreover,itwasrevealedthatthecoelomocyteETsarebuilt, amongothers,ofnuclearDNA,H3histones(Fig.2a–
g)andconservedheatshockproteinsHSP27(Homaetal.2016a ).However,itshouldbementionedthatthelackofspecificantibo diesmakesstudiesofinverte-brateETsverydifficult.
TheresultsindicateastrongsimilarityofinvertebrateETstoo riginallydescribedETsformedbyvertebrateneutrophils.More over,bothinstudiesofvertebrateandinvertebrateETs,inhibitor sofproteases,neutrophilelastaseandNADPHoxi-
dasewereusedtorevealthemechanismsresponsibleforETtrigg ering.Serineproteases,includingelastase-
likeproteasecalledearthwormfibrynolyticenzyme(EFE),hav ealsobeendescribedinAnnelida(Zhaoetal.2007).EFEdegrade sfibrin-
ogen,elastinandfibrin,butalsopartiallyconvertsplasmino- genintoactiveplasmin(Zhaoetal.2007).Inourexperimentsone arthwormETs,wefoundthatproteaseinhibitorsincludingserin eproteasesandelastaseinhibitETformationwhilethei n h i bi t o r so faut o p ha g yan dt hein h i b i t or so fapoptosis- promotingcaspasesdidnothinderthisprocess(Homaetal.20 16a).Surprisingly,itwasshownthatNETformationinhum anneutrophilsisdependentonautophagy(Remijsenetal.2011 a).
Intriguingly,P ieterseetal.
(2016)observedthat,inwholebloodculturesexvivoorinvitroint hepresenceofplatelets,allLPSserotypesinducedBvital^NETfo rmation.Thisplatelet-
dependentr eleaseo f N ETso ccurredr apidlyw ithoutneutrophilc elldeathandwasindependentof ROSformationanda utophagy butr equiredplateletTLR4-an dCD62P-
dependentplatelet–
neutrophilinteractions.Nevertheless,theinhibitionofROS(
withDPI)orautophagy(withwortmannin)didnotinfluenceBvita l^NETosisinducedbyLPS-
O111(Pieterseetal.2016).Moreover,itwasrecentlydemonstrate dthatLPS-
activatedplateletsinduceBvital^NETosisduringsepsis(Maand
CellTissueRes(2018)371:407–414 410
Kubes2008;YippandKubes2013).ThisformofNETreleaseisf undamentallydif-
ferentfromBsuicidal^NETosis;hence,Bvital^NETosisoccurs
Fig.2Earthworm(Eiseniaandre i)coelomocytesformextracellular traps(ETs)composedofextracell ularDNA(extDNA)andhistones.
aRepresentativeimagesoflivecoe lomocytesthatreleasedETsorarei naprocessoftheirrelease(ETting).
C oelomocytesretrievedfromE.an dreiwereseatedinslidechambersa ndstimulatedwithPMAand,after 24h,Sytoxorangewasaddedtostai ntheextDNA.bAutofluorescente leocytes(*,greenfluorecscenceis derivedfromriboflavin)andamoe bocytes(^),csomecoelomocytesi naprocessofextrudingtheirDNA(
ET).dRepresentativeimagesofim munofluorescencestainingofETs releasedbyE.andreicoelomocyt escollectedfromearthwormstreat edfor24hwithbacteriaX.bovienii .Retrievedcoelomocytesweresea tedinslidechambersandtheimmu nostainingwasperformedafter24 h;additionally,eSytoxorangewas usedtocounter-
stainextDNA.f,gImmunostainin gwithspecificantibodiesrevealed thatextDNA(red)isdecoratedwit hhistones3(H3,green).Scalebar 25μm
muchfaster,isnotdependentonautophagyorROS,andisnotasso ciatedwithdirectlyticcelldeath.Incontrasttoapoptoticcells,NE Tformationinvolveddifferentmechanismswithoutsignalssucha sphosphatidylserinebeforeplasmamembranedisruption(Remijs enetal.2011a).Moreover,caspaseactivityisonlydetectedduring spontaneousneutrophilapoptosis,butnotduring,e.g.,PMA- inducedNETosis(Remijsenetal.2011b).Furthermore,incoel omocytes,theNADPHoxidaseinhibitor,suppressingtherespirat oryburst,exertedaninhibito-
ryeffectontheETsformationincellsstimulatedwithPMAbutnot uponstimulationwithbacteria.Theseresultshaveconfir medearlierobservationsinvertebrates(Kolaczkowskaetal.2015
;Pijanowskietal.2013)thattheproductionofETsisnotalwaysR OS-dependent.
Asmentionedabove,theETscontainhistones,but,inter- estingly,partsofthemarecitrullinatedhistones.Itisknownthatt hepackingofnuclearchromatinisassociatedwiththepresence ofhistones,anditsdecondensationispartiallyde-
pendentonanappropriatemodificationoftheseconservative
proteins.Thereisalsoevidencethathistonesaresubjecttoanum berofpost-
translationalmodifications,fromwhichcitrullination(deimi nationofguanidineresiduesinarginines)inhistonesisessentia lforNETformation.Invertebrates,PAD4(peptidylargininede iminase4)istheenzymeresponsi-
bleforhistonecitrullination(Rohrbachetal.2012).As,todate ,PAD4hasnotbeendetectedinlowerorganisms(Bachand2 007),themodeofET-
containedhistonecitrullinationstillremainsunclear.Surprisi ngly,inourrecentstudy(Homaetal.2016a),aninhibitoryeff ectofa well-knownPAD4inhibitor(Cl-
amidine)onETformationinearthwormcoelomocytes,aswel lasthepresenceofcitrullinatedH3histoneswithintheETs,wa sfound.Theseresultssuggestthepotentialtocarryoutthepro cessofH3histonecitrullinationinearthworms,andthep ossibilityofthepresenceofanenzymethatplaysasimilarrolea ndshowssusceptibilitytothestandardPAD4inhibitor.Tosupp ortthisconclusion,itisworthnotingthatthemechanismoftheE Tformationininvertebrates,includingearthworms,exhibits
412 CellTissueRes(2018)371:407–414
manysimilaritieswiththemechanismdescribedinvertebrates(
Table1).Asmentionbefore,thesesimilaritiescanbefoundeven inthepresenceandactivityofserineproteases,produc- tionofROSandtheactivityofantioxidantenzymes.
Studiesconductedtodatehaveallowedscientiststoiden- tifyconsiderablesimilaritiesbetweentheformationandcom- positionofETsinearthwormsandstructuresformedbyver- tebrateneutrophils.Itshouldbenoted,however,thatmanyaspe ctsrelatedtotheinvertebrateETshavenotyetbeenverified.
Onemorequestionwhichhasnotbeenrevealeduntilnow istheinvolvementofETsintheprocessoftheeradicationoflarge rpathogens.Theimmunesystemofbothvertebratesandinverte bratescontrolspathogensofvaryingsizes,rangingfromsmallv irusesandbacteriatofungiandparasites.Largepathogens(e.g., parasites)avoidphagocytosisandthereforecanbedifficulttore move(Branzketal.2014).Asexplainedintheprevioussection,e ncapsulationandformationofbrownbodiesplayaparamountro leinremovingbiggerpathogens(e.g.,nematodes),andelimin atingbacteriaorthecellscontainedinthestructureofcapsule(V alemboisetal.1994).Withinsuchaggregates,activatedcoelom ocytesgenerateROS,andactivatetheproPOsystem.Thelatteri sdependentontheactionofproteases.Inturn,melanindepositio noccurswithinthebordersofbrownbodies.Themelaninisinvol vedintheseparationofpathogensfromthecoelom.Theidentity ofmechanisms/moleculesinvolvedintheformationofbrownbo diesandETssuggestthattheseareconnectedprocesses.And,i ndeed,itwasfoundthattheextracellularDNAmayfacilitatet heagglomerationofcellsandformationofbrownbodies(Homa etal.2016a).
Lifeisallaboutevolution:fromETstoNETs
Theearthwormsimmunesystemwhenstimulatedshowsphag ocytosis,encapsulation,agglutination,opsonization,clotting andlysis.Thelistofearthwormdefensemechanismsdemonstra tedthatcoelomocytescanalsoformETswhichsuccessfullyt rapbacteria.Similartovertebrates,earthworm
ETsareDNase-andheparin-
sensitive.ETsformationbycoelomocytesdependsonprotease activitybutisindependentofcoelomocyteapoptosisandNADP Hoxidase-independentinthecaseofbacteria-
inducedETs,incontrasttoROS- dependentETformationuponPMA-
stimulation.Moreover,coelomocyteETstrapbacteriaanda reinvolvedintheformationofcellaggregates(Homaetal.2016 a).Furthermore,theresultsobtainedonSentinelcellsofsociala moebae(Zhangetal.2016)arestrongevidencethatDNA- basedcell-
intrinsicdefensemechanismsemergedmuchearlierthanthoug ht,about1.3billionyearsago(ZhangandSoldati2016).Interest ingly,inplants,uponinfection,special-
izedcellsonthesurfaceofarootalsoreleasetheirchromatininapr ocessthatrequiresROSproduction(Hawesetal.2011).TheseN ET-
likestructureshavea defensefunction,asdegradingthemwit hDNasesmakestheplantmoresuscepti-bletofungalinfections.
Ininvertebrates,thereleasedchromatinparticipatesinde- fensenotonlybyensnaringmicroorganismsandalsobyex- ternalizingantibacterialhistonestogetherwithothercoel omocyte-/haemocyte-deriveddefensefactors,but,cru- cially,alsoprovidesthescaffoldonwhichintactcellsassem- bleduringencapsulation;aresponsethatsequestersandkillspot entialpathogensinfectingthebodycavity(Robbetal.2014 ).
WhatistheET/NETfunction,immobilizationoractive killing?
TheantimicrobialactivityofETsislikelytheresultacombinati onofthecomponents,andtheireffectsareen-
hancedbythehighlocalconcentrationsachievedintheNETstru cture.Lastly,antibodiesagainsthistonespreventNET- mediatedkillingofvariousmicroorganisms(Brinkman netal.2004),underliningthefindingthattheseabundantprote inskillmicrobesveryefficiently.Histonesareindispensablefor eukaryoticandarchaeallife.Histonesarehighlyconservedthr oughevolution,formthebasicunitofthechromatin,thenucl eosome,andhavebeenintensivelystudiedandarewellchara cterized(ThatcherandGorovsky1994;KornbergandLorch19 99).Inmammals,extranuclearhistonesarefoundinthecytoplas mandonthesurfaceofcellsandarereleasedabundantlyinNETs(
Urbanetal.2009;
Table1Summaryofsimilaritiesbet weene arthwormcoelomocytese x tracellulart rapsandv ertebraten eu trophilextracellulart raps
Neutrophilextracellulartrapsa Coelomocytesextracellulartrapsb
extDNA extDNA
Histones Histones(H3)
NeutrophilelastaseNE Elastase–likep roteases
MyeloperoxidaseMPO Proteases
PAD4/Cytrulination PAD4-notdetectedininvertebrates/cytrulination?
Cytoplasmic/granularproteins Cytoplasmic/granularproteins ROS-dependentornon-dependent ROS–dependentornon-dependent
CellTissueRes(2018)371:407–414 3
aB
rinkmannetal.2004;Papayann opoulosandZychlinsky2009
bH
omaetal.2016a
BrinkmannandZychlinsky2012).Invertebratehistonesalsosh owantimicrobialactivityagainstawiderangeofmicroor- ganisms:bacteriaandparasitesinvitroandinvivoandhavethea bilitytobindbacteriallipopolysaccharideandotherpathogen- associatedmolecules(Nikapitiyaetal.2013).Forexample,am ixofcorehistoneproteinsH2A,H2B,H3,andH4,isolatedfromt hehemocytesofthePacificwhiteshrimp,haveantimicrobialact ivityagainstMicrococcusluteus(Patatetal.2004).
Theexpulsionofchromatinasaweaponmightwellbeanancie nttoolconservedinevolutionintheformofETs.Exploringho wETsaremadeandtestingtheirrelevancedur-
ingdiseaseandinhealthcouldenhanceourunderstandingofthis novelaspectofimmunity.ETscould,onthehostside,helporg anismssurviveinanenvironmentwherepredationandparasiti smbymicrobesareathreat.However,ETsdrivetheevolutionar yselectionofmorepathogenicstrainsofmi-
croorganisms(BrinkmannandZychlinsky2012).
Suchatacticoffightpathogenshasalwaysbeenneeded,even intheworldofplants(Wenetal.2009;Hawesetal.2011).ETf ormationreliesoncommoncellularandmolecularmechanisms fromvertebratestoinvertebrates.
Inconclusion,theknowledgeabouttheproductionofETsini nvertebratesconfirmsthattheextracellularreleaseofchro- matinisanancientdefenseprocess,andhasbeenconservedthro ughevolution.
AcknowledgementThisstudywassupportedbytheNationalScienceCen treofPoland(grantnumber2014/15/B/NZ6/02519,Opus8)andK/ZD S/006311.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo m mo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecommo ns.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andr eproductioninanymedium,providedyougiveappro-
priatecredittotheoriginalauthor(s)andthesource,providealinktotheCreat iveCommonslicense,andindicateifchangesweremade.
References
AffarEB,DufourM,PoirierGG,NadeauD(1998)Isolation,purificationan dpartialcharacterizationofchloragocytesfromtheearthwormspeci esLumbricusterrestris.MolCellBiochem185:123–133
BachandF(2007)Proteinargininemethyltransferases:fromunicellulareu karyotestohumans.EukaryotCell6:889–898
BernardF,BrulleF,DumezS,LemiereS,PlatelA,NesslanyF,CunyD,Dera mA,VandenbulckeF(2015)Antioxidantresponsesofanne- lids,BrassicaceaeandFabaceaetopollutants:areview.EcotoxicolEn vironSaf114:273–303
BilejM,RossmannP,SinkoraM,HanusováR,BeschinA,RaesG,DeBaets elierP(1998)Cellularexpressionofthecytolyticfactorinearthwor msEiseniafoetida.ImmunolLett60:23–29
BilejM,DeBaetselierP,BeschinA(2000)Antimicrobialdefenseoftheeart hworm.FoliaMicrobiol(Praha)45:283–300
BilejM,DeBaetselierP,VanDijckE,StijlemansB,ColigeA,BeschinA(200 1)Distinctcarbohydraterecognitiondomainsofaninvertebrate
defensemoleculerecognizegram-negativeandgram-positivebac- teria.JBiolChem49:45840–45847
BilejM,ProcházkováP,ŠilerováM,JoskováR(2010)Earthwormim- munity.In:SöderhällK(ed)Invertebrateimmunity.Springer,NewY ork,pp66–79
BranzkN,LubojemskaA,HardisonSE,WangQ,GutierrezMG,Brown GD,PapayannopoulosV(2014)Neutrophilssensemicrobesizeands electivelyreleaseneutrophilextracellulartrapsinresponsetolargepat hogens.NatImmunol15:1017–1025
BrinkmannV,ReichardU,GoosmannC,FaulerB,UhlemannY,WeissDS, WeinrauchY,ZychlinskyA(2004)Neutrophilextracellulartrapsk illbacteria.Science303:1532–1535
BrinkmannV,ZychlinskyA(2012)Neutrophilextracellulartraps:isim- munitythesecondfunctionofchromatin?JCellBiol198:773–783 ChowOA,vonKöckritz-
BlickwedeM,BrightTA,HenslerME,ZinkernagelAS,CogenAL, Gal loRL,MonestierM,WangY,GlassCK,NizetV (2010)Statin sEnhanceFormationofPhagocyteExtracellularTraps.CellHostM icrobe8:445–454
ChuammitriP,OstojićJ,AndreasenCB,RedmondSB,LamontSJ,PalićD(
2017)Chickenheterophilextracellulartraps(HETs):novelde- fensemechanismofchickenheterophils.VetImmunolImmun opathol129:126–131
CooperEL,CossarizzaA,SuzukiMM,SalvoliS,CapriM,QuaglinoD,Fran ceschiC(1995)Autogeneicbutnotallogeneicearthwormef- fectorcoelomocyteskillthemammaliantumorcelltargetK562.CellI mmunol166:113–122
CooperEL,KauschkeE,CossarizzaA(2002)Diggingforinnateimmu- nitysinceDarwinandMetchnikoff.BioEssays24:319–333 CossarizzaA,CooperEL,SuzukiMM,SalvioliS,CapriM,GriG,
QuaglinoD,FranceschiC(1996)Earthwormleukocytesthatarenot phagocyticandcross-
reactwithseveralhumanepitopescankillhumantumorcelllines.Exp CellRes224:174–182
EngelmannP,MolnarL,PalinkasL,CooperEL,NemethP (2004)Earthw ormleukocytepopulationsspecificallyharborlysosomalen- zymesthatmayrespondtobacterialchallenge.CellTissueRes316:39 1–401
EngelmannP,PálinkásL,CooperE,NémethP(2005)Monoclonalanti- bodiesidentifyfourdistinctannelidleukocytemarkers.DevCompIm munol29:599–614
FischerE,MolnárL(1992)Environmentalaspectsofthechloragogenousti ssueofearthworms.SoilBiolBiochem12:1723–1727
FjøsneTF,StensethEB,MyromslienF,RudiK(2015)GeneexpressionofT LRhomologuesidentifiedbygenome-
widescreeningoftheearthwormDendrobaenaveneta.InnateImmu n21:161–166
GoldmannO,MedinaE(2013)Theexpandingworldofextracellulartrap s:notonlyneutrophilsbutmuchmore.FrontImmunol3:1–10 HawesMC,Curlango-
RiveraG,WenF,WhiteGJ,VanettenHD,XiongZ(2011)Extracellul arDNA:thetipofrootdefenses?PlantSci180:741–745
HomaJ,OrtmannW,KolaczkowskaE(2016a)Conservativemecha- nismsofextracellulartrapformationbyAnnelidaEiseniaandrei:ser ineproteaseactivityrequirement.PLoSONE11:e0159031 HomaJ,StalmachM,WilczekG,KolaczkowskaE(2016b)Effectiveacti
vationofantioxidantsystembyimmune-relevantfactorsre- verselycorrelateswithapoptosisofEiseniaandreicoelomocytes.JC ompPhysiolB186:417–430
HomaJ,ZorskaA,WesolowskiD,ChadzinskaM(2013)Dermalexposuretoi mmunostimulantsinduceschangesinactivityandproliferationofcoelo mocytesofEiseniaandrei.JCompPhysiolB183:313–322
KolaczkowskaE,JenneCN,SurewaardBG,ThanabalasuriarA,LeeWY,S anzMJ,MowenK,OpdenakkerG,KubesP(2015)Molecularmech anismsofNETformationanddegradationrevealedbyintravi- talimaginginthelivervasculature.NatCommun6:6673
KoiwaiK,AlentonRR,KondoH,HironoI(2016)Extracellulartrapfor- mationinkurumashrimp(Marsupenaeusjaponicus)hemocytesiscou pledwithc-typelysozyme.FishShellfishImmunol52:206–209
KornbergRD,LorchY(1999)Twenty-
fiveyearsofthenucleosome,fundamentalparticleoftheeukaryotec hromosome.Cell98:285–294
KurekA,HomaJ,PłytyczB(2007)Characteristicsofcoelomocytesof thes tubbyearthworm,A llolobophorac hlorotica(Sav.)E urJ S oilBiol 4 3:121–126
LangeMK,Penagos-TabaresF,Muñoz-CaroT,GärtnerU,MejerH, SchaperR,HermosillaC,TaubertA(2017)Gastropod-
derivedhaemocyteextracellulartrapsentrapmetastrongyloidlarvals tagesofAngiostrongylusvasorum,Aelurostrongylusabstrususa ndTroglostrongylusbrevior.ParasitVectors10:50
MaAC,KubesP(2008)Platelets,neutrophils,andneutrophilextracellu- lartraps(NETs)insepsis.JThrombHaemost6:415–420
NeeliI,DwivediN,KhanS,RadicM(2009)Regulationofextracellularchro matinreleasefromneutrophils.JInnateImmun1:194–201
NgTH,ChangSH,WuMH,WangHC(2013)Shrimphemocytesreleaseext racellulartrapsthatkillbacteria.DevCompImmunol41:644–651 NgTH,WuMH,ChangSH,AokiT,WangHC(2015)TheDNAfibersofshri
mphemocyteextracellulartrapsareessentialfortheclearanceofEsch erichiacoli.DevCompImmunol48:229–233
NikapitiyaC,DorringtonT,Gómez-ChiarriM(2013)Theroleofhis- tonesintheimmuneresponsesofaquaticinvertebrates.InvertebrateSu rvivJ10:94–101
OpperB,BognárA,HeidtD,NémethP,EngelmannP(2013)Revisinglysen inexpressionofearthwormcoelomocytes.DevCompImmunol39 :214–218
PatatSA,CarnegieRB,KingsburyC,GrossPS,ChapmanR,ScheyKL(200 4)Antimicrobialactivityofhistonesfromhemocytesofthepacific whiteshrimp.EurJBiochem271:4825–4833
PapayannopoulosV,ZychlinskyA(2009)NETs:anewstrategyforusingol dweapons.TrendsImmunol30:513–521
PapayannopoulosV,MetzlerKD,HakkimA,ZychlinskyA (2010)Neutr ophilelastaseandmyeloperoxidaseregulatetheformationofneutrop hilextracellulartraps.JCellBiol191:677–691
PieterseE,RotherN,YanginlarC,HilbrandsLB,vanderVlagJ(2016)Neutr ophilsdiscriminatebetweenlipopolysaccharidesofdifferentbacteri alsourcesandselectivelyreleaseneutrophilextracellulartraps.Fr ontImmunol7:484
PijanowskiL,GolbachL,KolaczkowskaE,ScheerM,Verburg-van KemenadeBM,ChadzinskaM (2013)Carpneutrophilicgranul ocytesformextracellulartrapsviaROS-dependentandinde- pendentpathways.FishShellfishImmunol34:1244–1252 PiresRH,FelixSB,DelceaM(2016)Thearchitectureofneutrophile
xtracellulartrapinvestigatedbyatomicforcemicroscopy.Nano8:1 4193–14202
PlytyczB,HomaJ,KoziolB,RozanowskaM,MorganAJ(2006)Riboflavi ncontentinautofluorescentearthwormcoelomocytesisspecies- specific.FoliaHistochemCytobiol44:65–71
PoirierAC,SchmittP,RosaRD,VanhoveAS,Kieffer- JaquinodS,RubioTP,CharrièreGM,Destoumieux-
GarzónD(2014)AntimicrobialhistonesandDNAtrapsininverte brateimmunity:evidencesinCrassostreagigas.JBiolChem28 9:24821–24831
QuaglinoD,CooperE,SalvioliS,CapriM,SuzukiM,RonchettiI ,FranceschiC,CossarizzaA(1996)Earthwormcoelomocytesinvi tro:cellularfeaturesand‘granuloma’formationduringcytotox- icactivityagainstthemammaliantumorcelltargetK562.EurJCellBio l70:278–288
RemijsenQ,KuijpersTW,WirawanE,LippensS,VandenabeeleP,Van denBergheT(2011a)Dyingforacause:NETosis,mechanismsbehind anantimicrobialcelldeathmodality.CellDeathDiffer18:581–588 RemijsenQ,VandenBergheT,WirawanE,AsselberghB,ParthoensE,
DeRyckeR,NoppenS,DelforgeM,WillemsJ,VandenabeeleP
(2011b)Neutrophilextracellulartrapcelldeathrequiresbothau- tophagyandsuperoxidegeneration.CellRes21:290–304
RobbCT,DyryndaEA,GrayRD,RossiAG,SmithVJ(2014)Invertebra teextracellularphagocytetrapsshowthatchromatinisanancientdef enceweapon.NatCommun5:4627
RohrbachAS,SladeDJ,ThompsonPR,MowenKA(2012)ActivationofP AD4inNETformation.FrontImmunol3:360
Saint-
DenisM,LabrotF,NarbonneJF,RiberaD(1998)Glutathione,gluta thione-relatedenzymes,andcatalaseactivitiesintheearth- wormEiseniafetidaandrei.ArchEnvironContamToxicol35:602 –614
SteinEA,CooperEL(1978)Cytochemicalobservationsofcoelomocytesfr omtheearthworm,Lumbricusterrestris.HistochemJ10:657–
678ŠkantaF,ProcházkováP,RoubalováR,DvořákJ,Bilej(2016)LBP/BP IhomologueinEiseniaandreiearthworms.DevCompImmunol54:
1–6
ŠkantaF,RoubalováR,DvořákJ,ProcházkováP,BilejM(2013)Molecul arcloningandexpressionofTLRintheEiseniaandreiearthworm.
DevCompImmunol41:694–702
SöderhällK(2010)Invertebrateimmunity.Advancesinexperimentalm edicineandbiology,vol708.Springer,NewYork
ThatcherTH,GorovskyMA(1994)Phylogeneticanalysisofthecorehist onesH2A,H2B,H3,andH4.NucleicAcidsRes22:174–179 UrbanCF,ErmertD,SchmidM,Abu-
AbedU,GoosmannC,NackenW,BrinkmannV,JungblutPR,Zychlin skyA(2009)Neutrophilextra-
cellulartrapscontaincalprotectin,acytosolicproteincomplexin- volvedinhostdefenseagainstCandidaalbicans.PLoSPathog5:e10 00639
ValemboisP,LassèguesM(1995)Invitrogenerationofreactiveoxygenspe ciesbyfreecoelomiccellsoftheannelidEiseniafetidaandrei:ananal ysisbychemiluminescenceandnitrobluetetrazoliumreduc- tion.DevCompImmunol19:195–204
ValemboisP,LassèguesM,RochP(1992)Formationofbrownbodiesinthe coelomiccavityoftheearthwormEiseniafetidaandrei:ananal- ysisbychemiluminescenceandnitrobluetetrazoliumreduction.De vCompImmunol16:95–101
ValemboisP,SeymourJ,LasséguesM(1994)Evidenceoflipofuscinandm elanininthebrownbodyoftheearthwormEiseniafetidaandrei.Cell TissueRes227:183–188
VorobjevaNV,PineginBV(2014)Neutrophilextracellulartraps:mech- anismsofformationandroleinhealthanddisease.Biochemistry(M osc)79:1286–1296
WenF,WhiteGJ,XiongX,VanEttenHD,HawesMC(2009)Extracellul arDNAisrequiredforroottipresistancetofungalinfec-
tion.PlantPhysiol151:820–829
WiesnerJ,VilcinskasA(2010)Antimicrobialpeptides:theancientarmofth ehumanimmunesystem.Virulence1:440–464
YildizK,GokpinarS,GazyagciAN,BaburC,SursalN,AzkurAK(
2017)RoleofNETsinthedifferenceinhostsu sceptibilitytoTox oplasmagondiibetweensheepandcattle.VetImmunolImmuno pathol189:1–10
YippBG,KubesP(2013)NETosis:howvitalisit?Blood122:2784–
2794YousefiS,GoldJA,AndinaN,LeeJJ,KellyAM,KozlowskiE,Schmi d
I,StraumannA,ReichenbachJ,GleichG J,S imonHU( 2008) Catapult-likereleaseofmitochondrialDNAbyeosinophilscontrib- utestoantibacterialdefense.NatMed14:949–953
ZhaoJ,XiaoR,HeJ,PanR,FanR,WuC,LiuX,LiuY,HeRQ(2007)Insitulocaliza tionandsubstratespecificityofearthwormprotease-IIandprotease-III- 1fromEiseniafetida.IntJBiolMacromol40:67–75
ZhangX,ZhuchenkoO,KuspaA,SoldatiT(2016)Socialamoebaetrapand killbacteriabycastingDNAnets.NatCommun7:10938
ZhangX,SoldatiT(2016)Ofamoebaeandmen:extracellularDNAtrapsasan ancientcell-intrinsicdefensemechanism.FrontImmunol7:269
