CD1d deficiency inhibits the development of abdominal aortic aneurysms in LDL receptor deficient

17 Pages·2017·2.66 MB·English

RESEARCHARTICLE
CD1d deficiency inhibits the development of
abdominal aortic aneurysms in LDL receptor
deficient mice
GijsH.M.vanPuijvelde1*,AmandaC.Foks1,RosemarieE.vanBochove1,IlzeBot1,Kim
L.L.Habets1,SaskiaC.deJager1,Marie¨tteN.D.terBorg1,PuckvanOsch1,LouisBoon2,
MariskaVos3,ViviandeWaard3,JohanKuiper1
1 DivisionofBiopharmaceutics,LeidenAcademicCentreforDrugResearch,LeidenUniversity,Leiden,The
Netherlands,2 BiocerosBV,Utrecht,TheNetherlands,3 DepartmentofMedicalBiochemistry,Academic
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MedicalCenter,UniversityofAmsterdam,Amsterdam,TheNetherlands
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a1111111111
*[email protected]
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Abstract
Anabdominalaorticaneurysm(AAA)isadilatationoftheabdominalaortaleadingtoserious
complicationsandmostlytodeath.AAAdevelopmentisassociatedwithanaccumulationof
OPENACCESS
inflammatorycellsintheaortaincludingNKTcells.Animportantfactorinpromotingthe
Citation:vanPuijveldeGHM,FoksAC,van
recruitmentoftheseinflammatorycellsintotissuesandtherebycontributingtothedevelop-
BochoveRE,BotI,HabetsKLL,deJagerSC,etal.
(2018)CD1ddeficiencyinhibitsthedevelopmentof mentofAAAisangiotensinII(AngII).WedemonstratethatadeficiencyinCD1ddependent
abdominalaorticaneurysmsinLDLreceptor NKTcellsunderhyperlipidemicconditions(LDLr-/-CD1d-/-mice)resultsinastrongdeclinein
deficientmice.PLoSONE13(1):e0190962.https://
theseverityofangiotensinIIinducedaneurysmformationwhencomparedwithLDLr-/-mice.
doi.org/10.1371/journal.pone.0190962
Inaddition,weshowthatAngIIamplifiestheactivationofNKTcellsbothinvivoandinvitro.
Editor:MichaelBader,MaxDelbruckCentrumfur
WealsoprovideevidencethattypeINKTcellscontributetoAAAdevelopmentbyinducing
MolekulareMedizinBerlinBuch,GERMANY
theexpressionofmatrixdegradingenzymesinvSMCsandmacrophages,andbycytokine
Received:June28,2017
dependentlydecreasingvSMCviability.Altogether,thesedataprovethatCD1d-dependent
Accepted:December22,2017 NKTcellscontributetoAAAdevelopmentintheAngII-mediatedaneurysmmodelby
Published:January18,2018 enhancingaorticdegradation,establishingthattherapeuticapplicationswhichtargetNKT
cellscanbeasuccessfulwaytopreventAAAdevelopment.
Copyright:©2018vanPuijveldeetal.Thisisan
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
Introduction
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare AccordingtotheWorldHealthOrganization,cardiovasculardiseasesaretheleadingcauseof
withinthepaperanditsSupportingInformation deathworldwide,responsiblefor17.7milliondeathseachyear,withatherosclerosis,achronic
files. inflammationofthevesselwall,asthemajorcause.Anothercommonvasculardisorder,linked
Funding:ThisstudywasfundedbyHartstichting, withagingandatherosclerosis,isthedevelopmentofanabdominalaorticaneurysm(AAA)
withthefollowinggrants:2007T039(Dr.GijsH.M. affecting2–8%oftheelderlypeople.AAA,achronicinflammatorydisease,isdefinedasalocal
vanPuijvelde);2008B048(Ph.D.AmandaC.Foks); permanentdilationoftheabdominalpartoftheaortatranscending1.5-timesthenormalaor-
2012T083(Ph.D.IlzeBot).LouisBoon,employed
ticdiameter.[1,2]AnAAAisoftenasymptomaticandundiagnoseduntilruptureoftheaorta
byBiocerosBV,Utrecht,TheNetherlands,
occurs.Uponrupturetheoverallmortalityisveryhigh(80%to90%).Althoughimproved
supportedthisstudyintheformofprovidingus
imagingtechniquessuchasX-ray,ultrasoundandechocardiogramresultinanearlierdetec-
withneutralizingα-IFNγ,α-IL4andα-IL10
antibodiesanddidnothaveanyroleinthestudy tionofAAA,surgicalinterventioniscurrentlytheonlyavailabletreatment.Sinceno
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CD1ddeficiencyinhibitsaneurysmdevelopment
design,datacollectionandanalysis,decisionto pharmacologicaltherapiesexist,thereisanurgentneedfornoveltherapeuticstrategiesto
publish,orpreparationofthemanuscript. inhibittheprogressionofAAA.
Competinginterests:Iwouldliketoconfirmthat Theinflammatoryresponse,akeyprocessinAAAdevelopment,contributestoan
Biocerosisonlyinvolvedinprovidinguswith increasedproductionofelastaseandseveralproteinases(matrixmetalloproteinases(MMPs),
materialswhichdoesnotalterouradherenceto serineproteinases,cathepsins),whicharemainlyresponsibleforthestructurallossofvessel
PLOSONEpoliciesonsharingdataandmaterials.
wallintegrityleadingtoAAAformation.[3]Locallyincreasedlevelsofchemokines(MCP-1[4,
5],CCL22[6],CXCL12[7]),growthfactors(GCSF,MCSF)[8]andcytokines(TNF-α,IL-6,IL-
1β)[8,9]causeattractionandaccumulationofdifferentleukocytessuchasmonocytes,macro-
phages,dendriticcells(DCs),NKcells,neutrophils,BandTcellsintheaneurysmalvessel
wall.[3,10]InmicethatweredepletedforCD4+Tcells,AAAformationdoesnotoccur.[11]
However,contradictoryresultsareobservedregardingtheroleofdifferentTcellsubsetsin
AAA.Foxp3expressingregulatoryTcellsarefoundtobeprotectiveinAAAformationin
mice,[12]whilebothpro-inflammatoryTh1cells(producingIL-1β,IL-6,TNF-αandIFN-γ)
andanti-inflammatoryTh2cells(producingIL-4,IL-5andIL-10)arelinkedtotheformation
of,aswellastheprotectionagainstAAA[13–19],confirmingthehighlycomplexinterplayof
differentimmunecellsandcytokinesinthepathogenesisofAAA.
NKTcells,anothersubsetofTcellsexpressinganinvariantTcellreceptor(TCR)and
markerscharacteristicofNKcells(NK1.1),arealsopresentinlargenumbersintheaneurys-
malvesselwall.[20]WhileTcellsareactivatedviapeptide-antigenpresentationonMHCmol-
ecules,NKTcellsareactivatedviaglycolipidpresentationonCD1d,anMHCclassI-like
molecule.TheseCD1d-dependentNKTcellscompriseaheterogeneouspopulationofcells
andbasedupondifferencesinTCRcharacteristics,CD1d-dependentNKTcellsaremainly
subdividedintotypeIortypeIINKTcells.ThemostprominentpopulationofNKTcellsin
micecomprisetypeINKTcells,alsocalledinvariantNKT(iNKT)cells,expressingalimited
diversityinTCRsandallrecognizingα-galactosylceramide(α-GalCer).TypeIINKTcells
expressamorediverserangeofTCRsanddonotrespondtoα-GalCer.UponTCRactivation
anddependingontheconditions,NKTcellsrapidlyandsimultaneouslyproducelarge
amountsofbothpro-inflammatory(IFN-γ,IL-2,TNF-α)and/oranti-inflammatorycytokines
(IL-4,IL-5,IL-10,IL-13).NKTcellsarealreadylinkedtothedevelopmentofatherosclerosis
[21–24],butwhetherthepresenceofNKTcellsintheaneurysmalvesselwallisdirectlyassoci-
atedwithAAAdevelopmentisstillunknown.NKTcellspresentinAAAtissuepredominantly
producepro-inflammatoryIFN-γ,whichmayleadtoanupregulatedexpressionofFasand
increasedFasL-mediatedapoptosisofvascularsmoothmusclecells(vSMCs).[20]However,
lateronitwasreportedthattheanti-inflammatoryIL-4,producedbythesameNKTcells,
mightberesponsibleforincreasedexpressionofMMPsbySMCsandmacrophages,thereby
possiblycontributingtothedevelopmentofAAA.[25–27]
InthecurrentstudyweestablishthatLDLr-/-micelackingCD1d-dependentNKTcells
demonstratereducedAAAseverityinthemostcommonlyusedmodeltostudythedevelop-
mentandpathogenesisofAAA,theangiotensinII(AngII)infusionmodel.Inaddition,in
vitrostudiesshowthattypeINKTcellscancontribute,inacytokinedependentway,toAAA
developmentbyincreasingtheexpressionofmatrixdegradingenzymesbymacrophagesand
vSMCs,andbydecreasingvSMCviability.Inconclusion,CD1d-dependentNKTcellsmaybe
atherapeuticallyinterestingtargettolimitAAAprogression.
Materialsandmethods
Animals
AllanimalworkwasapprovedbytheLeidenUniversityAnimalEthicsCommitteeandthe
animalexperimentswereperformedconformtheguidelinesfromDirective2010/63/EUofthe
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CD1ddeficiencyinhibitsaneurysmdevelopment
EuropeanParliamentontheprotectionofanimalsusedforscientificpurposes.MaleC57BL/6,
CD1d-/-andLDLr-/-miceonaC57BL/6backgroundwereobtainedfromourin-housebreeding
facility.LDLr-/-CD1d-/-miceweregeneratedbycrossingLDLr-/-micewiththeCD1d-/-mice.
TheoffspringwasintercrossedtoproducemicewithahomozygousdeletioninbothLDLrand
CD1d.Allmicewerekeptunderstandardlaboratoryconditions(conventionalopencages,
aspenbedding)ingroupsof2–4micepercageandwerefedaregularchowdietora‘Western-
type’diet(WTD)containing0.25%cholesteroland15%cocoabutter(SpecialDietServices,
Witham,Essex,UK).Allmiceusedinexperimentswere12–14weeksofageandofaverage
weight.Dietandwaterwereadministeredadlibitum.Atsacrifice,micewereanesthetizedbya
subcutaneousinjection(120μl)ofacocktailcontainingketamine(40mg/ml),atropine(50μg/
ml)andsedazine(6.25mg/ml).Subsequently,themicewereeuthanizedandexsanguinatedby
femoralarterytransectionfollowedbyperfusionwithPBSthroughtheleftcardiacventricle.
Mediaandreagents
NIH/3T3cellsconstitutivelyproducinggranulocyte-macrophagecolony-stimulatingfactor
(GM-CSF)[28](kindlyprovidedbyL.vanDuijvenvoorde,LUMC,Leiden,TheNetherlands),
andbonemarrowderivedmacrophagesanddendriticcells(DCs)wereculturedinIscove’s
ModifiedDulbecco’sMedium(IMDM)containinghighglucose,sodiumpyruvate,additional
aminoacidsandHEPES(Cambrex,Belgium)supplementedwith8%FCS,100U/mlpenicil-
lin/streptomycin(Pen/Strep,PAA,Germany),2mMGlutamax(Invitrogen,TheNetherlands)
and20μMβ-mercaptoethanol(SigmaAldrich,TheNetherlands).NKThybridomacells[29]
(DN32.D3,kindlyprovidedbyR.Raatgeep,ErasmusMC,Rotterdam,TheNetherlands)were
culturedinDMEMwithGlutamax(Cambrex,Belgium)supplementedwith2%FCS,100U/
mlPen/Strep,and1%non-essentialaminoacids(NEAA)(PAA,Germany).RPMI-1640
(Cambrex,Belgium)supplementedwith10%FCS,100U/mlPen/Strep,2mML-glutamine
and20μMβ-mercaptoethanolwasusedforspleencellcultures.Vascularsmoothmusclecells
(vSMCs),originallyisolatedfromaortasofmaleC57Bl/6miceasdescribedbefore[30],were
thawedandculturedinDMEMwith10%FCS,100U/mlPen/Strepand2mML-glutamine
andbonemarrowderivedmacrophageswereculturedinRPMIwith20%FCS,100U/mlPen/
Strep,2mML-glutamine,1%NEAAand1%sodiumpyruvate(PAA,Germany).Allcellswere
culturedat37˚Cand5%CO .
2
AAAinduction
AAAwasinducedbyusinganAngIIinfusionmodel.[31]Inthismodel,osmoticminipumps
(Model2004,Alzet,DURECTCorporation,Cupertino,USA)werefilledwithAngII(Sigma
Aldrich,TheNetherlands)resultinginareleaserateof1.44mgAngII/kg/day.Theosmotic
pumpsweresubcutaneouslyimplantedinage-matchedLDLr-/-(n=12)andLDLr-/-CD1d-/-
mice(n=11)afteranesthetizingthemicewithisoflurane.Basedupontheresultsofthestudy
byLiuetal.,themicewereputonaWTDoneweekpriortoimplementation.[32]Duetothe
infusionwithAngIIandthesubsequentdevelopmentofananeurysm,suddendeathcan
occurbecauseofaruptureoftheaorta.Thehealthofthemicewasmonitoredtwiceperday
duringtheexperiment.Ourhumanendpointcriteriaincludedweightchanges(measured
onceperday),abnormalbehavior,changesinthemobilityandruffledfur.Noneofthemice
reachedthesecriteriaduringthestudy.
AAAclassificationandquantification
Fourweeksafterplacementoftheosmoticpumpsmicewereanesthetized,euthanizedand
exsanguinated.Subsequently,themicewereperfusedthroughtheleftcardiacventriclewith
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CD1ddeficiencyinhibitsaneurysmdevelopment
PBSfor15min.Theentireaortaisharvestedandphotographed.Subsequentlyallthesamples
wereblindedbyacolleaguenotinvolvedintheprojectandtheextentofdilatationoftheaorta
wasdeterminedbymeasuringtheincreaseinthemaximalexternaldiameteroftheabdominal
(AAA)part,comparedwiththemaximaldiameterofthe“healthy”partproximaloftheaneu-
rysm.Theanalysiswasperformedtwicebytwoco-authors.Asdescribedbyothers,adiameter
of>150%comparedwiththehealthysituationisconsideredtobeananeurysmoftypeI,
>200%asatypeII,multipleaneurysmsand/ordissectionsastypeIIIandwhenamousedied
duetoaorticruptureitisconsideredasatypeIVaneurysm.[33–35]Accordingtothissystem,
aneurysmsvaryingfromtype0totypeIVwereassignedascorefrom0to4pointsinorderto
getameanscorepergroupofmice.
Atheroscleroticlesionquantification
Aftersacrifice,theheartswithaorticrootwereremovedand10μmcryosectionsoftheaortic
rootweremadeonaLeicaCM3050SCryostat(LeicaInstruments,UK).Thesectionswere
stainedwithOil-red-Oandhematoxylin,andplaquesizewasmeasuredonblindedslides
usingaLeicaDM-REmicroscopeandLeicaQwinsoftware(LeicaImagingSystems,UK).
Cholesterolassay
Todeterminethecholesterollevelsinserumofthemice,bloodwascollectedatdifferenttime
pointsduringtheexperimentbytailveinbleeding.Totalcholesterollevelswerequantified
spectrophotometricallyusinganenzymaticprocedure(RocheDiagnostics,Germany).Preci-
pathstandardizedserum(Boehringer,Germany)wasusedasaninternalstandard.
Immunohistochemistry
Aftervisualinspection,ahealthypartandanAAA-affectedpartoftheaortawereembedded
inparaffin,sectioned(7μm)andmountedonglassslides(Superfrost-Plus,VWR,TheNether-
lands).Subsequently,theslideswerestainedwithhematoxylin/eosin.Inaddition,todetectcol-
lagenandMMP-9theslideswerestainedwithaMasson’sTrichromestainingandananti-
MMP-9antibodyrespectively.AllimageswereanalyzedusingaLeicaDM-REmicroscope
(LeicaImagingSystems,UK).
Flowcytometricanalysis
TodeterminetheeffectsofAngIIinfusiononNKTcellnumbersandactivationinvivo,
osmoticminipumpsfilledwithAngIIwereimplantedinLDLr-/-mice,whichwerefedaWTD
foroneweek.Twoweeksafterpumpimplantation,themiceweresacrificedandperfusedas
described.Bloodwascollected,andliversandspleensweredissectedandmashedthrougha
70μmcellstrainer.Erythrocyteswereeliminatedbyincubatingthecellswitherythrocytelysis
buffer(0.15MNH Cl,10mMNaHCO ,0.1mMEDTA,pH7.3).Non-parenchymalcells
4 3
fromtheliverwereseparatedfromparenchymalcellsbycentrifugationatlowspeed.Thenon-
parenchymalcellswereputonaLympholytegradient(Cedarlane,Ontario,Canada)toisolate
liverlymphocytes.Singlecellsuspensionsofliverandspleenweresubsequentlystainedwith
APC-conjugatedα-GalCer/CD1dtetramer(1:800)providedbytheNIHtetramercorefacility
(Atlanta,GA)andPE-conjugatedanti-CD25(0.2μg/sample)mAb(eBioscience,Belgium)for
30min.CellswereanalyzedbyflowcytometryonaFACSCantoII(BectonDickinson,CA).
AlldatawereanalyzedwithFACSDivaandFlowJosoftware.
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CD1ddeficiencyinhibitsaneurysmdevelopment
InvitroNKTcellactivation
TodeterminetheeffectofAngIIonNKTcellactivation,bonemarrowcellswereisolated
fromthetibiaandfemursofLDLr-/-andLDLr-/-CD1d-/-miceaftereuthanizationasdescribed
above.Cellswereculturedfor10daysinIMDMinthepresenceofGM-CSF.After10days,the
resultingantigen-presentingcells(APCs)includingbothmacrophagesanddendriticcells
(DCs)werepulsedwithorwithoutα-GalCer(30ng/ml)andwithorwithoutAngII(100ng/
ml)addedtotheculturemedium.After4hincubation,theAPCswerewashedtwice.Subse-
quently,theAPCswereco-culturedwithNKThybridomacellsina1:5ratioandafter24hthe
IL-2concentrationinthesupernatantwasdeterminedbyELISAaccordingtothemanufactur-
er’sprotocol(eBioscience,Austria).
Real-timePCRassays
TodetermineeffectsofNKTcellactivationontheexpressionofproteinasesbyvSMCsand
macrophages,splenocytesfromLDLr-/-micewereculturedina96-wellsplate(2x105perwell)
andexposedtotheNKTcellspecificligandsα-GalCerorOCH(100ng/ml;EnzoLifeSciences,
TheNetherlands).Aftertwodaysthesupernatantofthesplenocyteswasaddedtobonemar-
row-derivedmacrophagesandvSMCs,whichwerethenculturedina6-wellsplate(1.8(cid:3)106
and2.5x105cellsperwellrespectively)infive-foldperconditionforthreedays.Subsequently,
mRNAwasextractedfromthemacrophagesandvSMCs,usingtheguanidiumisothiocyanate
(GTC)method,andreversetranscribed(RevertAidM-MulVreversetranscriptase).Quantita-
tivegeneexpressionanalysisforMMP-9,MMP-12,andCathepsinS,LandKwasperformed
onanABIPRISM7700sequencedetector(AppliedBiosystems,CA)usingSYBRgreentech-
nology.AcidicribosomalphosphoproteinPO(36B4),Hypoxanthinephophoribosyl-transfer-
ase(HPRT)andribosomalproteinS13(RPS13)wereusedastheendogenousreferencegenes.
TheprimerpairsusedareshowninTable1.
MTTassay
ToinvestigatetheeffectsofNKTcellspecificcytokinesonthevSMCviability,supernatantof
thesplenocytesculturedwithα-GalCerorOCH(50,100or200ng/ml)wasagainaddedto
vSMCculturesforthreedays.ViabilitywasassessedbytheamountofMTT[3-(4,5-dimethy-
lthiazole-2-yl)-2,5-diphenyltetrazoliumbromide]staining(SigmaAldrich,TheNetherlands).
CellsweretreatedwithMTTsolution(0.5mg/ml)for1handopticaldensitywasmeasured
usingaspectrophotometerat550nm.Inaddition,blockingantibodiesagainstIFN-γ,IL4or
IL-10(1,5,10and20μg/ml,providedbyLouisBoon)wereaddedtothesupernatantofthe
splenocytes30minbeforeculturingofthevSMCswiththissupernatant.AnincreaseinvSMC
deathwasassessedbyreductioninMTTstaining.
Table1. Primerpairsusedforquantitativegeneexpressionanalysis.
forward reverse
MMP9 5'-CTGGCGTGTGAGTTTCCAAAAT-3' 5'- TGCACGGTTGAAGCAAAGAA-3'
MMP12 5'-CCTGGGCTTCTCTGCATCTGT-3' 5'-CGACGGAACAGGGGGTCATATT-3'
CathepsinS 5'-GCCAGCCATTCCTCCTTCTTCT-3 5'-TGCCATCAAGAGTCCCATAGCC-3'
CathepsinK 5'-GGGAACGAGAAAGCCCTGAAGA-3' 5'-ACACTGCATGGTTCACATTATCACG-3'
CathepsinL 5'-TAGCAGCAAGAACCTCGACCAT-3' 5'-CCATACCCCATTCACTTCCCCA-3'
36B4 5'-GGACCCGAGAAGACCTCCTT-3' 5'-GCACATCACTCAGAATTTCAATGG-3'
HPRT 5'-TTGCTCGAGATGTCATGAAGGA-3' 5'-AGCAGGTCAGCAAAGAACTTATAG-3'
RPS13 5'-TGCTCCCACCTAATTGGAAA-3' 5'-CTTGTGCACACAACAGCATTT-3'
https://doi.org/10.1371/journal.pone.0190962.t001
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CD1ddeficiencyinhibitsaneurysmdevelopment
Statisticalanalysis
Alldataareexpressedasmean±SEM.Anunpairedtwo-tailedstudent’sT-testwasusedto
comparenormallydistributeddatabetweentwogroupsofanimals.Aone-wayANOVAwith
Dunnett’smultiplecomparisonpost-testwasperformedformultiplecomparisonsonthe
samesetofdata.TheMantel-Coxtestwasperformedtocomparethesurvivaldistribution
betweenLDLr-/-andLDLr-/-CD1d-/-mice.Probabilityvaluesof<0.05areconsideredsignifi-
cant.DatawereanalysedusingGraphPadPrismsoftware(GraphPadSoftware,LaJolla,CA,
USA).
Results
ReducedAAAformationinmicelackingCD1d-dependentNKTcells
TostudytheeffectofNKTcellsonAAAformation,theAngII-infusionmodelwasusedin
micewithorwithoutadeficiencyinCD1donanLDLr-/-background.TheincidenceofAAA
andaneurysmseverityinbothgroupsofmicewascompared.Duringtheexperiment,5outof
12LDLr-/-micediedduetoanaorticrupture(representativepictures,Fig1Aand1B),while
noneofthe11LDLr-/-CD1d-/-micedied(Fig1C,P<0.05).Fourweeksafterthestartofthe
AngIItreatment,thesurvivingmiceweresacrificedandtheaortaswereisolatedandanalyzed
(Fig2A).Thediameterofthehealthypartofboththethoracic(justproximaloftheaortic
arch)andabdominalaorta(justabovetheaorticbifurcation)didnotdifferbetweenboth
LDLr-/-andLDLr-/-CD1d-/-mice(S1Fig).Thedilatationoftheaortawasdeterminedbymea-
suringthemaximaldiameteroftheAAA-affectedpartandthemaximaldiameterofthe
“healthy”partjustproximaloftheaneurysm.Thisratiois49%lowerinLDLr-/-CD1d-/-mice
(1.35±0.14)whencomparedwithLDLr-/-mice(1.71±0.11,Fig2B,P<0.05),whilearatioof1
isthephysiologicalsituation.Classificationoftheaneurysmsusingthevisualmorphological
quantificationsystemforAngIIinducedaneurysmsinmice,[34]showedacleardifference
betweenbothgroups.Specifically,9outof11LDLr-/-CD1d-/-miceshowednodevelopmentof
Fig1.SurvivalcurveofangiotensinIItreatedLDLr-/-andLDLr-/-CD1d-/-mice.OsmoticpumpsfilledwithAngIIwereimplantedinLDLr-/-(■,n=12)and
LDLr-/-CD1d-/-(●,n=11)whichwerefedaWestern-typeofdietfor1week.Afterimplantation,LDLr-/-micestartedtodieduetoruptureoftheabdominal
aorta(AandB).Percentsurvivalpergroupisdepicted(C).StatisticalanalysiswasperformedusingtheMantel-Coxtest.(cid:3)P<0.05.
https://doi.org/10.1371/journal.pone.0190962.g001
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CD1ddeficiencyinhibitsaneurysmdevelopment
AA LLDDLLrr--//-- LLDDLLrr--//--CCDD11dd--//--
1100
BB CC 33..00 DD
11..7755
88 22..55
atio atio ce ce
orta rortar 11..5500 ** AAAAAAAA of miofmi 66 core core 22..00
urysm/Aurysm/A 11..2255 Number Number 44 AAA sAAAs 1111....0505 **
nene 22
AA 00..55
11..0000 00 00..00
LLDDLLrr--//-- LLDDLLrr--//-- 00 II IIII IIIIII IIVV LLDDLLrr--//-- LLDDLLrr--//--
CCDD11dd--//-- CCDD11dd--//--
Fig2.ReducedaneurysmformationinLDLr-/-CD1d-/-mice.OsmoticpumpsfilledwithAngIIwereimplantedinLDLr-/-(n=12)andLDLr-/-CD1d-/-
(n=11)micewhichwerefedaWesterntypeofdietfor1week.AneurysmformationinthesurvivingLDLr-/-mice(n=7)andLDLr-/-CD1d-/-mice(n=11)was
determinedbydissectingtheaorta(A)andmeasuringtheratiobetweenthemaximaldiameteroftheabdominalAAA-affectedpartandthemaximaldiameterof
the“healthy”thoracicpartoftheaorta(B).Scoringoftheseverityoftheaneurysmswasperformedusingthevisualdeterminationmethodasdescribedby
Daughertyetal.,2011(C,D).Allvaluesaremean±SEMandstatisticalanalysiswasperformedusingtheunpairedtwo-tailedstudent’sT-test.(cid:3)P<0.05.
https://doi.org/10.1371/journal.pone.0190962.g002
AAAwhileonly2outof12LDLr-/-micedidnotdevelopanAAA(Fig2C).Afterscoring
theaneurysms(Type0toIV)an84%reductioninaneurysmseveritywasobservedin
LDLr-/-CD1d-/-mice(2.25±0.63vs.0.36±0.24,Fig2D,P<0.05).Immunohistochemicalstain-
ingoftheAAAtissueshowedclearbreaksintheelasticlaminaandanincreasednumberof
MMP-9expressingcells(S2Fig).TotalcholesterollevelsdidnotdifferbetweenLDLr-/-and
LDLr-/-CD1d-/-micebefore(441±20vs.424±17mg/dl,respectively)andafterAngIIperfusion
(1416±104vs.1460±83mg/dl,respectively,S3Fig).LDLr-/-CD1d-/-micehadanotsignificant
lowerbodyweightatthebeginningoftheexperimentwhencomparedwiththeage-matched
LDLr-/-miceandduringtheexperimenttherewasnosignificantdifferenceinweightgain
betweenbothgroupsofmice(S3Fig).Additionally,a31%decreaseinatheroscleroticlesion
sizewasdetectedintheaorticrootofLDLr-/-CD1d-/-micecomparedwithLDLr-/-mice,
althoughthisdecreasewasnotsignificant(59085±6531μm2vs.85385±19987μm2,S3Fig,
P=0.157).
AngIIstimulatesNKTcellactivationinvivoandinvitro
ToinvestigatewhetherAngIIinfluencestypeINKTcellsinvivo,aFACSanalysiswasper-
formedonblood,liverandspleenofLDLr-/-miceafter2weeksofAngIIinfusion.FACSanal-
ysisshowedthatAngIIdidnotaffectthepercentagesofα-GalCer/CD1d-tetramer+NKTcells
inthecirculation,spleenandliver(Fig3A).However,AngIIinducedanactivationofthese
NKTcells.ThepercentageofsplenictypeINKTcellsexpressingCD25increasedwith21%
(76.0±2.1%vs.62.8±1.8%,Fig3B,P<0.01)whiletheexpressionleveloftheactivationmarker
CD25ontypeINKTcellsincreasedwith40%(MFIof1036±94vs.741±46,Fig3CandS3Fig,
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CD1ddeficiencyinhibitsaneurysmdevelopment
P<0.05).Additionally,circulatingIFN-γandIL-4levels,bothkeycytokinesproducedbyNKT
cells,weremeasuredinserumofsalineandAngIItreatedmicebutbothcytokinelevelswere
belowthedetectionlimitinbothgroupsofmice.ToconfirmtheenhancedNKTcellactivation
uponexposuretoAngII,DN32.D3NKThybridomacellswereco-culturedwithpre-treated
APCsobtainedfrombonemarrowofLDLr-/-orLDLr-/-CD1d-/-mice.Asignificantincrease
intheproductionofIL-2byNKTcellswasobservedafterexposuretoα-GalCerpulsedAPCs
(148.7±14.5pg/ml)whencomparedtounpulsedAPCs(53.0±2.2pg/ml,P<0.0001).Thiseffect
wassignificantlyamplifiedbytheadditionofAngII(275.4±5.0pg/ml,Fig3D,P<0.0001)
whileAngIIalonehadnoeffect(65.6±7.1pg/ml).Theseeffectswereabsentwhenα-GalCer
andAngIIpulsedLDLr-/-CD1d-/-APCswereco-culturedwithDN32.D3cells.
8 A B
**
75
n
6 o
s n ti
ell hiula
+ c witop 50
mer 4 +25 + pr
a De
tr Cm
e %a
T r 25
% 2 et
T
0 0
blood spleen liver control Ang II
C D
****
1,200 * 300

)
I
MF1,000
)
( ml
n
sio 800 g/ 200 ****
s p
pre 600 2 (
x -
e L
5 400 I 100
2
D
C 200
0 0
control Ang II control -GalCer Ang II -GalCer
+ Ang II
Fig3.IncreasedNKTcellactivityuponangiotensinIItreatment.OsmoticpumpsfilledwithPBS(n=5)orAngII(n=5)wereimplantedin
LDLr-/-micefedaWesterntypedietfor1week.Twoweeksafterpumpplacement,themiceweresacrificedandthepercentageofNKT(Tetramer+)
cellsinspleenandliver(A,whitebarsrepresentPBStreatedmice,blackbarsAngIItreatedmice)andtheactivationstatusofsplenicNKTcells(Band
C)weredeterminedbyFACSanalysis.Toconfirmtheseeffects,antigen-presentingcells(APCs)isolatedfrombonemarrowofLDLr-/-(whitebars)
andLDLr-/-CD1d-/-mice(blackbars)wereincubatedwithα-GalCer,AngIIoracombinationofboth.Fourhoursafterincubation,theAPCswereco-
culturedwithDN32.D3hybridomacells.After24hours,theIL-2concentrationinthesupernatantwasdetermined(D).Allvaluesaremean±SEMand
statisticalanalysiswasperformedusingtheunpairedtwo-tailedstudent’sT-test(A-C)orone-wayANOVA(D)(cid:3)P<0.05,(cid:3)(cid:3)P<0.01,(cid:3)(cid:3)(cid:3)(cid:3)P<0.0001.
https://doi.org/10.1371/journal.pone.0190962.g003
PLOSONE|https://doi.org/10.1371/journal.pone.0190962 January18,2018 8/17
CD1ddeficiencyinhibitsaneurysmdevelopment
4 A B C
**** *
5 1.5
*
****
on 3 4
essi **** *** 1.0
pr 3
x 2
e
e
v 2
ati 0.5
el 1
R 1
0 0 0.0
control -GalCer OCH control -GalCer OCH control -GalCer OCH
D E F
*** *** *
4
3
n
o 2
si
s 3
e
r
p 2
x
e
e 2
v 1
ati
el 1
R 1
0 0 0
control -GalCer OCH control -GalCer OCH control -GalCer OCH
Fig4.IncreasedexpressionofmatrixdegradingmoleculesbyvSMCsandmacrophagesaftertypeINKTcellactivation.SplenocytesofLDLr-/-micewereincubated
withorwithouttypeINKTcellspecificligandsα-GalCerandOCHfor48hours.Subsequently,supernatantofthesplenocyteswasaddedtovSMCs(A-C)or
macrophages(D-F)for72hoursafterwhichthemRNAexpressionofthematrixdegradingmoleculesCathepsinS(AandD),MMP-12(BandE),CathepsinK(C)and
CathepsinL(F)wasdetermined.Allvaluesaremean±SEMandstatisticalanalysiswasperformedusingone-wayANOVA.(cid:3)P<0.05,(cid:3)(cid:3)(cid:3)P<0.001,(cid:3)(cid:3)(cid:3)(cid:3)P<0.0001.
https://doi.org/10.1371/journal.pone.0190962.g004
IncreasedexpressionofproteasesafterNKTcellactivation
ToinvestigatehowtypeINKTcellscouldcontributetothedevelopmentofAAA,splenocytes
ofLDLr-/-micewereculturedfor48hinthepresenceofthetypeINKTcellspecificligandsα-
GalCerorOCH.α-GalCerisknowntoinduceamixedTh1/Th2(especiallyIFN-γ)cytokine
profile[36],whileOCHmorespecificallyinducesNKTcellstoproduceTh2cytokines(IL-4
andIL-10).[37,38]Subsequently,vSMCsandmacrophageswereexposedtosupernatantof
thesesplenocytesfor3days,afterwhichtheexpressionofseveralmatrixdegradingproteinases
wasdetermined.IncubationofvSMCswithconditionedmediumoftheα-GalCer-orOCH-
treatedsplenocytescausedasignificantincreaseinmRNAexpressionofCathepsinS,MMP-
12andCathepsinK(Fig4A–4C,respectively).Incubationofbonemarrow-derivedmacro-
phageswithconditionedmediumofOCH-treatedsplenocytesincreasedtheexpressionof
CathepsinS,MMP-12,andCathepsinL(Fig4D–4F,respectively).Thesesignificanteffectson
macrophageswerenotobservedaftertheadditionofconditionedmediumfromα-GalCer-
treatedsplenocytes,indicatingthatespeciallyTh2cytokines(producedafterOCH)inducethe
expressionofproteasesbymacrophages.
PLOSONE|https://doi.org/10.1371/journal.pone.0190962 January18,2018 9/17
CD1ddeficiencyinhibitsaneurysmdevelopment
IncreasedvSMCapoptosisafterNKTcellactivation
TofurtherstudytheeffectoftypeINKTcellactivationonthedevelopmentofAAA,spleno-
cyteswereculturedwithdifferentconcentrationsofα-GalCerorOCHfor48h.Subsequently,
vSMCswereexposedtoconditionedmediumfromthesesplenocytesandafter72h,theviabil-
ityofthevSMCswasassessedusinganMTTassay.Additionofconditionedmediumfrom
α-GalCer-andOCH-treatedsplenocytesdecreasedtheviabilityofvSMCsinadose-dependent
manner(Fig5A).Thisdecreaseinviabilitycouldbecounteracteddose-dependentlyby
addingincreasingconcentrationsofIFN-γ(Fig5B)andIL-4(Fig5C)blockingantibodies,
3 A 2.0 B
*
*

)
m 1.5
n
2 *
0
5
5
( **
n 1.0
tio ***** *** ***
p
r 1
o
bs 0.5
A
0 0.0
-GalCer OCH -GalCer + -IFN-
C D
1.5 ** 2.0 *
*

m) 1.5
n
1.0
0
5
5
( 1.0

n
o
ti
p 0.5
r
o 0.5
s
b
A
0.0 0.0
OCH + -IL-4 OCH + -IL-10
Fig5.CytokinedependentdecreaseinvSMCviabilityaftertypeINKTcellactivation.SplenocyteswereculturedinpresenceoftypeI
NKTcellspecificligandsα-GalCerorOCH(50,100and200ng/ml)for48hours.Subsequently,supernatantofthesplenocyteswasadded
tovSMCsfor72hoursandviabilityofthevSMCswasassessedbyanMTTassay(A).Inaddition,supernatantoftheα-GalCer(200ng/ml)
stimulatedsplenocyteswaspre-incubatedwithα-IFN-γantibodies(B)andsupernatantofOCH(200ng/ml)stimulatedsplenocyteswithα-
IL4(C)andα-IL10(D)antibodiespriortoadditionofthesupernatanttovSMCs.Allvaluesaremean±SEMandstatisticalanalysiswas
performedusingone-wayANOVA.(cid:3)P<0.05,(cid:3)(cid:3)P<0.01,(cid:3)(cid:3)(cid:3)P<0.001,(cid:3)(cid:3)(cid:3)(cid:3)P<0.0001.
https://doi.org/10.1371/journal.pone.0190962.g005
PLOSONE|https://doi.org/10.1371/journal.pone.0190962 January18,2018 10/17