The Placental Protein Syncytin-1 Impairs Antiviral Responses and Exaggerates Inflammatory ...

18 Pages·2015·0.69 MB·English

RESEARCHARTICLE
The Placental Protein Syncytin-1 Impairs
Antiviral Responses and Exaggerates
Inflammatory Responses to Influenza
JorgeM.Tolosa1,KristyS.Parsons2,PhilipM.Hansbro2,RogerSmith1,Peter
A.B.Wark2,3*
1 MothersandBabiesResearchCentreandHunterMedicalResearchInstitute,TheUniversityofNewcastle,
Newcastle,NSW,Australia,2 CentreforAsthmaandRespiratoryDiseaseandHunterMedicalResearch
Institute,TheUniversityofNewcastle,Newcastle,NSW,Australia,3 DepartmentofRespiratoryandSleep
Medicine,JohnHunterHospital,NewLambton,NSW,Australia
* [email protected]
Abstract
OPENACCESS
Background
Citation:TolosaJM,ParsonsKS,HansbroPM,
SmithR,WarkPAB(2015)ThePlacentalProtein Pregnancyincreasessusceptibilitytoinfluenza.Theplacentareleasesanimmunosuppres-
Syncytin-1ImpairsAntiviralResponsesand siveendogenousretroviralproteinsyncytin-1.Wehypothesisedthatexposureofperipheral
ExaggeratesInflammatoryResponsestoInfluenza.
monocytes(PBMCs)tosyncytin-1wouldimpairresponsestoH1N1pdm09influenza.
PLoSONE10(4):e0118629.doi:10.1371/journal.
pone.0118629
AcademicEditor:MircoSchmolke,Universityof
Geneva,SWITZERLAND MethodsandFindings
Received:September4,2014
Recombinantsyncytin-1wasproduced.PBMCsfromnon-pregnantwomen(n=10)were
Accepted:January21,2015 exposedtoH1N1pdm09inthepresenceandabsenceofsyncytin-1andcomparedtore-
Published:April1,2015 sponsesofPBMCsfrompregnantwomen(n=12).PBMCswerecharacterisedusingflow
cytometry,releaseofinterferon(IFN)-α,IFN-λ,IFN-γ,IL-10,IL-2,IL-6andIL-1βweremea-
Copyright:©2015Tolosaetal.Thisisanopen
accessarticledistributedunderthetermsofthe suredbycytometricbeadarrayorELISA.ExposureofPBMCstoH1N1pdm09resultedin
CreativeCommonsAttributionLicense,whichpermits thereleaseofIFN-α,(14,787pg/mL,95%CI7311-22,264pg/mL)IFN-λ(1486pg/mL,95%
unrestricteduse,distribution,andreproductioninany CI756-2216pg/mL)andIFN-γ(852pg/mL,95%CI193-1511pg/mL)after48hours.This
medium,providedtheoriginalauthorandsourceare
wassignificantlyimpairedinpregnantwomen(IFN-α;p<0.0001andIFN-λ;p<0.001).Fur-
credited.
thermore,inthepresenceofsyncytin-1,PBMCsdemonstratedmarkedreductionsinIFN-α
DataAvailabilityStatement:Allrelevantdatais
andIFN-λ,whileenhancedreleaseofIL-10aswellasIL-6andIL-1β.
includedinthemanuscriptorthesupplementarydata.
Funding:ThisworkwassupportedbytheNational
HealthandMedicalResearchCouncilofAustralia
APP1008924.Thefundershadnoroleinstudy
Conclusions
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. Ourdataindicatesthataplacentalderivedprotein,syncytin-1mayberesponsibleforthe
heightenedvulnerabilityofpregnantwomentoinfluenza.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0118629 April1,2015 1/18
PlacentalSyncytin-1ImpairsResponsestoInfluenza
Introduction
Theadvantageofgeneticdiversityhasensuredthesuccessofsexualreproductioninevolution,
butinplacentalmammalsitcomeswiththeinherentdilemmathatthematernalimmunesys-
temmusttoleratetheexistenceofthesemi-allogenicfoetus/placentathatexpressesbothself
(maternal)andnon-self(paternal)antigens[1].Itisalsoclearthattopreventmaternalrejection
ofthefoetusandtosustainasuccessfulpregnancy,potentmechanismsofimmuneregulation
mustbeactivated,suchassuppressionofTcellactivationandfunction[2,3]andthegeneration
ofsuppressorTregulatory(TReg)cellsthatfunctioninanantigenspecificmanner[4].Recent
advanceshavedemonstratedtheimportanceofco-ordinatingimmuneresponsesbetween
pathogendetection(non-selfantigens)bytheinnateimmunesystemandactivationorsup-
pressionofadaptiveimmuneresponses.Inthisregarddendriticcells(DCs)areanessential
linkincoordinatingtheseimmuneresponsesinhealthandinpregnancy[5,6].Theplacenta
alsoappearstoplayanimportantthoughpoorlyunderstoodroleinpromotingtheimmunoto-
lerantstate[2].
Thehumanplacentaisuniqueamongsttissuesinhavingthehighestexpressionofhuman
endogenousretroviruses.Theseareevolutionaryfossils,whichatsomeancienttimepointhave
infectedgermlinecells.Theirongoinghighlevelexpressionintheplacentaimpliesthatthey
haveimpartedanevolutionaryadvantageforthehost[7].Syncytin-1isanaturallyoccurring
placentalproteinthatisencodedbyoneoftheseretrovirusesandhasbeenshowntofuse
humancytotrophoblastcellsandpromotesyncytialisationthatiscrucialforplacentalforma-
tion[8].Recentlywealsodeterminedthathumansyncytin-1releasedfromtheplacentasup-
pressesmaternalimmuneresponsesdemonstratingit’spotentialtoplayaroleinmaternal/
foetaltolerance[9].
Maternalimmunetolerancemaycomeatthecostofincreasedsusceptibilitytoinfection,
withpregnantwomenatgreaterriskofinfectionfromintracellularbacteriaandviruses[10].
Theemergenceofanewpandemicstrainofswineinfluenzain2009(hereinreferredtoas
H1N1pdm09)recentlyrefocussedattentiononthisissue.Inthispandemic50%ofthe
womenwhodiedwerepregnant.Thisisastartlingstatisticthatemphasisesthatpregnant
womenareatincreasedriskofdevelopinginfectionwithinfluenzaandarealsomorelikely
todevelopseveredisease[11].Thisincreaseinmortalityandmorbidityisalsoobserveddur-
ingseasonalinfluenza;withnumerousstudiesreportingincreasedratesofinfluenza-
inducedrespiratoryhospitalisationsinpregnantcomparedtonon-pregnantwomen[12–
15].Whileseveralcomorbiditieshavebeenidentifiedthatpredisposedtosusceptibilityto
H1N1pdm09infection,pregnancyisthemostintriguingasitimpartsatemporarysuscepti-
bilityofthematernalimmunesystemtoinfection.Tofurtherunderstandthisphenomenon,
itisofparticularnotethatthosepregnantwomenwithsevereH1N1pdm09infectionpre-
sentedwithanacutesyndromeofsevererespiratorycompromiseandsystemicinflamma-
tion,associatedwithaberrantimmuneresponses.Thisischaracterisedbyintenseacute
inflammation(increasedlevelsofthecytokineIL-6),butalsoreducedantiviralT-helper
(TH)-1responses(reducedIFN-γ)andheightenedsuppressorTcellsignals(IL-10)[16].
Theimplicationsoftheseobservationsarethatthedevelopmentofamaternalstateofim-
munetolerancemayparticularlyimpairtheirimmuneresponsetonovelpathogenssuchas
H1N1pdm09.
Tofurtherexplainthesusceptibilityofpregnantwomentoinfluenzawehypothesisedthat
thehumanendogenousretroviralenvelopeproteinsyncytin-1suppressesmaternalcellmediat-
edimmuneresponsestoinfluenzaviruses,impairingantiviralDCs,promotingthedevelop-
mentofsuppressorTRegcellsandinhibitingthedevelopmentofrobustanti-viralTH-1
immuneresponses.
PLOSONE|DOI:10.1371/journal.pone.0118629 April1,2015 2/18
PlacentalSyncytin-1ImpairsResponsestoInfluenza
Methods
Subjects
Toassessthenaïveinnateresponsetoviruswerecruitedwomenwhohadneverbeenvaccinat-
edtoH1N1pdm09.Twelvepregnantwomen(non-vaccinated)(PNV)and10healthynon-
pregnantwomen(non-vaccinated)(NPNV),recruitedfromOctober2012-October2013,
participatedinthisstudy.PregnantwomenwererecruitedfromtheJohnHunterHospital
(JHH)antenatalclinicsandnon-pregnantwomenwererecruitedbyadvertisement.Inclusion
criteriaforallparticipantswerefemalesofchild-bearingage(18–40years).Pregnantwomen
wererecruitedfromanytrimester.Womenwereexcludediftheyhadanyconcomitantchronic
medicalillness,drugoralcoholdependence,currentlysmoking,iftheyhadasthmaorotherre-
spiratoryconditionsoriftheyhadcold/flusymptomswithinthefourweekspriortosample
collection.Writteninformedconsentwasobtainedfromallparticipantspriortosamplecollec-
tionandethicsapprovalwasobtainedfromtheHunterNewEnglandHumanResearchEthics
CommitteeandtheUniversityofNewcastleResearchEthicsCommittee.Pregnantandnon-
pregnantwomenattendedasinglestudyvisitatwhichbaselinecharacterisationwasassessed
andvenepuncturewasperformed.
VirusStocks
Astrainof2009pandemicswineflu(H1N1A/Auckland/3/2009)wasobtainedfromthe
WorldHealthOrganization(WHOMelbourne)in2010.Viralstockswerepropagatedin
Madin-DarbyCanineKidney(MDCK)cells(ATCC,Manassas,VA,USA),aspreviouslyde-
scribed[17–19].Stockviralconcentrationsweremeasuredusingplaqueassays;whichdeter-
mineslivevirionsbasedonplaqueformingunitsperml(pfu/ml)[17–19].Ultra-violet(UV)
inactivationofliveviruseswasachievedbyplacinglivevirusesdirectlyunderaUVlamp
(254nm)for3hrs.Successfulinactivationwasconfirmedbyplaqueassays.
GenerationofSyncytin-1Ectodomain
Syncytin-1recombinantectodomainproteinwasproducedandpurifiedfollowingpreviously
statedmethods[9,20].Syncytin-1recombinantectodomainwasclonedintothepET-28bvector
(Novagen)usingBspH1,Nco1andXho1restrictionenzymes.Forscaleupproduction,thepro-
teinwasexpressedinE.coliBL21(DE3)cells(Stratagene)in10LofLuriaBrothculturemedia
(Sigma)andexpressionwasinducedwith1mMIPTG(Sigma).Theproteinwasextractedfrom
thesolublecytoplasmicmaterialusingBugBusterMasterMix(MerckMillipore,Novagen).
Theclarifiedsupernatantwaspurifiedtwiceonnickel-coupledHitrapIMACHPcolumns
(threeHitrapIMACHP5mLcolumns(GE)connectedinseries).Afterconcentrationand
bufferexchangeusingAmiconUltracentrifugalfilter10K(Millipore),theproteinwaspre-
purifiedonapreparativeSephadexG75(Sigma-Aldrich)2.5x100cmcolumn(Econo-
Column,Bio-Rad)withaflowrateof0.7mL/min.Thebioactiveoligomericfractionwasthen
furtherpurifiedonananalyticalSephadexG751.5x120cmcolumn(Econo-Column,Bio-
Rad)withaflowrateof0.2mL/min.in25mMHEPES,150mMNaCl,pH7.4.Theproteinwas
concentratedusinganAmiconUltracentrifugalfilter10K(Millipore)andfiltersterilized
(0.2μm).Toensurenoendotoxinwaspresentinthepreparation,thesyncytin-1stockwastest-
edusingtheLimulusAmebocyteLysate(LAL)QCL-1000kit(LONZA)followingthe
manufacturer’sinstructions.
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
Syncytin-1StockPreparation
Theproteinstockwasstoredat-20°Cataconcentrationof100μMin25mMHEPES,150mM
NaCl,pH7.4.A20μMworkingsolutionwaspreparedbydiluting1volumeof100μMstock
with4volumesofbuffer(25mMHEPES,150mMNaCl,pH7.4).FiftyμLof20μMsyncytin
workingsolutionwasaddedtoformafinalconcentrationof1μM.
PeripheralBloodMononuclearCell(PBMC)IsolationandCulture
PBMCswereisolatedfromwholebloodbydensitycentrifugationusingFicoll-PaquePLUS
(GEHealthcareUppsala,Sweden)andresuspendedinRoswellParkMemorialInstitutemedia
(RPMI;Invitrogen,AustraliaPtyLimited)in10%foetalbovineserum(FBS;SAFCBiosciences,
Lenexa,Kansas,USA).PBMCswereculturedin24wellplatesatafinalconcentrationof
2.0x106cells/ml(NUNC,Denmark).Cellswerestimulatedwith1μMsyncytin-1for2hrsbe-
forevirusstimulation.AfterthisthecellswerestimulatedwithH1N1atMOI0.1(2.0x105pfu/
ml),orculturedinmediaalonefor48hrs,35°Cand5%CO2.Theseconcentrationswerebased
ontheabilityofthevirustoinducemaximalIFNproductionwithminimalcelldeathat48hrs
(S3A-CFig.).Afterculture,cellularsuspensionswerecentrifugedat550xgfor10min.Cellly-
sateswerestoredinBufferRLT(QIAGENPtyLtdDoncaster,VIC)andsupernatantswere
storedat-80°Cforsubsequentanalysis.
ELISA
IFN-λ1proteinwasmeasuredfromculturesupernatantsbyELISAfollowingthemanufactur-
er’sinstructions(R&DSystems,MN,USA)withanalysisusingonaFluorostarOptimamicro-
platereader(BMGLabtech,Ortenberg,Germany).
CytometricBeadArray(CBA)
IFNγ,IFNα,IL-10,IL-6,andIL-1βweremeasuredinculturesupernatantsusingCBA(BD
BioscienceCA,USA).ThesampleswererunonaBDFACSCantoIIFlowCytometerand
analysedwithBDFCAPArraySoftware(BDBioscienceCA,USA),accordingtothe
manufacturer’sinstructions.
AntibodiesforFlowCytometry
Thereweretwoflowcytometrypanelsused;aDCandalymphocytepanel.Thesepanelswere
furthersubdividedintocytokinemeasurestoidentifywhichcellswerereleasingcytokinesas
wellasmarkersofcellactivation.Thefluorochrome-conjugatedantibodiesusedintheDCcy-
tokinepanelwere:FixableViabilityStain450,CD45BV510,HLA-DRBV605,CD56
PerCP-Cy5.5,CD123PE,CD14Pe-Cy5,CD19PE-Cy5,CD11cAF700,CD3APC-H7,IL-6
FITC,IFN-γPE-Cy7,IFN-αAF647andIL-10PE-CF594.AntibodiesusedintheDCactivation
markerpanelwere:FixableViabilityStain450,CD45-BV510,CD56PerCP-Cy5.5,CD123PE,
CD14Pe-Cy5,CD19PE-Cy5,CD11cAF700,CD3APC-H7,HLA-DRAPC,CD80PE-Cy7,
CD86PE-CF594andHLA-A,B,CFITC.Antibodiesusedinthelymphocytecytokinepanel
were:FixableViabilityStain450,CD45BV510,CD56BV605,CD4PerCP-Cy5.5,FoxP3PE,
CD25APC,CD8AF700,CD3APC-H7,IL-6FITC,IFN-γPE-Cy7andIL-10PE-CF594.Anti-
bodiesusedinthelymphocyteactivationmarkerpanelwere:FixableViabilityStain450,CD45
BV510,CD56BV605,CD4PerCP-Cy5.5,FoxP3PE,CD25APC,CD8AF700,CD3APC-H7,
CD103PE-Cy7,CTLA-4PE-CF594andGITRAF488.AllwerefromBDBioscience,CA,USA
exceptCD14,CD19,GITRandCD103(ebioscience,USA).Appropriatecontrolswereusedin
allexperiments.
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
FlowCytometryGeneralGatingforallPBMCExperiments
Tofurtherelucidatethecelltypesthatwereproducingthecytokinesofinterest,additionalin-
tracellularcytokinestainingwasperformedoncellsfromtwo(n=2)nonpregnant,notvacci-
natedwomen.AllsampleswererunonaBDFACSARIAFlowCytometerandFlowJo
software(TreeStarInc,USA).Gatingstrategiesforexcludingdoublets,cellclumps,deadcells,
intracellularandsurfacemarkerstainingwerebasedoninstructionsfromBDBioscience.Dou-
bletsandcellclumpswereexcludedatthesametimeasgatingthePBMCpopulationbycreat-
ingscatterplotsforFSC-HvsFSC-A,FSC-AvsFSC-W,SSC-AvsFSC-A,andCD45+/FVS450
negative.Thecellpopulationsthatfellwithinthesefourplotswereconsideredthesinglecellvi-
ablePBMCpopulation.Atotalof5x105–1x106eventswerethencollectedwithinthese
intersectedgates.
IsolatedPBMCswerefirstculturedfor44hrsbeforeaddinga1:10dilutionoftheprotein
blocker,brefeldinA(BDBioscience,CA,USA)andthenculturedforafurther4hours.Aftera
totalof48hours,PBMCswerecentrifugedat300xgfor10minutesandresuspendedinPBS
bufferatafinalconcentrationof1x106/mlPBMCs.FixableViabilityStain450protocolwas
performedaccordingtothemanufacturer’sinstructions.PBMCswerethenstainedforeither
theDCpanelorthelymphocytepanelforcytokinesoractivationmarkers.
ExpressionofCytokinesinDCsandLymphocytes
TheDCpanelemployedfollowedtheBDBiosciencecytofix/cytopermmanufacturer’sinstruc-
tions.PBMCswerestainedwithCD123forpDCsandCD11cformDC.PBMCswerealso
stainedwithCD14andCD19toexcludemonocytesandBcells,respectively.Todeterminethe
DCpopulationswithinthelivesinglecell(FVS450-)PBMCpopulations(CD45+),Bcellsand
monocyteswerefirstexcluded.ThiswasdonebyconstructingascatterplotforCD14/CD19vs
CD3.LymphocyteswerethengatedastheCD3-population.ToexcludebasophilsandNK
cells,anHLA-DRvsCD11cplotwascreatedandDCswereconsideredHLA-DRpositive.
FromtheHLA-DRpositivecells,mDCcellswereconsideredtheCD11c+populationwhilst
pDCswereconsideredtheCD123+fromaCD123vsCD11cscatterplot.IL-10,IL-6,IFN-γ
andIFN-αintracellularcytokineexpressionofeachDCpopulationwasdeterminedbyplotting
eachandmeasuringthepercentageofpositivecellsoftheparentpopulation.
ThelymphocytepanelemployedfollowedtheBDBioscienceTranscriptionfactorbufferset
manufacturer’sinstructions.PBMCswerestainedwithCD4forThelpercells,CD8forcyto-
toxicTcells,CD56forNKcellsandFoxP3/CD25forTregulatorycells.TodeterminetheT
cellpopulationswithinthelivesinglecell(FVS450-)PBMCpopulations(CD45+)ascatterplot
forCD56vsCD3wascreatedandTlymphocyteswerethengatedastheCD3+population.T
helpercellswereconsideredtheCD4+populationwhilstcytotoxicTcellswereconsideredthe
CD8+fromaCD8vsCD4scatterplot.TheCD4+cellswerethenplacedonaFoxP3vsCD25
scatterplotandTregulatorycellswereconsideredFoxP3/CD25+.NKcellsweregatedas
CD56+.IL-10,IL-6andIFN-γcytokineexpressionofeachlymphocytepopulationweredeter-
minedbyplottingeachandmeasuringthepercentageofpositivecellsoftheparentpopulation.
ExpressionofActivationMarkersinDCsandLymphocytes
FortheDCpanel,tomeasureactivationmarkerexpressiononpDCsandmDCs,PBMCswere
stainedwithantibodiesforCD86,HLA-DR,CD80andHLA-A,B,C.Followingincubationfor
30minutesat4°C,PBMCswerewashedtwicewithflowstainbuffer,centrifugedandresus-
pendedatafinalvolumeof350μlinflowstainbuffer.Thesamegatingstrategywasfollowedas
above.However,inthiscase,todeterminethepercentageofpDCs/mDCspositiveforCD86,
HLA-DR,CD80andHLA-A,B,C,scatterplotswereconstructedforeachmarkeragainstthe
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
CD123+(pDC)orCD11c+(mDC)population,gatedontheparentpopulation.Meanfluores-
cenceintensitywasalsomeasuredforeachactivationmarker.
Forthelymphocytepanel,tomeasureactivationmarkerexpressiononTlymphocytes,
PBMCswerestainedwithantibodiesforGITR,CTLA-4andCD103.Thelymphocytepanel
usedfollowedtheBDBioscienceTranscriptionfactorbuffersetmanufacturer’sinstructions.
Thesamegatingstrategywasfollowedasabove.However,inthiscase,todeterminetheper-
centageofCD4/CD8/Treg/NKcellspositiveforGITR,CTLA-4,andCD103,scatterplotswere
constructedforeachmarkeragainsttheCD4+(CD4),CD8+(CD8),CD4+/FoxP3+/CD25+
(Treg),CD3-/CD56+(NKcells)population,gatedontheparentpopulation.Meanfluores-
cenceintensitywasalsomeasuredforeachactivationmarker.
StatisticalAnalyses
Dataareexpressedasmean±standarderrorofmean(SEM)fortwelvesubjects(n=12)with
tworeplicatesforeachsubjectforpregnantwomenandtensubjects(n=10)withtworepli-
catesforeachsubjectfornon-pregnantwomen(exceptfortheflowcytometryanalysiswhich
wascarriedoutonn=2nonpregnantwomen).Statisticalsignificancewasassessedbyone-
wayanalysisofvariance(ANOVA),post-hocanalysiswasperformedusingtheHolm-Sidak’s
correctionusingGraphPadPrismSoftware.APvalueof<.05wasconsideredsignificant.For
NPNV,H1N1wascomparedtoH1N1+Syncytin,mediaaloneandtoH1N1forPNV.Alsofor
NPNV,syncytinalonewascomparedtomedia.AcomparisonwasmadebetweenNPNV
H1N1+SyncytinandPNVH1N1.AlsoforPNV,H1N1wascomparedtoH1N1+Syncytinand
mediaalone.
Results
PBMCsfromnon-pregnantwomenhavearobustIFNresponseto
H1N1pdm09infection,whichisreducedinpregnantwomen
PBMCsfrompregnantandnon-pregnantwomenwereculturedwithastrainofinfluenzaiso-
latedduringthe2009swineflupandemic(H1N1pdm09)orculturedinmediaonly.PBMCs
fromnon-pregnantwomendemonstratedarobustIFN-α(14,787pg/mL,95%CI7311–22,264
pg/mL)andIFN-λ(1486pg/mL,95%CI756–2216pg/mL)responsetoH1N1pdm09
(Fig.1A-C,)comparedtomediaaloneafter48hoursofexposure.Conversely,PBMCsfrom
pregnantwomen,followingH1N1pdm09stimulation,producedsignificantlylessIFN-α
(Fig.1A)(3661pg/mL,95%CI2382–4939pg/mL)andIFN-λ(Fig.1B)(558pg/mL,95%CI
407–710pg/mL)comparedtoPBMCsfromnon-pregnantwomen(p<0.0001andp<0.001,
respectively).However,thiswasnotthecasewithIFN-γ(691pg/mL,95%CI340–1043
pg/mL)(Fig.1C).
TreatmentofPBMCsfromnon-pregnantwomentreatedwithsyncytin-1
priortoinfectionwithH1N1pdm09reducedIFNresponses,tolevels
similartothoseinPBMCsfrompregnantwomen
PBMCsfromnon-pregnantwomenwerepre-treatedwith1μMsyncytin-1andthencultured
withH1N1pdm09ormediaonly.Thisdoseofsyncytin-1wasselectedbyperformingadose
responseonarangeofbiologicallyrelevantdoses(1nMto5μM).1μMhadtheabilitytore-
duceIFN-αandIFN-λandinduceIL-10(S1A-CFig.).Cytotoxicityofsyncytin-1hasalso
previouslybeenmeasuredandconfirmedtohavenoeffectoncellviabilityorcytokinerelease
[9].Furthermore,thesyncytin-1preparationtechniquedidnotresultinanyendotoxininthe
finalprotein.Usingsizeexclusionchromatographywewereabletoseparatesyncytin-1into
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
Fig1.PBMCsfromnon-pregnantwomenhavearobustIFNresponsetoH1N1pdm09.TheresponseofPBMCsfrompregnantandnon-pregnant
womentoH1N1pdm09wasassessedafter48hoursofexposure.PBMCsfromnon-pregnantwomenwerepre-exposedtosyncytin-1(1μM).PBMCswere
infectedwithH1N1MOI0.1for48hrs.LevelsofIFN-α(A),IFN-λ(B)andIFN-γ(C)incellculturesupernatantsweremeasuredbycytometricbeadarray
(IFN-αandIFN-γ)orELISA(IFN-λ).NPNV=Non-pregnantandnon-vaccinatedn=10,PNV=Pregnantandnon-vaccinatedn=12.ANOVA(Holm-Sidak)
****P<0.0001,***P<0.001,**P<0.01,*P<0.05
doi:10.1371/journal.pone.0118629.g001
twomajorpeaks,onecorrespondingtoalowleveloligomerizationandanotherasamulti-
mericcomplex.Themultimericpeakcorrespondstothebioactiveconformationoftheprotein
andhasbeenusedinallexperiments.Thelowleveloligomerizationpeakwasalsousedbut
foundnottobebioactive.Theadditionofsyncytin-1significantlyreducedIFN-α(6083pg/
mL,95%CI2848–9319pg/mL)andIFN-λresponses(539pg/mL,95%CI255–823pg/mL)
(p<0.001Fig.1A-B).Infacttreatmentwithsyncytin-1inducedaresponsefromthesecells
thatmirroredwhatwasseeninPBMCsfrompregnantwomenwithinfectionalone(H1N1
PNV)(Fig.1A-C).
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
Fig2.PBMCsfromnon-pregnantwomentreatedwithsyncytin-1priortoinfectionhaveaheightened
IL-10response.PBMCswereinfectedwithH1N1MOI0.1for48hrswithandwithoutsyncytin-1(1μM).
LevelsofIL-10weremeasuredinculturesupernatantsbycytometricbeadarray.Non-pregnantPBMC
demonstratedaheightenedIL-10responsewhenexposedtoH1N1pdm09infectionandsyncytin-1.
NPNV=Non-pregnantandnon-vaccinatedn=10,PNV=Pregnantandnon-vaccinatedn=12.ANOVA
(Holm-Sidak)****P<0.0001,***P<0.001,**P<0.01.
doi:10.1371/journal.pone.0118629.g002
PBMCsfromnon-pregnantwomentreatedwithsyncytin-1priorto
infectionhaveaheightenedIL-10response
H1N1pdm09infectionalonedidnotinduceanIL-10responsefromeitherpregnantornon-
pregnantPBMCswhencomparedtomedia(Fig.2)(75pg/mL,95CI34–114pg/mL).Howev-
er,therewasasubstantialincreaseinIL-10innon-pregnantPBMCswithpre-treatmentwith
syncytin-1,p<0.0001(372pg/mL,95%CI230–514pg/mL).
TheplasmacytoidDCsarethesourceofthereleasedIFN-α/λandIL-10
inresponsetoH1N1pdm09
Wethenusedflowcytometryandsurfacestainingtoidentifythecellsresponsiblefortheob-
servedresponses.TheDClineagewasidentifiedasCD3-/HLA-DR+,andPBMCswerefurther
characterisedasplasmacytoidDCs(pDCs)(CD123+)ormyeloidDCs(mDCs)(Fig.3A).
IntracellularcytokinestainingofthepDCpopulationshowedthatpDCswereresponsible
forthereleaseofIFN-α.ThepDCshadincreasesinthepercentageofIFN-αpositivecellsin
thepresenceofH1N1pdm09comparedtomedia(Fig.3BPanel1and3).Whiletheadditionof
syncytin-1andH1N1pdm09,reducedpDCIFN-αexpression(Fig.3BPanel2).Furthermore,
intracellularcytokinestainingoftheCD4lymphocytesshowedthattheCD4+populationwas
responsibleforthereleaseofIFN-γ,withthepercentageofpositivecellsreducedinthepres-
enceofsyncytin-1(S2AFig.),though,attheearlytime-pointofassessment,therelease
wassmall.
IntracellularcytokinestainingofthepDCpopulationalsoshowedthatthesecellswerere-
sponsiblefortheincreasedreleaseofIL-10inthepresenceofH1N1pdm09whencomparedto
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PlacentalSyncytin-1ImpairsResponsestoInfluenza
Fig3.FlowcytometricanalysisofPBMCsshowingthatpDCsareresponsibleforthereleaseofIFN-α
andIL-10.PBMCswereinfectedwithH1N1pdm09MOI0.1for48hrswithandwithoutsyncytin-1.Thecells
werethensubjectedtosurfaceandintracellularcytokinestainingandflowcytometrytodeterminecell
populationsandcytokinerelease.ThepDCpopulationwasdeterminedusingCD3-/CD45+cellswhichwere
alsoHLA-DR+andCD123+(A).Thispopulationwasanalysedforcytokinereleaseusingintracellular
PLOSONE|DOI:10.1371/journal.pone.0118629 April1,2015 9/18
PlacentalSyncytin-1ImpairsResponsestoInfluenza
stainingandthepercentageofcellspositiveforIFN-α(B)andIL-10(C)wascalculated.Surfacestainingof
pDCsfortheactivationmarkersHLA-DRandCD86(D)wasalsomeasured.NPNV=Non-pregnantandnon-
vaccinatedn=2,ANOVA(Holm-Sidak)*P<0.05.
doi:10.1371/journal.pone.0118629.g003
media(Fig.3CPanel1and3).Whilethecombinationofsyncytin-1andH1N1pdm09,further
increasedpDCexpressionofIL-10(Fig.3CPanel2).AlthoughthemajorityoftheIL-10was
frompDCs,therewasalsoasmallamountexpressedbyCD4TRegcells(S2BFig.).
ToconfirmthatthesepDCswereactive,surfaceexpressionofCD86andHLA-DRwere
measured.InfectionwithH1N1pdm09increasedtheexpressionofHLA-DRandCD86
(Fig.3D).Thisexpressionwasfurtheramplifiedwithsyncytin-1pre-treatment.
PBMCsfromnon-pregnantwomentreatedwithsyncytin-1priorto
infectionhaveaheightenedreleaseofIL-6andIL-1β
H1N1pdm09infectionalonedidnotinducereleaseofIL-1βorIL-6PBMCsfromeitherpreg-
nantornon-pregnantwomenwhencomparedtomedia.However,therewasasubstantialin-
creaseinIL-1βfromnon-pregnantPBMCspretreatedwithsyncytin-1(p<0.0001Fig.4A)
(124pg/mL,95%CI76–172pg/mL).Further,therewasalsoanexaggeratedIL-6releasefrom
non-pregnantPBMCspretreatedwithsyncytin-1(p<0.0001Fig.4B)(3985pg/mL,95%CI
2659–5310pg/mL).Bothoftheseresponsesweresignificantlyelevatedcomparedtothoseseen
inpregnantPBMCswithinfectionalone(p<0.0001)(4.3pg/mL,95%CI1–7pg/mL;140pg/
mL,95%CI63–217pg/mLrespectively).
Discussion
Impairedimmuneresponsestointracellularpathogens,suchasinfluenzarepresentanongoing
andimportantriskforotherwisehealthypregnantwomen.Forthefirsttimewehavedemon-
stratedthataplacentalderivedprotein,syncytin-1,impairsearlyinnateimmuneresponsesto
influenza.ExposureofPBMCsfromNPNVwomentoH1N1pdm09,resultedinarobustanti-
viralresponse,characterisedbythereleaseofIFN-αandIFN-λ.Howeverthisresponseiscon-
siderablyreducedinPBMCsfromPNVwomen.FurthermoreexposureofPBMCsfrom
NPNVwomentosyncytin-1remarkablyalterstheirnormalantiviralresponse;impairingthe
releaseofIFN-α/λ,andpromotingthereleaseofIL-10aswellasthepro-inflammatorycyto-
kinesIL-1βandIL-6.Wewentontodemonstratethatthisearlyantiviralresponseispredomi-
nantlymediatedbypDCs,confirmingourpreviousdatathatthereareimpairedearlyinnate
immuneresponsestoinfluenzainpregnancy[21],butnowdemonstratingthatthiscanbein-
ducedbyexposureofpDCstosyncytin-1.
Sequencesofretroviraloriginmakeupasubstantialpartofthemammaliangenome.Analy-
sisofthehumangenomerevealedthataround8%ofhumanDNAisderivedfromretrovirus-
likeelements[22].HumanEndogenousRetroviruses(HERVs)arederivedfromexogenous
retroviruses,which,atsomeancienttime-point,haveinfectedgermlinecells[7,23]and,there-
fore,arepassedontoallhumancells.AHERVenvelopegenebelongingtotheHERV-Wfami-
lyandencodingaproteinexpressedinthesyncytiotrophoblastwasfirstdescribedin1999[24]
andwasdenotedsyncytin-1[8].Syncytin-1wasshowntofusehumancytotrophoblastcellsand
maybeakeyfactorinregulatingsyncytializationduringplacentalformation[8,25].Inaddition
tothefusogenicpropertyofsyncytin-1anditsroleinsyncytialization,thepresenceofanim-
munosuppressiveregion(ISD)intheaminoacidsequencewasalsoidentified[8,24].Syncytin-
1isnormallyproducedandsecretedbytheplacentainexosomes.Duringpregnancy,maternal
plasmaexosomalproteinconcentrationsaverage0.61±0.14;0.94±0.41and1.4±0.11mg/mlof
PLOSONE|DOI:10.1371/journal.pone.0118629 April1,2015 10/18