STING agonists enable antiviral cross-talk between human cells and confer protection against

22 Pages·2017·11.8 MB·English

RESEARCHARTICLE
STING agonists enable antiviral cross-talk
between human cells and confer protection
against genital herpes in mice
MortenK.Skouboe1,AliceKnudsen1,LineS.Reinert1,CedricBoularan2,ThierryLioux2,
EricPerouzel2,MartinK.Thomsen3☯*,SørenR.Paludan1☯*
1 DepartmentofBiomedicine,AarhusUniversity,Denmark,2 InvivoGen,ToulouseFrance,3 Departmentof
ClinicalMedicine,AarhusUniversity,Denmark
a1111111111
☯Theseauthorscontributedequallytothiswork.
a1111111111
*[email protected](SRP);[email protected](MKT)
a1111111111
a1111111111
a1111111111
Abstract
Inrecentyears,therehasbeenanincreasinginterestinimmunomodulatorytherapyasa
meanstotreatvariousconditions,includinginfectiousdiseases.Forinstance,Toll-likerecep-
OPENACCESS tor(TLR)agonistshavebeenevaluatedfortreatmentofgenitalherpes.However,although
theTLR7agonistimiquimodwasshowntohaveantiviralactivityinindividualpatients,nosig-
Citation:SkouboeMK,KnudsenA,ReinertLS,
BoularanC,LiouxT,PerouzelE,etal.(2018) nificanteffectswereobservedinclinicaltrials,andthecompoundalsoexhibitedsignificant
STINGagonistsenableantiviralcross-talkbetween sideeffects,includinglocalinflammation.CytosolicDNAisdetectedbytheenzymecyclic
humancellsandconferprotectionagainstgenital
GMP-AMP(2’3’-cGAMP)synthase(cGAS)tostimulateantiviralpathways,mainlythrough
herpesinmice.PLoSPathog14(4):e1006976.
https://doi.org/10.1371/journal.ppat.1006976 inductionoftypeIinterferon(IFN)s.cGASisactivateduponDNAbindingtoproducethecyclic
dinucleotide(CDN)2’3’-cGAMP,whichinturnbindsandactivatestheadaptorproteinStimu-
Editor:EdwardMocarski,EmoryVaccineCenter,
UNITEDSTATES latorofinterferongenes(STING),thustriggeringtypeIIFNexpression.IncontrasttoTLRs,
STINGisexpressedbroadly,includinginepithelialcells.Herewereportthatnaturalandnon-
Received:August30,2017
naturalSTINGagonistsstronglyinducetypeIIFNsinhumancellsandinmiceinvivo,without
Accepted:March17,2018
stimulatingsignificantinflammatorygeneexpression.Systemictreatmentwith2’3’-cGAMP
Published:April2,2018 reducedgenitalherpessimplexvirus(HSV)2replicationandimprovedtheclinicaloutcomeof
Copyright:©2018Skouboeetal.Thisisanopen infection.Moreimportantly,localapplicationofCDNsatthegenitalepithelialsurfacegave
accessarticledistributedunderthetermsofthe risetolocalIFNactivity,butonlylimitedsystemicresponses,andthistreatmentconferred
CreativeCommonsAttributionLicense,which
totalprotectionagainstdiseaseinbothimmunocompetentandimmunocompromisedmice.In
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal directcomparisonbetweenCDNsandTLRagonists,onlyCDNsacteddirectlyonepithelial
authorandsourcearecredited. cells,henceallowingamorerapidandIFN-focusedimmuneresponseinthevaginalepithe-
DataAvailabilityStatement:Allrelevantdataare lium.Thus,specificactivationoftheSTINGpathwayinthevaginaevokesinductionoftheIFN
withinthepaperanditsSupportingInformation systembutlimitedinflammatoryresponsestoallowcontrolofHSV2infectionsinvivo.
files.
Funding:ThisworkwasfundedbyTheDanish
MedicalResearchCouncil(grantsno:12-124330
andDFF–6110-00068),TheLundbeckFoundation
Authorsummary
(grantnoR198-2015-171)andanunrestricted
researchgrantfromInvivoGen(alltoSRP).MKT Herpessimplexvirus(HSV)-2istheleadingcauseofgenitalulcers,andHSV-2infection
wasfundedbyafellowshipsponsoredby
hasalsobeenreportedtoamplifyHIV-transmission.Sofar,allattemptsatmakinganeffec-
InvivoGen,Danishcancersociety(R146-A9394-
tiveanti-HSV2vaccinehavefailed.Inrecentyears,therehasbeenanincreasinginterestin
16-S2)andAUFFNOVA(E-2o15-FLS-9-8).AKis
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 1/22
STINGagonistsconferprotectionagainstgenitalherpes
recipientofaPhDscholarshipfromtheGraduate
School,HEALTH,AU.Thefundershadnorolein immunomodulatorytherapyasameanstotreatinfections.AlthoughtheTLR7agonistimi-
studydesign,datacollectionandanalysis,decision quimodhasbeenshowntohaveantiviralactivityinindividualpatients,nosignificant
topublish,orpreparationofthemanuscript.
effectswereobservedinclinicaltrials,andthecompoundalsoexhibitedsignificantside
Competinginterests:Ihavereadthejournal’s effectsincludinglocalinflammation.TypeIinterferon(IFN)sarekeyplayersinantiviral
policyandtheauthorsofthismanuscripthavethe defense,anditisnowknownthattheDNAsensorcyclicGMP-AMPsynthaseproduces
followingcompetinginterests:Invivogenhaspartly
thecyclicdi-nucleotide(CDN)2’3’-cyclicGMP-AMP(cGAMP),whichactivatestheadap-
fundedthisworkandhasafinancialinterestin
torproteinSTINGtoinduceIFNexpression.Inthisworkweshowthatnaturalandnon-
Stingagonists.CB,TL,andEPareemployeesof
InvivoGen.Thisdoesnotalterouradherencetoall naturalCDNsactivatestrongtypeIIFNresponsesinvivowithoutstimulatingsignificant
PLOSPathogenspoliciesonsharingdataand expressionofgenesdrivenbythetranscriptionfactorNF-κB,whichinducesinflammation.
materials. ApplicationofCDNsatepithelialsurfacesgaverisetolocalIFNactivity,butonlylimited
systemicresponses.Importantly,alltestedtreatmentregimens,stronglyreducedreplica-
tionofHSV-2inamodelforgenitalherpes,andsignificantlyreduceddevelopmentofdis-
ease.Finally,whencomparingtoTLRagonists,CDNsshowedthebestprofilewithstrong
IFNresponsespecificallyintheepithelialcellsandlimitedinductionofinflammation.
Introduction
Virusinfectionsmaycauseacuteandchronicaldiseases,andthereisthereforeaneedfor
developmentofefficienttreatments.Significantimprovementshavebeenmadeinthedevelop-
mentoftherapeuticsthattargetspecificviralmolecules,suchastheHIVreversetranscriptase
andthehepatitisCvirusNS5Aprotein[1,2].Despitethis,satisfactorytreatmentsarenotavail-
ableformanyvirusinfections,andthereisalsoaneedfortreatmentsactinginabroaderman-
ner,andwhicharelesssensitivetoviraldevelopmentofresistance.Herpessimplexvirus
(HSV)-2istheleadingcauseofgenitalulcers[3,4]withanestimated500millioninfectedpeo-
pleglobally[5].Althoughintensivelypursued,allattemptsatmakinganeffectiveanti-HSV2
vaccinehavefailed[4].Thecurrentstandardtreatmentisacyclovirorderivatives,whichtarget
theviralthymidinekinase[6],andalthoughgenerallyefficientiftreatmentisinitiatedearly,
therearereportsofdevelopmentofresistanceinimmunosuppressedpatientsreceivinglong-
termtreatment[6].Theneedfornewandbetteranti-HSV2treatmentsisunderpinnedbysev-
eralfactors,includingtheabilityofthisvirustocauseneonatalherpes[3],theroleofHSV2in
amplifyingHIV-transmission,whichhasbeenreportedtoaccountforuptohalfofallnew
transmissionsinareasofhighHSV2seroprevalence[7,8],andtherecentlyreportedassocia-
tiontoincreasedratesofautism-spectrumdisorders[9].
Inadditiontodirectlytargetingthevirus,antiviraltreatmentscanstimulatehostimmune
responses.Previouslytestedexperimentalimmunemodulatorytherapiesforvirusinfections
havemainlyfocusedonagonistsforToll-likereceptors(TLRs).TheTLR7-agonistimiquimod
andthemixedTLR7/8-agonistresiquimodinduceinterferon(IFN)α,andimiquimodisthe
firstapprovedtopicallyactiveTLR7agonistusedtotreathumanpapillomavirus(HPV),but
hasfailedtoshowsignificantefficacyagainstHSV2infection[10–12],althoughcaseshave
beenreportedwithbenefitofimiquimod5%creamfortreatmentofherpeslabialisandgenital
herpes[13,14].Furthermore,pretreatmentofmicewitholigodeoxynucleotideTLR9agonists
hasbeenshowntolowertheviralloadinthebraininanHSV-1encephalitismodel[15].Com-
montotheaforementionedTLRsistheirroleininnaterecognitionofforeignnucleicacidsin
theendosomalcompartment[16].However,TLRsaremainlyexpressedinleukocytes,andto
amuchlesserextentinepithelialcells[17].Anotherproteininvolvedinnucleotidesensingis
thecyclicGMP-AMPsynthase(cGAS),whichislocalizedinthecytoplasm,andhenceisasen-
sorofmislocalizedendogenousorexogenousDNA[18].Weandothershaveshownthat
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 2/22
STINGagonistsconferprotectionagainstgenitalherpes
cGASplaysanintrinsicroleinmountingprotectiveimmuneresponsesagainstDNAviruses,
includingHSV-1[19–21].WhencGASsensesdsDNAinthecytosol,itproducesthesecond
messenger2’3’-cyclicGMP-AMP(2’3’-cGAMP)whichactivatestheadaptorproteinStimula-
torofIFN-genes(STING)ontheER[18,22,23].STINGdimerizesandtrafficstotheER-Golgi
intermediatecompartment(ERGIC)whereitrecruitstheTANK-bindingkinase1(TBK1),
whichinturnphosphorylatesIFN-regulatoryfactor3(IRF3)thattranslocatesasadimertothe
nucleuswhereitinitiatestranscriptionoftypeIIFNgenes[24].TypeIIFNsaresecretedcyto-
kines,whichworkinauto-andparacrinemannersviatheIFNαreceptor(IFNAR)toupregu-
lateIFN-stimulatedgenes(ISGs)thattargetspecificstepsinthevirallifecycletoinhibit
replication[24].
Inadditiontotheactionof2’3’-cGAMPinsidetheDNA-stimulatedcell,thisCDNisalso
abletoexerteffectsinsideothercellsthroughatleasttwodistinctmechanisms,eitherjuxtacri-
nelybydiffusingthroughgapjunctions[25]orendocrinelybybeingpackagedintonewlyform-
ingvirions[26,27].Serumcontainstheectonucleotidepyrophosphatase/phosphodiesterase
(ENPP)1,whichmetabolizes2’3’-cGAMP[28],thushinderingextensivecirculationandendo-
crinestimulationbycell-free2’3’-cGAMP.However,free2’3’-cGAMP(andothercyclicdinucle-
otides(CDNs))hasthecapacitytopassthecellmembraneandactivateSTING[29].Therefore,
CDNscouldpotentiallystimulateimmuneresponseslocallyandoverlongerdistances.
TheuseofSTINGagonistsfortreatmentofdiseasehasbeentestedinaseriesofmodels.The
smallmoleculeDMXAA(vadimezan)wasshowntohaveanti-tumoreffectsinamousemodel
[30]beforeitsmechanismofactionasaSTINGligandwasdiscovered[31],anditwastestedin
aclinicalphaseIIIefficacytrialoftreatmentofadvancednon-small-celllungcancer[32].Itwas
subsequentlyshownthatDMXAAdoesnotstimulatehumanSTING,asopposedtomurine
STING,duetoasingleaminoaciddifferencebetweenhumanandmurineSTINGataresidue
mediatinginteractionbetweenmurineSTINGandDMXAA[31,33].Anotherinterestingdif-
ferencebetweenSTINGinhumansandmiceisthathumanSTINGisactivatedmorepotently
by2’3’-linkedCDNs(producedinmetazoancells)than3’3’-linkedCDNs(producedinnon-
metazoancells),whilemurineSTINGisactivatedtocomparabledegreesbythetwotypesof
CDNs[29].Inthepastfewyears,STINGligandshavebeeninvestigatedforanti-tumoractivity
inavarietyofmousemodels[34,35],anti-inflammatoryeffectsinanexperimentalautoim-
muneencephalitismousemodel[36],aswellasforpotentialasadjuvantsinvaccines[37–41].
However,whetherSTINGagonistscouldhaveantiviraltherapeuticeffectshasremainedunder-
explored.Inthisstudy,weinvestigatetheantiviralactionofnaturalandnon-naturalCDNsina
murinemodelofgenitalHSV2infection.WedemonstratethatdifferentCDNsstimulatethe
IFNpathwaytovaryingdegrees,andthatintraperitoneal(i.p.)andlocaldeliverystimulates
rapidIFNresponsewithassociatedantiviralfunction.Lastly,weshowthatlocalapplicationofa
CDNtothevaginalmucosaconfersfullprotectionagainstgenitalHSV2infectioninbothwild
typeandimmunodeficientcGas-/-mice.TheeffectsofCDNtreatmentaresuperiortoTLR7
and9agonists,basedonhighantiviralactivity,IFN-biasedresponsewithlowTNFexpression,
andtargetedstimulationofepithelialcells.
Results
STINGagonistsdifferentiallystimulatehumancelltypes,whichenables
cross-talk-mediatedprotectionagainstHSV2infection
TotestwhetherSTINGagonistscaninduceantiviralresponseagainstHSV2,wetreated
humankeratinocytes(HaCaT)withfivedifferentSTINGagonists.Keratinocytesarespecial-
izedepithelialcellsandaretheprimarycellsinvolvedinclinicallesionscausedbyHSV2.The
STINGagoniststestedwereDMXAA(5,6-dimethylxanthenone-4-aceticacid,vadimezan),
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 3/22
STINGagonistsconferprotectionagainstgenitalherpes
2’3’-cGAMP;3’3’-cyclicdi-adenosinemonophosphate(3’3’-c-di-AMP),andtwocyclicdinu-
cleotideanalogs,namelytheRp,Spisomerofthebisphosphorothioateanalogof2’3’-cGAMP
(2’3’-cGAM(PS) (Rp/Sp)),and3’3’-cyclicadenosinemonophosphate-inosinemonophos-
2
phate(3’3’-cAIMP).Asexpected,DMXAAdidnotinduceexpressionoftheISGsviperinand
ISG15orphosphorylationofSTAT1(Fig1A),asmeasured6hafterstimulation.Forthe
CDNs,weobservedphosphorylationofSTAT1,upregulationofviperin,andlowerlevelsof
STING,indicatingitsactivationandsubsequentdegradation(Fig1A).Despitetheobserved
phosphorylationofSTAT1andinductionofviperin,wedidnotfinddetectablelevelsoftypeI
IFNinthesupernatantsofHaCaTcellsstimulatedwith2’3’-cGAM(PS) (Rp/Sp)(Fig1B).In
2
sharpcontrasttoHaCaTcells,thehumanmonocyte-likecelllineTHP-1respondedto2’3’-
cGAM(PS) (Rp/Sp)stimulationwithaverystronginductionoftypeIIFNproduction,but
2
limitedinductionofISGs,andwasalsolessresponsivetoIFNαor-βtreatment(Fig1B,
1D–1F).SinceHaCaTcellswerehighlyresponsivetoIFNαor-βtreatment(Fig1Eand1F),
wecannotexcludethatthesecellsdidinducetypeIIFNlevelsbelowthedetectionlimit,which
stimulatedsignificantIFNsignalingandgeneexpression.Ofnote,forthedatashowninFig
1B,1Dand1H,cellswerenotpermeabilizedbeforetreatmentwith2’3’-cGAM(PS) (Rp/Sp),
2
thussuggestingdirectcellularentrywithoutadditionaldeliverysystems.Collectively,thedata
suggestthatmonocytesproducemuchhigherlevelsoftypeIIFNinresponsetoSTINGago-
niststhankeratinocytes,whichhoweveraremoreresponsivetoIFNstimulationthan
monocytes.
ToinvestigatewhethertheCDNstimulationcouldprotectagainstanHSV2infection,we
treatedHaCaTcellswith2’3’-cGAM(PS) (Rp/Sp)either24hor30minbeforeinfection.Incells
2
treatedwithCDN24hpriortoinfection,weobservedstronglyreducedlevelsofthemajorcapsid
protein,viralprotein5(VP5)(Fig1C),indicatingaprotectionofthecellsfrominfection.The
reducedaccumulationofviralproteinswasalsoseenincellstreatedwithhighdoseof2’3’-cGAM
(PS) (Rp/Sp)30minbeforeinfection.Since,THP-1cellsproducedhighlevelsoftypeIIFNupon
2
CDNstimulation,wewantedtotestwhetherthiscouldcontributetoantiviralactivityinasetting
withcrosstalkbetweendifferentcelltypes.Therefore,supernatantsfrom2’3’-cGAM(PS) (Rp/Sp)
2
-treatedTHP-1cellsweretransferredtoWTandIFNAR2-/-HaCaTcells,whichweresubsequently
infectedwithHSV2(Fig1G).Interestingly,treatmentwiththesupernatantsfromstimulated
THP1cellsledtoastrongreductionofHSV2replicationinWT,butnotIFNAR2-/-HaCaTcells
(Fig1Hand1I).Collectively,thesedatasuggestthatCDNsdifferentiallystimulatekeratinocytes
andmonocytes,andthatcrosstalkbetweenthecell-typesenablesthefullantiviralresponse,which
isdependentontypeIIFNs.
SystemicdeliveryofCDNsstimulatesIFNresponsesinseveraltissuesin
mice
TodeterminetheeffectandpotencyofSTINGagonistsinanimals,weinjectedthecom-
poundsi.p.intowildtype(WT)miceinequimolardoses(Fig2).At6hposttreatment,we
observedphosphorylationofSTAT1andupregulationofviperinandISG15(Fig2A).This
wasmostpronouncedinthemicetreatedwiththenon-endogenousCDNs,andtoamuch
lesserextentwith2’3’-cGAMP.Verystrongresponseswereobservedinserum,spleen,and
thevagina(epithelialsurface),whereassystemictreatmentwiththecompoundsgaveriseto
verylowresponsesinthebrain(Fig2A–2C).ThelackofaresponsetoDMXAAisexplained
bythelowdoseusedinthissetting,aswefoundthatDMXAApotentlystimulatedanIFN
responsewhenthedosewasincreased(S1AFig).Furtheranalysisoftheinductionof
expressionofIFNsandISGsbysystemicallydeliveredSTINGagonistsconfirmedthatthe
non-endogenousCDNspotentlystimulatedtheIFNpathwayinthevaginaandthespleen,
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 4/22
STINGagonistsconferprotectionagainstgenitalherpes
Fig1.STINGagonistsinducestypeIIFNandanti-viraleffectinhumancells.(A)HaCaTcellswerepermeabilizedwithdigitoninbeforestimulatedwithdifferent
STINGagonistsatalow(L:10μg/ml)orhigh(H:100μg/ml)concentrationfor24hours.CelllysateswereusedforWesternblottingforpSTAT1,STINGandviperin.
(B)HaCaTandTHP-1cellswerestimulatedwith2’3’-cGAM(PS) for24hoursandtypeIIFNactivitywasmeasuredinthesupernatant(n=3,(cid:3)=p<0,05).(C)
2
HaCaTcellswerepermeabilizedwithdigitoninandtreatedwith2’3’-cGAM(PS) beforeorafterinfectionwithHSV2(MOI0.1)for24hours.Celllysateswereused
2
forWesternblottingforVP5,STINGandviperin(n=3).(D,E,F)HaCaTandTHP-1cellswerestimulatedwith2’3’-cGAM(PS),IFNαorIFNβfor24hoursand
2
levelsofviperinandISG15weredeterminedbyWesternblotting.(G,H,I)THP-1cellswerestimulatedwith2’3’-cGAM(PS) for24hoursbeforethemediawere
2
transferredtowildtypeorIFNAR2-/-HaCaTcells.HaCaTcellswereinfected24hourslaterwithHSV2(MOI0.1)for24hours.CelllysateswereusedforWestern
blottingforVP5andISG15andthemediawereusedtodeterminetheHSV-2virusload.ForallWesternblots,Vinculinwasusedasloadingcontrol.n=3,a
representativesampleisshowed.NT,non-treated.Statistics,(B,I)Two-wayANOVAwithSˇida´k’smultiplecomparisonstest;p(interaction)<0.05.
https://doi.org/10.1371/journal.ppat.1006976.g001
andthatDMXAAand2’3’cGAMPweremuchlesspotentinvivo(Fig2C).Wealsofound
thatCDNsstimulatedmodestinductionofthenuclearfactor(NF)κB-inducedgenesA20,
Il6,andTnfa (S1BFig),thussuggestingthatdirectactivationoftheSTINGpathwayinmice
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 5/22
STINGagonistsconferprotectionagainstgenitalherpes
Fig2.STINGagonistsinducestypeIIFNexpressinginvivo.Equimolar(1.687x10-7mol)dosesofSTINGagonists2’3’-cGAMP(121μg/mouse),3’3’c-diAMP
(119μg/mouse),2’3’-cGAM(PS)2(125μg/mouse),3’3’-cAIMP(111μg/mouse)orDMXAA(95μg/mouse,STINGactivationbyDMXAArequirestwomolecules
(3.374x10-7mol))wereadministratedtomicei.p.Sampleswerecollected6hourslaterforfurtheranalysis.(A)LevelsofphosphorylatedSTAT1(pSTAT1),STING,
viperinandISG15weredeterminedbyWesternblottingofspleen,vaginaandbraintissues.Vinculinwasusedasloadingcontrol.n=5,tworepresentativesamplesare
shown.(B)IFNαandIFNβintheserumdeterminedbyELISA.n=3–5.(cid:3)=p<0.05comparedtomock.(C)ExpressionforIfnbandMx1mRNAintissuessamples
fromvagina,spleenandbrain,normalizedtoGAPDH.n=3–5.(cid:3)=p<0,05comparedtomock.(D)Micewereperfusedpriortoisolationofbrainsamplesandgene
expressionsofIfnbandMx1mRNAweremeasured.n=3–5.(cid:3)=p<0,05comparedtomock.Statistics,(B-D)Kruskal-WallistestwithDunn’smultiplecomparisons
test.
https://doi.org/10.1371/journal.ppat.1006976.g002
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 6/22
STINGagonistsconferprotectionagainstgenitalherpes
doesnotleadtostrongactivationoftheNFκBpathway.Furthermore,micedeficientfor
STINGdidnotrespondto2’3’-cGAM(PS) (Rp/Sp)treatment,underliningthatSTINGis
2
thetargetforthemoleculestested(S1CFig).However,sinceanNFκBresponsivereporter
genewasreadilyactivatedbySTINGagonistsinTHP1cells(S1DFig),wearereluctantto
concludethatNFκBisnotinvolvedinthegeneinductionprograminduceduponSTING
activation.
Toensurethatthesmall,butsignificant,2’3’-cGAM(PS) (Rp/Sp)-inducedIFNresponse
2
weobservedintheCNSwasnotderivedfrombloodleftoverinthevasculaturefollowinghar-
vestofbraintissue,weinjectedWTmicei.p.with2’3’-cGAM(PS) (Rp/Sp)andperformeda
2
whole-animaltranscardialperfusionofPBStoremoveanyresidualbloodfromtheperipheral
vasculaturebeforetakingoutthebrainoftheanimals.Followingthisprocedure,westillfound
asignificantupregulationoftheexpressionofIfnbandMx1inthebrain(Fig2D).Thisindi-
catesthattheIFNresponseinthebraintissueoriginatesfromresidentcells,suggestingthat
amongtheCDNstested,2’3’-cGAM(PS) (Rp/Sp)maycrosstheblood-brain-barrierandexert
2
aneffectdirectlyintheCNS.Takentogether,thesedatasuggestthatsystemicadministration
ofnon-endogenouslyproducedCDNsyieldsanIFNresponseinseveraldifferenttissues,
includinglymphoidorgans,epithelialsurfaces,andtheCNS.
STINGagonistsprovokeanISG-responseinthevaginalmucosa
ToinvestigatehowtheSTINGagonistsaffectedtheIFNresponseinthetissue,wechoseto
lookmorecloselyatthevagina,whichisoneofthemainportalsofentryforviralinfections.
Usingimmunohistochemicalstainingofvaginaltissues,wefoundthatsystemicdeliveryof
2’3’-cGAM(PS) (Rp/Sp)inducedstrongexpressionoftheISGviperinbycellsinthestroma
2
(Fig3).Furthermore,epithelialcellslocatedclosetothebasalmembranewerepositivefor
viperin,whileepithelialcellslocatedtotheluminalsidehadlessexpression.Vaginaltissue
frommiceinfectedlocallywithHSV2showedstrongviperinexpressionincellslocatedtothe
areaofinfection.Fewcellsco-expressedviperinandHSV2antigens,butcellsaroundthe
HSV2-infectedcellsexpressedhighlevelsofviperin.Thisincludedarangeofcelltypes,includ-
ingepithelial,stroma,andpotentiallyalsoimmunecells.
SystemicadministrationofSTINGagonistsimprovesthesurvivalofmice
infectedwithHSV2
ToexplorewhethertheupregulationofISGsinthevaginalmucosacouldplayaroleincon-
trolofinfectioninvivo,wesetupthreedifferenttreatmentregimenswithi.p.injectionsof
2’3’-cGAM(PS) (Rp/Sp)beforeorafterHSV2infection.Theregimenswere(CDNtreat-
2
mentrelativetoinfection):onepretreatment,onepost-treatment,ortwopost-treatments
(Fig4A).WhentreatingWTmice,theCDNpretreatmentandtwo-timepost-treatments
showedsignificantlyimprovedsurvivalascomparedtonon-treatedcontrols,andtheone-
timepost-treatmentgroupsuggestedimprovedsurvival,butthedatadidnotreachstatisti-
calsignificance(p=0.06)(Fig4B).WealsotestedcGas-/-mice,sincetheycannotproduce
2’3’-cGAMPuponimmunerecognitionofHSV2,butcanbestimulatedbyexternaldelivery
ofSTINGagonists.WhentreatingcGas-/-mice,however,onlythepretreatedgrouphada
100%overallsurvival,whilealmostallthepost-treatedmiceeventuallysuccumbedtothe
infection,althoughthetwo-timespost-treatmentledtosignificantlyimprovedsurvival,(Fig
4C).Thedifferencesintheeffectsoftwo-timeposttreatmentonsurvivalbetweenWTand
cGas-/-micemaybeexplainedbytheroleofendogenousactivationofSTINGsignaling
upongenitalHSV2infection,asreportedpreviously[42].AllgroupsreceivingCDNtreat-
ment,regardlessofthegenotype,hadsignificantlylowerviralloadsinthevaginawhen
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 7/22
STINGagonistsconferprotectionagainstgenitalherpes
Fig3.HSV2andSTINGagonistsinduceantiviralgenesinvaginalepithelium.Micewereinjectedi.p.with2’3’-cGAM(PS)2(125μg/mouse)or
infectedwithHSV2(6.7×104p.f.u.).Tissueswereisolatedfrommice6hand24hafterCDNstimulationandHSV2infection,respectively.Paraffin
sectionsofthevaginaltissueswerestainedforviperin(red)andHSV2(green).DAPI(blue)marksthenucleiandthedottedwhitelinesmarkbasal
membranebetweentheepitheliumandstroma.Whitearrowshighlightexamplesofviperinpositivecells,andarrowheadsmarkexamplesof
HSV2-infectedcells.L=lumen,E=epithelium,S=stroma.n=3.Onerepresentativepictureisshownforeachstainingandtreatmentgroup.
https://doi.org/10.1371/journal.ppat.1006976.g003
comparedtotheirrespectivecontrols(Fig4D).Takentogether,thesedatademonstratethat
activationoftheSTINGpathwaywiththenon-endogenousCDN2’3’-cGAM(PS) (Rp/Sp)
2
mountsapotentantiviraldefense,theefficacyofwhichdependsonthetimingandrepeti-
tionoftreatmentaswellastheimmunocompetenceofthemouse.
LocaladministrationofSTINGagonistsyieldsarobustlocalandaweaker
systemicIFNresponsewhichconfersprotectionagainstHSV2infection
GiventheclearantiviralactionofCDNsinthevaginatogetherwiththesystemicimmuneacti-
vationafteri.p.treatment,wewantedtoevaluatewhetherdirectadministrationofCDNsto
epithelialsurfaceswouldalsostimulateaprotectiveresponseandwithlesssystemiceffects.For
theseexperiments,weused3’3’-cAIMP,whichshowedinvivoresponsesverysimilarto2’3’-
cGAMP(PS) (Rp/Sp)andhasahighsolubility,thusallowing250μgtobeappliedin20μl
2
saline.Sixhoursafterapplicationof3’3’-cAIMPtothevaginalmucosalsurface,weobserved
verystrongstainingforviperinthroughouttheepitheliallayer(Fig5A).Interestingly,thiswas
incontrasttomicereceivingsystemicCDNtreatment,whereviperinstainingwasstrongestin
cellslocalizingtothestromaandinnerepithelium(Fig3).Invaginaltissuefromthemice
receivinglocalCDNtreatment,astrongupregulationofbothIfnbwasobservedandmodest
inductionofTnfa, Il6,andA20,(Fig5B).Incontrasttothis,noIFNsignaturewasobservedin
thespleen,whichhasaveryhighexpressionofSTING(Fig2A).However,wediddetectele-
vatedIFNβproteinlevelsinserumfromsome,butnotallthemicetreatedwith3’3’-cAIMPin
thevagina(Fig5C).Toexaminetheantiviralresponsestimulatedbylocallyadministered
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 8/22
STINGagonistsconferprotectionagainstgenitalherpes
Fig4.SystemictreatmentwithSTINGagonistsconfersprotectionagainstgenitalHSV2infection.WildtypeandcGas-/-miceweretreatedwith2’3’-cGAM
(PS)2(Rp/Sp)(125μg/mouse)andinfectedintravaginallywithHSV2(6.7×104p.f.u.).(A)Illustrationofthetimelineforthetreatmentregimens.(B,C)Overall
survivalforHSV2-infectedandtreatedwildtype(B)orcGas-/-(C)mice.n=6–10.(cid:3)=p<0,05comparedtomock.(D)HSV2titer(TCID50)invaginalwashes
collected48hourspostinfection.n=6–10.(cid:3)=p<0,05comparedtomockinthesamegenotype.Statistics,(B,C)Log-ranktestwithHolm-Bonferroni
correction.(D)One-wayANOVAoflog -transformeddatawithDunnett’smultiplecomparisonstest.
10
https://doi.org/10.1371/journal.ppat.1006976.g004
CDN,WTorcGas-/-miceweretreatedinthevaginawith3’3’-cAIMPandinfectedwithHSV2.
Followingthistreatment,bothgenotypesshowedcompleteprotectionagainstdisease,andno
detectablevirusinthevaginallavage(Fig5D–5F).Stainingoftissuesectionsfrom3’3’cAIMP-
treatedHSV2-infectedmicefailedtorevealvirus-infectedcells,whichwasreadilyseeninthe
absenceofCDNtreatment(Figs5Gand3).Finally,weexaminedhowlongtheCDNtreatment
couldbeseparatedfromHSV2infectiontemporally.Localtreatmentwith3’3’-cAIMPupto
72hpriortoHSV2infectionconferredfullprotectionagainstgenitalherpesandsuppression
ofviralreplication(Fig5Hand5I).Takentogether,thesedatasuggestthatlocaladministration
ofaSTINGagonisttoamucosalsurfaceconferslocalprotectionagainstviralinfectionswith
onlymildsystemicactivationoftheIFNsystem.
STINGagonistsinduceIFNresponsesinthevaginalepitheliumfasterand
moreefficientlythanTLRagonists
Previousattemptstotakeadvantageofinnateimmuneactivationtocontrolgenitalherpes
infections,showedpromisingresultsinmice,butonlylimitedclinicaleffectswereobtained,
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 9/22
STINGagonistsconferprotectionagainstgenitalherpes
Fig5.LocalapplicationofSTINGagonistsprotectsagainstgenitalHSV2infection.Micewereanesthetizedfor30minand250μg3’3’-cAIMPwas
appliedtothevagina.(A)Tissueswereisolatedfrommice6hafterCDNstimulation.Paraffinsectionsofthevaginaltissueswerestainedforviperin(red)
andHSV2(green).DAPI(blue)marksthenucleiandthedottedwhitelinesmarkbasalmembranebetweentheepitheliumandstroma.Whitearrows
highlightexamplesofviperinpositivecells.L=lumen,E=epithelium,S=stroma.n=4.Onerepresentativepictureisshownforeachstaining.(B)After6
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006976 April2,2018 10/22