TheJournalofImmunologyPegylatedBisacycloxypropylcysteineaDiacylatedLi - PDF document

TheJournalofImmunologyPegylatedBisacycloxypropylcysteine,aDiacylatedLipopeptideLigandofTLR6,PlaysaHost-ProtectiveRoleagainstExperimentalLeishmaniamajorSuryaPrakashPandey,*HimanshuSinghChandel,*SunitSrivastava,*SathishkumarSelvaraj,*MukeshKumarJha,*DivanshuShukla,*ThomasEbensen,CarlosA.Guzman,andBhaskarSaha*TLRsrecognizepathogen-expressedAgsandelicithost-protectiveimmuneresponse.AlthoughTLR2formsheterodimerswithTLR1orTLR6,recognizingdifferentligands,differencesinthefunctionsoftheseheterodimersremainunknown.Inthisstudy,wereportthatinLeishmaniamajor-infectedmacrophages,theexpressionofTLR1andTLR2,butnotTLR6,increased;TLR2–TLR2associationincreased,butTLR2–TLR6associationdiminished.Lentivirus-expressedTLR1–shorthairpinRNA(shRNA)orTLR2–shRNAadministrationreduced,butTLR6–shRNAincreasedL.majorinfectioninBALB/cmice.Corroboratively, *NationalCentreforCellScience,Ganeshkhind,Pune411007,India;andofVaccinologyandAppliedMicrobiology,HelmholtzCentreforInfectionResearch,Braunschweig,38124Braunschweig,GermanyReceivedforpublicationMarch26,2014.AcceptedforpublicationJuly30,2014.ThisworkwassupportedinpartbytheDepartmentofBiotechnologyandIndianCouncilofMedicalResearch,GovernmentofIndia,andtheHelmholtzAssociation(Indo–GermanScienceCentreforInfectiousDiseases). L.major(11)andL.donovani(23)infections.AlthoughTLR1–TLR2andTLR2–TLR6heterodimersareknownforrecognitionoftriacylatedlipopeptidesordiacylatedlipopeptides,respectively(4,5),whethertheyfunctiondifferentlyisnotknown.Antileishmanialhost-protectiveTcellresponseislimitedbyregulatoryT(Treg)cells(24–28),whichcanbemodulatedbyIL-2,IL-10,andCD40.Infact,TLR2hasbeenimplicatedintheregu-lationofTregcellsindifferentdiseases(29,30).Altogether,thesepreviousreportsthusimpliedthatTLR2playsanimportantroleininfectionandcanpossiblybetargetedfordevisingtherapeuticorprophylacticstrategies.Therefore,basedonthesendings,weexaminedtheroleofTLR1–TLR2–TLR6systemofTLRsinL.majorinfectionandtestedwhetherBPPcysMPEGtreatmentL.major-infectedBALB/cmicecanresultinaTLR6-targetednoveltherapyorprophylaxis.Inthisstudy,wereportthatBPPcysMPEGincreasedTLR9ex-pressioninBALB/c-deriveduninfectedandL.major-infectedmac-rophagesandinducedahigherIL-12:IL-10ratiothanthatinducedbyPamCSKandpeptidoglycan(PGN).TLR1shorthairpinRNA(shRNA)orTLR2shRNAreduced,butTLR6shRNAaggravatedL.majorinfectioninBALB/cmice.PamandPGNaugmented,whereasBPPcysMPEGreducedamastigotenumberinmacrophages.BPPcysMPEGinducedhigherp38MAPKandactivatingtranscrip-tionfactor2(ATF2),whereasPamandPGNinducedmoreofERK1/2andELK-1activation.ThesecontrastingeffectsofTLR2heterodimerswithTLR1orTLR6revealaTLR2functionalduality.MaterialsandMethodsParasites,mice,peritonealmacrophages,andreagentsL.major(strainMHOM/Su73/5ASKH)wasmaintainedinRPMI1640with10%FCS(LifeTechnologies-BRL,GrandIsland,NY).TheparasiteswerepassagedthroughBALB/cmicetomaintainthevirulence.C57BL/6,inducibleNOsynthase(iNOS)–decient(C57BL/6background),BALB/c,andIL-12–decient(BALB/cbackground)mice,originallyfromTheJacksonLaboratory(BarHarbor,ME),weremaintainedintheexperi-mentalanimalfacilityoftheNationalCentreforCellScience.AnimalswereusedaccordingtotheInstitute’sethicalanimalusecommittee–ap-provedanimaluseprotocol.TheelicitedperitonealmacrophageswereisolatedasadherentcellsfromC57BL/6,iNOS-decient,IL-12–decient,andBALB/cmice5dafterthioglycolateinjection(3%thioglycolate,2ml;i.p.).Purityofmacrophageswastestedroutinely,asdescribedearlier(31),anditwasalwaysbetween97and98.5%(datanotshown).MacrophageswereculturedinRPMI1640supplementedwith10%FCS.After6h,nonadherentcellswerewashedoutandcellswereincubatedfor36hat37Ccontaining5%COandhumidiedatmosphere.PamCSK,PGN,andCpGODN1826werepurchasedfromInvivogen(SanDiego,CA).BPPcysMPEGwassynthesizedattheHelmholtzCentreforInfectionResearch(Braunschweig,Germany).TheprimersforTLRsandcytokines(TableI)werefromIntegratedDNATechnologies(SanDiego,CA).AbsandstandardsforcytokineELISAwerefromBDPharmingen(SanDiego,CA).ImmunoblottingAbsforp-IKK,totalIKK,p-NF-B,totalNF-B(CellSignaling,Danvers,MA),p-ATF2,totalATF2,p-ELK1,totalELK1,p-p38MAPK,totalp38MAPK,p-ERK1/2,totalERK1/2,TLR-1,TLR-6,MyD88,Toll/IL-1Radaptorprotein(TIRAP),TNFR-–associatedfactor6(TRAF6),-actin(SantaCruzBiotechnology;SantaCruz,CA),andTLR-2(Imgenex,SanDiego,CA)wereobtained,asindicated.L.majorinfectionofmacrophagesC57BL/6,iNOS-decient,IL-12–decient,andBALB/c-derivedperitonealmacrophageswereinfectedwithstationaryphase

L.majorpromastigotesataratioof1:10for6h,andextracellularparasiteswerewashedout.Macro-phagesweretreatedwithdifferentdosesofTLRligandsorleftuntreatedfor60h.MacrophageswerexedwithchilledmethanolandstainedwithGiemsastain.Amastigoteswerecountedunderthemicroscope,asreportedearlier(11).RT-PCRandreal-timePCRRNAwasextractedusingTRI-Reagent(Sigma-Aldrich,St.Louis,MO).ForcDNAsynthesis,2gtotalRNAfromeachsamplewasincubatedwith0.6grandomprimer,0.1MDTT,10mMdNTPs,40URNaseout,and200UMoloneymurineleukemiavirus–reversetranscriptase,andwasusedforlreactionmixture(InvitrogenLifeTechnologies,Carlsbad,CA).Sam-pleswereincubatedat37Cfor1h,followedby10-minheatinactivationat65C.cDNAfromeachsamplewasampliedwithTaqDNApolymerase(InvitrogenLifeTechnologies)in25lreactionmix,underthefollowingconditions:94Cfor1min,annealingfor1minatatemperaturebetween53and60Cdependinguponthegene-specicprimers(TableI),and72Cfor1minforatotalof28–30cycles.EachsamplewasampliedformouseGAPDHor-actintoensureequalcDNAinput,asdescribedearlier(11).AmpliedPCRproductswererunin1.2%agarosegel.Theexpressionlevelsoftheabove-mentionedgeneswerequantiedusingtheQuantityOne-4.6.1(basic)software(Bio-Rad,Hercules,CA).Real-timePCRwasperformedontheEppendorfRealPlexmastercyclerusing2XIQSYBRGreensupermix(5l;Bio-Rad),10ngcDNAastemplate,2ngforwardprimer,and2ngreverseprimer,andthereactionswereperformedinthin-wall0.2-mlstriptubes(Axygen,UnionCity,CA)foratotalof10lreactionmix.Relativequantitationwasdoneusingthecomparativethreshold()method.ThemRNAexpressionlevelsofthetargetgeneswerenormalizedagainstthoseofGAPDHlevelsandexpressedasrelativefoldchangecomparedwithuntreatedcontrols(32).ImmunoprecipitationandWesternblottingForimmunoprecipitation,cellswerelysedinlysisbufferbyincubationonicefor1h.Equalamountsofproteins(500g)wereincubatedwith1ganti–TLR-2Abfor8–10hat4CandpulledwithproteinA/G–agarosebeads(Pierce,Rockford,MI)at4Cfor3h.ImmunoprecipitateswerewashedtwiceincoldlysisbufferandonceincoldTBST.PelletswereresuspendedinSDSsamplebufferandsubjectedtoWesternblotanalysis(32).ForWesternblotting,cellsweretreatedwithindicatedreagentsandlysedwithlysisbuffer(20mMTris[pH7.5],150mMNaCl,10%glycerol,1mMEDTA,1mMEGTA,and1%NonidetP-40),proteaseinhibitormixture(RocheAppliedScience,Mannheim,Germany),andphosphataseinhibitormixture(Pierce)byincubationonicefor1h.Lysateswerecentrifuged(12,000rpm,15min),andsupernatantswerequantiedbybicinchoninicacidkit(Pierce,Rockford,Michigan).EqualamountofproteinwasloadedonSDS-PAGE,andresolvedproteinwastransferredtopolyvinylidenediuoridemembrane(Millipore,Billerica,MA)andblockedwith5%nonfatdriedmilkinTBST(25mMTris[pH7.6],137mMNaCl,and0.2%Tween20).MembraneswereincubatedwithprimaryAbat4Covernight,washedwithTBST,andincubatedwithHRP-conjugatedsec-ondaryAb.ImmunoreactivebandswerevisualizedwiththeLuminolreagent(SantaCruzBiotechnology,SantaCruz,CA)(32).DensitometricanalysesofbandswereperformedusingQuantityOnesoftware.FACSreagentsandTregcellanalysesAnti-CD3–PE-Cy7(553062),anti-CD25–allophycocyanin-Cy7(557658),anti-GITR–PE(12-5874-82),andanti–IL-10–FITC(554466)Abswerepur-chasedfromBDBiosciences(SanJose,CA);anti-CD127–allophycocyanin(17-1271-82)AbwaspurchasedfromeBioscience(SanDiego,CA);andanti-CD4–PerCP-Cy5.5(100434)andanti-Foxp3–PacicBlue(126409)AbswerepurchasedfromBioLegend(SanDiego,CA).FormulticolorFACSanalyses,cellswerestained(afterblockingwith5%FCS)withuorescentlylabeledAbsanti-CD3–PE-Cy7,anti-CD4–PE-Cy5,anti-CD25–allophycocyanin-Cy7,anti-GITR–PE,andanti-CD127–allophycocyaninfor45minat4CindarkandwashedtwicewithFACSbuffer(1PBS,10mMHEPESbuffer,and3%FCS).IntracellularcytokinestainingwasperformedusingaCytox/Cytoperm-PlusKitwithGolgiPlug(555028;BDPharmingen),asperthemanufacturer’sinstructions.CellswereacquiredintheCD3gatebyFACSCantoIIowcytometer(BDBiosciences)andanalyzedforexpressionofGITRandFoxp3orFoxp3andIL-10usingBDFACSDivasoftware(version5.2;BDBiosciences)(28).Macrophage–TcellcocultureBALB/c-derivedperitonealmacrophagesfromnaivemicewereinfectedL.majorpromastigotesataratioof1:10for6h.Theextracellularparasiteswerewashedout,andmacrophagesweretreatedwiththeindi-cateddosesofBPPcysMPEGfor24h.Thepopliteallymphnodewascollectedfromthe21-dL.major-infectedBALB/cmice.Infectedmacro-phageswerecoculturedwithlymphnodeCD4Tcellsat1:3ratio(macrophage:Tcells).After60h,supernatantswerecollectedforcytokineassaysandcellsforTreganalyses.Foramastigotecount,macrophageswerewashed,

xedwithchilledmethanol,stainedwithGiemsastain,andcountedunderalightmicroscope(E-600;Nikon).ProductionoflentiviralparticlesforTLRshRNAandcontrolshRNATLR1andTLR6shRNA(GenBankaccessionnumberNM_030682,http://www.ncbi.nlm.nih.gov/nuccore/NM_030682,andNM_011604,TheJournalofImmunology http://www.ncbi.nlm.nih.gov/nuccore/NM_011604)inpLKO.1lentivirusvector;controlshRNAinpLKO.1lentivirusvector;TLR2shRNA(Gen-BankaccessionnumberNM_011905,http://www.ncbi.nlm.nih.gov/nuccore/NM_011905)inpGIPZlentivirusvector;andcontrolshRNAinpGIPZlentivirusvectorwerepurchasedfromOpenBiosystems(Huntsville,AL).TheseshRNAwereusedforlentiviralproductionusingTrans-Lentiviralpackagingsystem(replication-incompetentHIV-1–basedlentivirus;OpenBiosystems)inHEK293Tcelllinefollowingthemanufacturer’sprotocol.Briey,5.5cellswereseededin100-mmpetriplates.Nextday(day1),Trans-Lentiviralpackagingmix(28.5g)andTLRshRNA(9g)orcontrolshRNA(9g)plasmidDNAweremixedtogether.Transfectionwascarriedoutinserum-freemediausingArrest-Intransfectionreagent(OpenBiosystems)ata1:5DNA/transfectionreagentratio.After6h,mediumwasreplacedwithcompletegrowthmedium(DMEMsupplementedwith10%FCS),andcellswereincubatedinaCOincubatorat37C.Virus-containingsupernatantwasharvestedafter48and72h,lteredthroughamlter(Millipore),andconcentrated100-fold(32).InvitrolentiviraltransductionPeritonealmacrophagesweretransducedwithlentivirallyexpressedTLR1shRNA(TLR1-Lv),TLR2shRNA(TLR2-Lv),TLR6shRNA(TLR6-Lv),andcontrolshRNA(control-Lv)particles(2transductionunits/cell)for8hinRPMI1640supplementedwithFCS(0.5%)andpolybrene(8(Sigma-Aldrich).Cellswerewashedtoremoveresidualviralparticles.After48-htransduction,uninfectedandL.major-infectedcellsweretreatedwithBPPcysMPEGandCpGorleftuntreatedfor48hforamas-tigotecountandELISA(32).Generationofbonemarrowmonocyte–deriveddendriticcellsDendriticcells(DCs)wereculturedfrombonemarrowprogenitorcellsusingamodiedprotocolofapreviouslydescribedmethod(33).Inbrief,femoralcellswerefractionatedonadensitygradient(Histopaque-1077;Sigma-Aldrich)toisolatemononuclearcells,whichwereincubatedforplasticadherencefor90min.Nonadherentcellswerewashedoutwith1PBS.DCculturemedium(RPMI1640,10%FCS,100U/mlpenicillin,100g/mlstreptomycin,50M2-ME,20ng/mlGM-CSF,and10ng/mlIL-4)wasaddedtotheadheredmonocytes(1cells/ml).Culturemediumwasreplacedwithfreshculturemediumevery3d.Onday6,dislodgedcellswereusedasbonemarrow–derivedDCs.PurityoftheDCwasalways85%(datanotshown)(27,28,33).DCandTcellcocultureBonemarrow–derivedDCswereinfectedwithL.majorpromastigoteswitharatioof1:10for3h.Theextracellularparasiteswerewashedout,andDCsweretreatedwiththeindicateddosesofBPPcysMPEGfor24h.Thepopliteallymphnodewascollectedfromthe21-dL.major-BALB/cmice.InfectedDCswerecoculturedwithlymphnodeCD4Tcellsata1:3ratio(DC:Tcells).After60h,supernatantswerecollectedandquantiedforcytokineresponse.CellswereharvestedandwereusedforTreganalysesbyowcytometry(28).InvivoL.majorBALB/candIL-12–decientmicewereinfectedintheleftfootpadswithL.majorpromastigotes(s.c.,2in50lsaline/mouse).MiceweretreatedwithBPPcysMPEG(1g/mouse)foralternating3dstartingfromthethirddaypostinfection.Insomeexperiments,2dpriortoL.majorinfection,micewerepretreatedwith5transductionunits(in50HBSS;LifeTechnologies,GrandIsland,NY)ofTLR1shRNA,TLR2shRNA,TLR6shRNA,orcontrolshRNAexpressinglentivirusesinthesamefootpadtobeinfected.SomemiceweretreatedwithBPPcysMPEGg/mouse)orCpG(10g/mouse)orboth(s.c.intothefootpadfor3dalternativelystartingfromthethirddaypostinfection).Sevenmicewereusedineachgroup.Thefootpadswellingwasscoredusingadigitalmi-crometer(Mitituyo).Miceweresacriced5wkaftertheinfection;pop-liteallymphnodeswerecollectedforassessingparasiteload(34)andforcytokineproductionbyELISA(32).TestingtheadjuvanticityofBPPcysMPEGinantileishmanialThreegroupsofmice,eachcontaining10BALB/cmice,wereimmunizeds.c.intothehindfootpadwith1formalin-xedL.major.Fixationoftheparasiteswasconrmedbythefailureofparasitestogrowinvitro.ImmunizedmiceweretreatedtwicewitheitherPamg/mouse)orBPPcysMPEG(1g/mouse)intothesamefootpad,onalternatedays,startingfromtherstdayofimmunization,andonegroupwasleftun-treatedasacontrol.After5wk,allimmunizedandcontrolmicewereinfectedwith2L.majorpromastigotes.Fiveweeksafterthein-fection,miceweresacricedandpopliteallymphnodeswerecollectedforparasiteenumeration(34),cytokineproductionassay,andTregcellana-lyses(28).StatisticalanalysisTheinvitroculturesweresetintriplicates.Weus

ed5–10micepergroupforinvivoexperiments,asindicated.ErrorbarssignifySEMfromonerepresentativefrommultiplesetsofexperiments.Studenttestwasusedtoassessthesignicanceofthedifferencesbetweenthemeanvaluesforcontrolandexperimentalgroups. TableI.TheforwardandreverseprimersusedinRT-PCRandreal-timePCRGenePrimerSequence(5)GenePrimerSequence(5RT-PCRPrimersGAPDHF:GAGCCAAACGGGTCATCATCTLR5F:CAACCAAACCAACGTCACCCTGTTCCTGCTTCACCACCTTCTTGTGGCTTCCTCTTCATCGCAGTCTT-actinF:GTCCCTGTATGCCTCTGGTCTLR6F:TTGTTTGCGCCCTGGCCTTAATCAAGAAGGAAGGCTGGAAAAGTGTTCTTGGTGGCAGGTCTTTGiNOSF:CAGAGGACCCAGAGACAAGCTLR7F:TGCAACTGTGATGCTGTGTGGTAAGACCAGAGGCAGCACATCTTTGACCTTTGTGTGCTCCTGGTLR1F:CAATGTGGAAACAACGTGGATLR8F:TGCCAAACAACAGCACCCAATGTAACTTTGGGGGAAGCTGTCAAAGACTCAGGCAACCCATLR2F:AAGAGGAAGCCCAAGAAAGCTLR9F:ACTGAGCACCCCTGCTTCTACGATGGAATCGATGATGTTGAGATTAGTCAGCGGCAGGAATLR3F:TCCTTGCGTTGCGAAGTGAATLR11F:CTGAAGGTGAAGGCTTGAGGTTTTGGGCGTTGTTCAAGAGGACCTGTCATTTCCAATCCCAAATLR4F:ACCTGGCTGGTTTACACGTCTLR12F:TTGAGCTGCAGGGCTTGATTGTCTGCCAGAGACATTGCAGAAGCTTGGTTAGGCCAGTGCTTGAAATLR13F:ACACAGTGAATGGTGCCGACTTAAAGTGGCTGCTGGTGAACACTReal-TimePCRPrimersTLR1F:CAATGTGGAAACAACGTGGATLR9F:ACTGAGCACCCCTGCTTCTATGTAACTTTGGGGGAAGCTGAGATTAGTCAGCGGCAGGAATLR2F:AAGAGGAAGCCCAAGAAAGCiNOSF:GTGACACACAGCGCTACAACGATGGAATCGATGATGTTGGAGCACAGCCACATTGATCTTLR6F:TTGTTTGCGCCCTGGCCTTAATGAPDHF:ATGGACTGTGGTCATGAGCCTGTTCTTGGTGGCAGGTCTTTGATTGTCAGCAATGCATCCTGF,forward;R,reverse.3634ANTILEISHMANIALFUNCTIONOFBPPcysMPEG TLR1,TLR2,andTLR6aredifferentiallyexpressedonL.major-infectedmacrophagesBecauseseveralTLRsareimplicatedinthecontrolofinfection(11,12,14,23,35–39),wecheckedtheexpressionofallTLRsinuninfectedandL.major-infectedmacrophagesbyRT-PCR(TableI).WeobservedanincreasedexpressionofTLR1andTLR11/TLR12,butdecreasedexpressionofTLR4andTLR9(Fig.1A).AmongthesealteredTLRs,TLR4andTLR9promotehost-protectiveantileishmanialimmuneresponses(23,40–42)andTLR11andTLR12arenotyetimplicatedinHowever,theroleofTLR1ininfectionremainedtobeelucidated.AsTLR1functionsasaheterodimerwithTLR2,whichcanalsobindtoTLR6,werstconrmedTLR1,TLR2,andTLR6expressioninuninfectedandL.major-infectedmac-rophages.Thereal-timePCRandWesternblotdataconrmedthatTLR1expressionwasincreasedinL.majorinfection(Fig.1B).Next,wetestedtheassociationbetweenTLR1andTLR2andbetweenTLR2andTLR6inuninfectedandL.major-macrophages.ItwasobservedthatTLR1–TLR2andTLR2–TLR2associationswereenhanced,buttheTLR2–TLR6associationwasreducedinL.major-infectedmacrophages(Fig.1C).Becausetheligandscanpromoteheterodimerizationofreceptors,wetestedwhetherprovidingextraneousTLR2–TLR6-ligandBPPcysMPEGwouldrestoreTLR2–TLR6associationintheinfectedmacrophages.ItwasobservedthatBPPcysMPEGenhancedtheassociationbe-tweenTLR2andTLR6inbothuninfectedandL.major-infectedmacrophages(Fig.1D).TheseresultssuggestthatL.majorselec-tivelyaugmentsTLR1expression;theparasiteenhancesTLR1–TLR2andTLR2–TLR2association,butreducesTLR2–TLR6as-sociation.ItisplausiblethattheparasitereducestheTLR2–TLR6associationasanimmuneevasionstrategy.Inthatcase,bettingtheprincipleofparasitism,thereducedassociationbetweenTLR2andTLR6impliesahost-protectiveroleinL.majorinfection.TLR1andTLR2shRNAreducedL.majorinfection,butTLR6shRNAincreasedtheinfectionTotesttheplausiblerolesforTLR1,TLR2,andTLR6inL.majorinfection,theexpressionofeachoftheseTLRswassilencedusingrespectiveshRNA,followedbyL.majorinfectionoftheBALB/cmice.WeobservedthatTLR1andTLR2shRNAreducedtheparasiteloadinmacrophages(Fig.2A);apartoftheTLR2shRNA-inducedreductioninparasiteloadcouldbeduetolessinternalization(Fig.2B).InBALB/cmice,TLR1shRNAandTLR2shRNAreducedparasiteburden,whereasTLR6shRNA-treatedBALB/cmiceincreasedtheparasiteloadcomparedwiththatobservedincontrolshRNA-treatedmice(Fig.2C).These FIGURE1.L.majorinfectiondifferentiallyregulatestheexpressionofTLR1,TLR2,andTLR6andassociationofTLR1orTLR6withTLR2.BALB/c-derivedthio-glycolate-elicitedperitonealmacrophageswerekepteitheruninfected(UIM)orinfectedwithL.majorpromastigotesat1:10ratiofor72h(IM).()ThecellswerelysedandthemRNAlevelswereanalyzedforallTLRsbyRT-PCR.DensitometricquantitationwasperformedusingtheQuan-tityOnesoftware(Bio-Rad),andthevaluesforgeneswerenormalizedagainst-actin(rightpanel).()cDNAfromtheaboveexperimentswasusedtochecktheexpressionofTLR1,TLR2,andTLR6byreal-timePCR.mRNAex-pressionlevelsofthetargetgeneswerenormalizedagainstGAPDHandexpressedasrelativefoldchangecomparedwithuninfectedcontrols.Thecellsfromtheaboveexperi-me

ntswerelysed,andproteinswereusedforWesternblottingforTLR1,TLR2,andTLR6(lowerpanelThedensitometricquantitationwasperformedusingtheQuantityOnesoftware,anddataareshown(lowerrightpanel).()Equalamountofproteinwasimmuno-precipitatedwithanti-TLR2Ab,andtheassociationofTLR1,TLR2,andTLR6wascheckedbyimmunoblottingusingrespectiveAbs.DensitometricvaluesforTLR1,TLR2,andTLR6expressionwerenormalizedagainstthevaluesof-actin(rightpanel).()Macrophageswerestim-ulatedwithBPPcysMPEG(100ng/ml)for15minandlysed;equalamountofproteinwasimmunoprecipitatedwithanti-TLR2;andtheassociationofTLR6wascheckedbyimmunoblotting.DatashownaredensitometricunitforTLR6(rightpanel).Fromthesamesetoftheexperi-ment,equalamountofproteinwasresolvedandblottedfor-actintoensuretheequalloading.Theexperimentswereperformedthrice.Resultsfromonerepresentativeexperi-mentareshown.ThedensitometricvaluesrepresentmeanSEM.*0.05,**0.01,***0.001.D-Units,den-sitometricunits.TheJournalofImmunology observationssuggestthatTLR1andTLR2promoteL.majorfection,whereasTLR6playsanantileishmanialrole.BecauseTLR2–TLR6associationwasreducedinL.major-fectedmacrophages,butrestoredbyitsligandBPPcysMPEG,wetestedtheeffectofitsligandonparasitegrowthinBALB/c-derivedperitonealmacrophagesandcomparedwiththeeffectsofPam,aligandforTLR1–TLR2heterodimer,andPGN,aligandforTLR2ontheinfection.ItwasobservedthatPamPGNincreasedtheinfection,butBPPcysMPEGreducedthein-fectioninadose-dependentmanner(Fig.2D).Corroboratingwiththisobservation,amongthesethreeligands,BPPcysMPEGappearedtobethestrongestinducerofIL-12(Fig.2E),ahost-protectivecytokine(13,22);iNOS(Fig.2F),theenzymethatcatalyzesthegenerationofNO,whichkills(43);andTLR9(Fig.2G),whichisalsoreportedtoplayahost-protectiverolebyinducingIL-12(44).Thus,BPPcysMPEGisobservedtoenhancekeyhost-protectivefunctionsinvitro.BPPcysMPEGactivateshigherp38MAPKandATF2BecausePam,PGN,andBPPcysMPEG,theligandsforTLR1,TLR2,andTLR6,respectively,induceddifferentialef-fectorfunctionsinmacrophages,weexaminedthesignalingin-ducedbytheseligands.AsTLR2signalsthroughtheadaptormoleculesMyD88,TIRAP,andTRAF6(44),TLR2wasimmu- FIGURE2.TLR1andTLR2increased,whereasTLR6reducedL.majorinfection.BALB/c-derivedthioglycolate-elicitedperitonealmacrophagesweretransducedwithlentivirallyexpressedTLR1,TLR2,TLR6shRNA-Lv,orcontrolshRNA-Lv(twotransductionunitspercell)for48h.()UntransducedorTLR-speciclentivirallytransducedcellswereinfectedwithL.majorfor6h.Extracellularparasiteswerewashedout,andparasiteburdenwasassessedbyGiemsastaining.RT-PCRwasperformedtochecktheinhibitionofTLR1,TLR2,andTLR6expressionbyrespectiveTLRshRNALvorcontrolshRNALv,transducedinmacrophages(lowerpanel).()InternalizationofL.majorparasitesinperitonealmacrophageswaschecked1hpostinfection.BALB/cmice(=7)wereinjecteds.c.with5transductionunits(in50lHBSS)TLR1,TLR2,TLR6shRNALv,orcontrolshRNALvintohindfootpadandwereinfecteds.c.with2L.majorinsamehindfootpad3dafterlentiviraltreatment.SomemiceleftuntreatedwereinfectedwithL.majorwerekeptascontrols.Footpadthicknesswasmeasuredweeklytoassesstheseverityofdisease(upperpanel).Fiveweeksaftertheinfection,miceweresacricedandparasiteswereenumeratedfromthepopliteallymphnodecells(lowerpanel),asdescribedearlier.Theexperimentswererepeatedtwice.TheerrorbarsrepresentmeanThioglycolate-elicitedBALB/c-derivedmacrophageswereeitherleftuninfected(UIM)orinfected(IM)withL.majorpromastigotesataratioof1:10for12h,followedbywashingoutextracellularparasitesandincubationforanadditional12h.ThecellsweretreatedwiththeindicateddosesofPamC),PGN,orBPPcysMPEG(BPP)foranother48h.ParasiteburdenwasassessedbyGiemsastaining.)IL-10andIL-12(p70)fromtheculturesupernatantsofuninfectedandL.major-infectedmacrophagestreatedwiththeindicateddosesofPC,PGN,orBPPwereassessedbyELISA.ThesignicancetestswereperformedtocomparetheIL-12productionsinducedbyBPPcysMPEGandtheothertwoligands.()Uninfectedand64-hL.major-infectedmacrophagesweretreatedwiththeindicateddosesofPC,PGN,orBPPforanother8h,followedbyassessmentofiNOSexpressionbyRT-PCR()andTLR9expressionbyRT-PCRandreal-timePCR().Forreal-timePCR,dosesusedwerePC(100ng/ml),PGN(10g/ml),orBPP(100ng/ml)(rightpanel).Theexperimentswereperformedthreetimes.TheerrorbarsshowmeanSEM.*0.01,***3636ANTILEISHMANIALFUNCTIONOFBPPcysMPEG noprecipitatedfromthePam-,PGN-,andBPPcysMPEG-stimulatedmacrophages,followedbyimmunoblottingforMyD88,TIRAP,andTRAF6.WeobservedthatTLR2recruitedmoreMyD88inresponsetoPGNorBPPcysMPEGthantoPam

CSKonlyBPPcysMPEGdrasticallyincreasedtheTIRAPrecruitmenttoTLR2,whereasPamCSKshowedatendencytoincreasedre-cruitmentofTRAF6toTLR2(Fig.3A).Assignalsfrommembranereceptorsaretakentothenucleusviap38MAPKandERK1/2(45,46)andtranscriptionfactors,itwaspossiblethattheobserveddifferencesbetweenTLR2ligandsmightoccurduetodifferentialphosphorylationandactivationofthesesignalingintermediates.WeobservedthatPam,PGN,andBPPcysMPEGalsodif-feredintheirabilitytoactivatep38MAPK,ERK1/2,andtranscrip-tionfactors.ComparedwithPamandPGN,BPPcysMPEGactivatedmorep38MAPKandATF2thanERK1/2andELK1(Fig.3B).ItwasreportedthattheL.donovani-infectedTHP-1cells,amacrophage-likecellline,respondedtoPamCSKbyincreasinghigherERK1/2andIL-10production(14).Takentogether,theseobservationssuggestthatBPPcysMPEGsignalsthroughp38MAPKandATF2,whereasPamandPGNsignaledprimarilythroughERK1/2andELK1.TheexpressionofTLR1,TLR2,andTLR6;theassociationsbetweenthesethreeTLRs;andtheeffectsoftherespectiveTLRshRNAsandligandsonvariouseffectorfunctions,includingtheL.majorinfection,identifyTLR2–TLR6anditsligandBPPcysMPEGasthehost-protectivereceptor–ligandpair.Therefore,wefurtherassessedthemechanismofthehost-protectivefunctionofBPPcysMPEG.BPPcysMPEGinducesTcell–mediatedTh1responsebothinvitroandinasusceptiblehostAsBPPcysMPEGshowedantileishmanialeffectsinmacrophagesinvitro,wetesteditseffectontheinductionofTcell–mediatedhostprotectioninamacrophage–Tcellcoculturesystem.WhenL.majorinfectedmacrophageswerecoculturedwiththelymphnodeTcellsfromL.major-infectedBALB/cmice,theamastigotenumbersin-creasedinmacrophages,butBPPcysMPEGtreatmentreducedtheparasitecount(Fig.4A).AlowerdoseofBPPcysMPEGincreasedbothIL-4andIFN-production,butIL-4productionwasreduced,whereasIFN-productionincreased,withthehigherdosesofBPPcysMPEG(Fig.4B);aresultanteffectofthesechangeswassignicantlyenhancedIFN-production(Fig.4B,inset).Theseob-servationssuggestedthatBPPcysMPEGenhancedtheantileish-manialeffecteveninpresenceofthedisease-exacerbatingTcells,whichpredominatedinthedraininglymphnodeofaL.majorinfectedsusceptiblehost.Therefore,wenextexaminedtheanti-leishmanialeffectofBPPcysMPEGtreatmentonL.majorinfectioninBALB/cmice,whichreceivedthreeBPPcysMPEGtreatmentsonalternatedaysstartingfromthethirddaypostinfection.Weob-servedasignicantreductioninparasiteloadinthelymphnodeoftheBPPcysMPEG-treatedBALB/cmice(Fig.4C,ThisantileishmanialeffectwasaccompaniedbyheightenedIFN- FIGURE3.BPPcysMPEGactivateshigherp38MAPKandATF2.()Thioglycolate-elicitedBALB/c-derivedmacrophagesweretreatedwiththein-dicateddosesofPam,PGN,orBPPfor15minandlysed,andanequalamountofproteinwasimmunoprecipitatedwithanti-TLR2AbandblottedwithAbsagainstMyD88,TIRAP,andTRAF6.Densitometricvalueswerenormalizedagainstthevaluesof-actin(lowerpanel).Densitometryshownisthecollateddatafromtwoexperiments.ErrorbarsshowmeanSEM.()Celllysateswereimmunoblottedforp-p38MAPK,p-ERK1/2,p-IKKB,p-ATF2,p-ELK1ortotalp38MAPK,ERK1/2,IKK,NF-B,ATF2,andELK1.Densitometricvalueswerenormalizedagainstthevaluesofrespectivetotalproteins,asindicated(lowerpanel).Thedatafromtworepeatexperimentsareshowninthedensitometry.Theratiosofp-p38/ERK1/2orp-ATF2/p-ELK1werecalculatedfromthemeanvaluesoftheirrespectivelanes.TheJournalofImmunology production,butloweredIL-4production(Fig.4D).AnapparentdifferenceintheproleofBPPcysMPEG-inducedchangesinIL-4andIFN-productionsinvitro(Fig.4B)andinBALB/cmice(Fig.4D)wasreconciledastheratioofIFN-toIL-4(Fig.4B,),whichshowedadrasticincreaseinIFN-productioncom-paredwithIL-4.AnotherplausibleexplanationfortheapparentdiscrepancyismorecomplexinteractionbetweenAPCsandTcellsinvivothanthatobservedindenedmacrophage–Tcellco-culture.Thus,theTLR2/TLR6ligand,BPPcysMPEG,establisheshost-protectiveTcellstoexertitsantileishmanialfunctions.TLR6shRNAsignicantlyabrogatedtheantileishmanialeffectsofBPPcysMPEGBecauseBPPcysMPEG,aTLR2/6ligand,increasedTLR9ex-pression,westudiedthesynergismbetweenTLR2/TLR6andTLR9.BALB/c-derivedthioglycolate-elicitedmacrophagesweretrans-ducedwithTLR6shRNAorcontrolshRNA-expressinglentivirus,followedbyL.majorinfectionandtreatmentwithBPPcysMPEGandCpG,orleftuntreatedascontrol.ItwasobservedthatTLR6shRNA-treatedcellsreversedtheantileishmanialresponsetoBPPcysMPEGorCpGaloneorincombination(Fig.5A).TheTLR6shRNA-transducedmacrophagesproducedlessIL-12,buthigherIL-10inresponsetotheseligands(Fig.5B).Next,BALB/cmiceweretreatedwithlentivirallyexpressedTLR6shRNAorcontrolshRNA,followedby

L.majorinfectionandtreatmentwithBPPcysMPEGorCpG,orwereleftuntreated.ItwasobservedthatTLR6shRNAsignicantlyabrogatedtheantileishmanialeffectofBPPcysMPEGandCpG(Fig.5C).WhereasBPPcysMPEGandCpGincreasedproductioninthesemice,TLR6shRNAreducedIFN-production,butincreasedIL-4production(Fig.5D).Thesere-sultsclearlysuggestedthattheobservedantileishmanialeffectsofBPPcysMPEGwereindeedduetoTLR6andthatCpGandBPPcysMPEGsynergizetoeffecttheobservedantileishmanialfunctions.BPPcysMPEGreducedTregcellsincocultureswithmacrophagesorDCsTregcellspromotesurvivalandgrowthinasuscepti-blehost,asthenumbersofTregcellsincreaseinprogressivein-fection(24–28),butdepletionofTregcellsreducesthedisease(26,47).AsBPPcysMPEGreducedL.majorinfectioninBALB/cmice(Fig.4C),weexaminedwhetherBPPcysMPEGwouldde-creasethenumberofTregcells.Totestthehypothesis,weco-culturedtheBPPcysMPEG-treatedoruntreated,L.majormacrophagesorbonemarrow–derivedDCwithCD4Tcellsfromthedrainingpopliteallymphnodesof21-dL.major-infectedBALB/cmice.WeobservedthattheBPPcysMPEGtreatmentreducedthenumberofCD3cellsinbothmacrophage–CD4TcellandDC–CD4Tcellcocul-tures(Fig.6)andreducedthenumberofIL-10cellsinDC–CD4Tcellcocultures(Fig.6B).TheculturesupernatantswerecollectedfromDC–CD4Tcellcocultures,andthelevelsofIL-12,IL-10,IL-4,andIFN-wereassessed.WeobservedthatareducednumberofTregcellswasassociatedwithhigherIL-12andIFN- FIGURE4.BPPcysMPEGelicitshost-protectiveimmuneresponse.BALB/c-derivedperitonealmacrophageswereinfectedwithL.majorfor72h.Afterwashingouttheextracellularparasites,macrophagesweretreatedwiththeindicateddosesofBPPandwerecoculturedwiththeCD4TcellsisolatedfromthelymphnodesofL.major-infected(21-d–infected)andnaiveBALB/cmiceat1:3ratio(M:Tcells).()After72-hinfection,macrophageswerewashed,xed,stainedwithGiemsastain,andevaluatedfortheamastigotesper100macrophages.()IL-4andIFN-wereassessedfromculturesupernatants,asdescribedinMaterialsandMethods)FoldoverchangeincytokineIL-4andIFN-wasdeterminedfromthevaluesofBPPtreatedagainstthevaluesofinfectedmacrophagescoculturedwithinfectedTcells.Datashownaretherepresentativeofoneexperiment,repeatedtwice.ErrorbarsshowmeanSEM.()BALB/cmicewereinfecteds.c.inhindfootpadwith2L.majorpromastigotes.MiceweretreatedwithBPP(1intotheL.major-infectedfootpadalternativelyfor3dstartingfromthethirddaypostinfectionorwereinjectedwithsterilePBSascontrol.Diseaseprogressionwasscoredweeklybyevaluatingthenetfootpadswelling(thethicknessoftheuninfectedleftfootpadsubtractedfromtheinfectedrighfootpad)bydigitalmicrometerfor5wk.()Miceweresacriced5wkaftertheinfection,andparasiteburdeninlymphnodecellswasassessed.(Lymphnodecellsfrommice,asindicated,ineachgroup(=7)werepooledandstimulatedwithanti-CD3(0.5g/ml)andanti-CD28(2g/ml)for60h.CulturesupernatantswereassessedfortheproductionofthecytokinesIL-4andIFN-,asdescribedinMaterialsandMethods.Theexperimentwasrepeatedtwice.Datashownaretherepresentativeofoneexperiment,anderrorbarsshowthemeanSEM.*0.05,**3638ANTILEISHMANIALFUNCTIONOFBPPcysMPEG butreducedIL-10andIL-4levels(Fig.6B,),suggestingthatBPPcysMPEG-inducedhostprotectionwasassociatedwithde-creasedTregcellsandincreasedIL-12induction.BPPcysMPEG-inducedhostprotectionisIL-12dependentBecauseBPPcysMPEGincreasedtheproductionofIL-12thatplayedahost-protectiveroleagainstinfection(13,22),theuninfectedandL.major-infectedmacrophagesfromIL-12–decientorBALB/cmiceweretreatedwithBPPcysMPEG.ItwasobservedthatBPPcysMPEGreducedthenumberofamastigotesinwild-type,butnotIL-12–decientmacrophages(Fig.7A),com-mensuratingwithitseffectoniNOSexpression(Fig.7B).Cor-roboratingwiththeseobservations,theantileishmanialeffectofBPPcysMPEGwassignicantlyreducediniNOS-decientmice(Fig.7C).Togethertheseobservationsindicatethattheanti-leishmanialeffectofBPPcysMPEGispartlyiNOSmediated.ToconrmtheroleofIL-12inBPPcysMPEG-inducedantileishmanialdefense,wetestedtheeffectsofBPPcysMPEGtreatmentonparasiteload,IL-4/IFN-production,andTregcellnumbersinIL-12–decientandwild-typeBALB/cmice.WeobservedthatBPPcysMPEGtreatmentreducedtheseverityofthedisease(Fig.7D)andparasiteloadinBALB/cmice,butnotintheIL-12–decientmice(Fig.7E);theantileishmanialeffectofBPPcysMPEGwasaccompaniedbyhighIFN-,butlessIL-4productions(Fig.7E)andreducedTregcellnumbers(Fig.7F)inBALB/c,butnotinIL-12–decientmice.TheseobservationsindicatethattheBPPcysMPEG-inducedantileishmanialdefenseisIL-12depen-dent,butTregsensitive. FIGURE5.TLR6shRNAsignicantlyabrogatedtheantilei

shmanialeffectsofBPPcysMPEG.Thioglycolate-elicitedBALB/c-derivedmousemacro-phagesweretransducedwithTLR6shRNA(TLR6Lv)orcontrolshRNA(controlLv)lentiviralparticles.Forty-eighthourslater,lentivirallytransducellswereeitherleftuninfectedorinfectedwithL.majorataratioof1:10,followedbytreatmentwithBPP(50ng/ml)andCpG(0.12M)foratotal72-hinfection.()CellswerestainedwithGiemsa,andamastigoteswereenumerated.TheamastigotenumberscountedinthecontrolCpGorcontrolshRNA-Lvwere565.527.5and684.519.5,respectively.()CulturesupernatantscollectedwereassessedforIL-10andIL-12p70byELISA.RatiobetweenIL-12:IL-10wasdeterminedfromtherespectivetreatments.TheIL-12:IL-10ratiosinthecontrolCpG-andcontrolshRNA-Lv–treatedcultureswere0.03and0.250.05,respectively.()TLR6lentivirusreversedtheCpG-inducedantileishmanialimmuneresponseinsusceptibleBALB/cmice.SomegroupsofBALB/cmiceweretreatedwithTLR6shRNALv,2dlaterinfectedwithL.major,andtreatedwithBPP(1g/mouse)andCpG(10mouse)togetheroraloneonalternating3d,startingfrom2dpostinfection.()Parasiteloadwasdetermined5wkaftertheinfection.NumberofparasitesenumeratedfromcontrolCpG-orcontrolshRNA-Lv–treatedmicewere(69.75and(88.333,respectively.(ImmunoblotanalysisofTLR6expressionfromfootpadlesion(FP)andlymphnode(LN)ofL.major-infectedBALB/cmicetreatedwithcontrolshRNA-LvandTLR6shRNA-Lv5wkafterL.majorinfection.()Totallymphnodecellsfromvariousmicegroupswerestimulatedwithanti-CD3Ab(0.5andanti-CD28Ab(2g/ml)for60h.CulturesupernatantsfromdifferentgroupswereassessedforIL-4andIFN-productionbyELISA.ThevaluesforIL-4:controlCpG(478.533.5pg/ml),controlshRNA-Lv(913.08.0pg/ml);thevaluesforIFN-:controlCpG(38412pg/ml),controlshRNA-Lv90.5pg/ml).Theexperimentswereperformedtwice,andonerepresentativedataareshown.ErrorbarsrepresentmeanSEM.*0.05,**0.01,***TheJournalofImmunology BPPcysMPEGinduceshost-protectiveprophylacticantileishmanialeffectsAsBPPcysMPEGreducedparasiteload,accompaniedbyanIFN-–dominatedresponseininvitroandinBALB/cmice,weex-aminedtheadjuvanticityofBPPcysMPEGinestablishinghost-protectiveantileishmanialmemoryresponse.TheBPPcysMPEGtreatmentduringprimingwiththexedL.majorresultedinsignicantlyreducedparasiteloadinthesemice,comparedwiththeuntreatedcontrolsorunprimedcontrols,fol-lowingachallengeL.majorinfection(Fig.8A).TheprotectionwasaccompaniedbyhighIFN-,butlowIL-4(Fig.8B).Theprotec-tionwasassociatedwithreducedTregcellsintheBPPcysMPEG-treatedgroup(Fig.8C).TheseobservationstogetherindicatethatBPPcysMPEGpossessesastrongadjuvanticityagainstL.majorinfection.Thisworkdocumentsseveraluniqueobservations.First,theexpressionoftheTLR2associates,TLR1,TLR2,andTLR6,andtheirdimerizationsisdifferentiallymodulatedbyL.majortioninmacrophages.ThereducedTLR2–TLR6associationinL.major-infectedmacrophageswasrestoredbyitssyntheticli-gandBPPcysMPEG.Second,TLR1orTLR2silencingreducedL.majorinfection,butTLR6silencingincreasedtheinfection.Conversely,theligandsPamandPGNexacerbatedtheinfec-tion,butBPPcysMPEGreducedtheinfectioninmacrophages.Third,thesehost-protectiveeffectsofBPPcysMPEGwereaccompaniedbyincreasedTLR9expression,enhancedIL-12production,andhigheriNOSinduction.Fourth,asapossiblemechanismforitshost-protectiveeffects,BPPcysMPEGinducedleishmanicidalactivitiesinTcellsfromtheL.major-infectedmiceandreducedTregcellnumbers.Thesehost-protectiveeffectswereTLR6mediated,IL-12dependent,iNOSdependent,andTregsensitive.Finally,targetingTLR2–TLR6ledtosignicantreductioninparasiteloadinboththerapeuticandprophylacticmodes,accompaniedbymodulationofTcellresponses.Thus,theroleforTLR2inselectivehetero-dimerizationswitheitherTLR1orTLR6inL.majorassumessignicance,whichwasindicatedbythestronganti-leishmanialeffectoftheTLR2–TLR6ligand,BPPcysMPEG,butnotbytheligandoftheTLR1–TLR2heterodimer.Altogether,toourknowledge,thisistherstdemonstrationofTLR2functionalduality,contingentuponitsdimerizationwitheitherTLR1orTLR6.TheincreasedTLR1–TLR2orTLR2–TLR2association,butreducedTLR2–TLR6association,impliesselectivetargetingofTLR2partnersbytheparasite.Itispossiblethatdueto-inducedchangesinmembraneanisotropyoftheinfectedmacrophages(48),theinteractionbetweenthehydrophobicdomainsoftheseTLRs(49)isdifferentiallyaffected,resultinginalteredassociationsinL.major-infectedmacrophages.Incontrast,TLR1orTLR2silencingreduces,butTLR6silencingincreases,L.majorinfection.ItispossiblethatsilencingofoneTLRalterstheothertwoTLRs’heterodimerizingpropensity.Thus,theinitialdifferencebetweenTLR1–TLR2,TLR2–

TLR2,andTLR2–TLR6 FIGURE6.BPPcysMPEGtreatmentdepletesTregcellsinvitro.Thioglycolate-elicitedperitonealmousemacro-phagesorbonemarrowDCsderivedfromBALB/cmice,infectedwithL.majorpromastigotes,andtreatedwithindicateddosesofBPPfor24handstimulatedwithcrudesolubleleishmaniaAgs(CSA).CD4TcellswereisolatedfrompoplitealdraininglymphnodesofL.majorBALB/cmice21dpostinfection.CD4TcellswereusedforthecoculturewithmacrophagesorDCsfromtheabove-describedculturefor60h.UpperpanelofdatarepresentsthegatingstrategyfortheFACSanalyses.(CoculturedmacrophageCD4TcellswereusedtochecktheCD3Foxp3cells.(CoculturedDCCD4TcellswereusedtocheckFoxp3andIL-10cellsinCD3cells.()CulturesupernatantswerecollectedfromthecoculturedDCCD4TcellsandassayedforIL-10,IL-12,IL-4,andIFN-cytokines,asdescribedinandMethods.FoldoverchangeatthelevelofcytokineswasdeterminedfromthevaluesofBPP-treatedDCCD4cocultureagainsttheuntreatedDCCD4Tcells.3640ANTILEISHMANIALFUNCTIONOFBPPcysMPEG dimerscanbeinducedeitherbyalterationofthephysicalprop-ertiesofthemacrophagemembraneorbyalterationoftheratiosamongthesethreeTLRs.Throughacascadeofevents,theseinitialchangesledtocontrastingoutcomesofL.majorThecascadeofeventsmayberepresentedbythedifferentialsignalingtriggeredbytheligand-inducedhomotypicorheterotypicassociationofTLR2.Forexample,PamandPGNtriggerprimarilyERK1/2andELK1activation,whereasBPPcysMPEGinducesp38MAPKphosphorylation,leadingtoactivationofC-Jun(50)thatcandimerizewiththeBPPcysMPEG-activatedATF2toinduceinammatoryresponse(51,52).Incontrast,ERK1/2activatesELK1viaAP-1,whichbindstoactivatedc-Fos(53,54).HowthesesignalingpathwaysarereciprocallyrelatedtotheTLR1–TLR2–orTLR2–TLR6–inducedcontrastingoutcomes—diseasepromotionorhostprotection—ofL.majorinfectionawaitselucidation.Onemechanismofthehost-protectiveantileishmanialfunctionofBPPcysMPEGisrelatedtotheenhancedexpressionofTLR9,IL-12,andiNOS.iNOScatalyzestheformationofNO(11,43),whichkills(11,32).AsTLR6silencingsignicantlycompromisedtheantileishmanialfunctionofBPPcysMPEG,thefailureofIL-12–decientmacrophagestoreduceL.majortioninresponsetoBPPcysMPEGtreatmentsuggestsasignicantroleforautocrineIL-12inTLR2/TLR6-dependentkilling.Finally,asTLR9recognizesL.majorDNAandinducesIL-12production(41),itispossiblethattheleishmanialDNA-activatedTLR9-inducedIL-12mayexertantileishmanialfunc-tionsinmacrophages.AsTcellswereabsentinalltheseassays,thismodeofantileishmanialfunctionofBPPcysMPEGcanbeattributedtoinnateimmunemechanisms.Besidestheinnateimmunemechanisms,BPPcysMPEGisalsoshowntoaffectTcellsinthecoculturewithmacrophagesandDCs.ItispossiblethattheTcell–dependentantileishmanialfunc-tionsofBPPcysMPEGbuildupfromitsinnateimmuneeffects.Forexample,theTLR2–TLR6–activatedorTLR9-activatedIL-12cansetaTh1bias,asBPPcysMPEGfailedtoexertitsanti-leishmanialeffectsinIL-12–decientmice.Inadifferentstudy,BPPcysMPEG,alongwithIFN-,isshowntoreducetheallergicairwayinammation(55).BecausetheallergicmanifestationsintheairwaymayindicateexaggeratedTh2response(21),there-ductionintheairwayallergybyBPPcysMPEGimpliespossibleantagonismofTh2differentiation.AsTh2responsesexacerbateL.majorinfection(56),itispossiblethatBPPcysMPEGexertstherapeuticandprophylacticeffectsbyfacilitatingTh1response(57).TheenhancedproductionsofIL-12inmacrophagesinvitroandofIFN-inBPPcysMPEG-treatedmiceindicatetheTh1response-facilitatingroleforTLR2/TLR6heterodimersandtherebyitshost-protectiveroleagainstL.majorinfection.Incontrast,oneofthedisease-promotingTcellsinL.majorfectionisTregcells,asthereductioninparasiteburdenhasbeencorrelatedwithreducednumberofTregcells(24–28).ToexploretheprophylacticroleforTLR2–TLR6heterodimer,weprimedBALB/cmicewithxedL.majorandBPPcysMPEG,followedbychallengeinfectionwithL.major.BPPcysMPEG,butnotPam,theligandfortheTLR1–TLR2heterodimer,establishedahost-protectiveantileishmanialimmuneresponse,accompanied FIGURE7.BPPcysMPEG-inducedhostprotectionagainstL.majorinfectionisIL-12dependent.Thioglycolate-elicitedperitonealmacrophagesderivedfromBALB/corIL-12knockout(IL-12KO)micewereeitherinfectedwithL.majorpromastigotesorleftuninfected.()Twenty-four-hour–infectedmacrophageswerestimulatedwithBPPforanother48h,cellswerestainedwithGiemsastain,andamastigoteswereenumerated.()MacrophageswerestimulatedwithindicateddosesofBPPfor8h.CellswerelysedforthesynthesisofcDNAandwereusedfortheamplicationofiNOSbyreal-timePCR.Datashownrepresenttherelativefoldchangebetweenuntreatedandtreatedcells.()Macro

phagesderivedfromC57BL/6oriNOSknockout(iNOS-KO)micewereinfectedwithL.majorpromastigotesandweretreatedwiththeindicateddosesofBPP.Seventy-twohourspostinfection,amastigoteswerecounted.()BALB/candIL-12KOmicewereinfectedwith2L.majorpromastigotes.Twodayspostinfection,miceweretreatedwithBPPg/mouse)alternativelyfor3d.()Thefootpadthicknesswasmeasuredweekly.()Fiveweeksaftertheinfection,miceweresacricedandparasiteswereenumeratedfromthepopliteallymphnodecells.Lymphnodecellspooledfrommice(=7)ofeachgroupwereculturedintoanti-CD3Ab(0.5ml)-precoatedplatefor60h.Supernatantswerecollected,andcytokineELISAwasperformedforIFN-andIL-4(rightpanel).()Foxp3TregcellswereanalyzedbyFACSgatedonCD3upperpanelofdatarepresentsthegatingstrategy.Datashownarerepresentativeofoneexperiment,andtheexperimentswererepeatedtwice.ErrorbarsrepresentmeanSEM.*0.05,**0.01,***TheJournalofImmunology bysignicantlyreducedTregcells.BecauseTregcelldifferentia-tiondependsonTGF-(58),itispossiblethatPaminducedmuchhigherTGF-thanthatinducedbyBPPcysMPEG(datanotshown).Thus,theeffectsofBPPcysMPEGonTregcellsrevealauniquefacetofTLR2-regulatedantileishmanialimmuneresponse.ThedecreasedTLR2–TLR6associationinL.major-infectedmacrophagesindicatesaparasite-devisedimmuneevasionstrategyunfoldinganintriguingTLR2-targetedhost–parasiteinteractionthathasnotbeenreportedearlier.Incaseofintracellularpathogens,suchasLeishmaniaSalmonellaListeria,andMycobacterium,thepathogensneedtosurvivewithinthehostcellsandalsotoevadeadaptiveimmuneresponsesthatcaneliminatethesepathogens.Therefore,itwaslogicaltohypothesizethat,toensuretheirintra-cellularsurvival,thepathogenwouldmodulatetheexpressionoftheintracellularTLRsasaconsequenceoftheirinitialinterac-tionswiththecellsurfaceTLRs.Indeed,theligandsfortheTLR1–TLR2andTLR2–TLR6heterodimersdifferentiallymodulatedtheexpressionofTLR9,anintracellularTLR,clearlysuggestingthesignicanceoftheselectiveTLR2heterodimerizationwithTLR1orTLR6.Asanoveltherapeuticstrategy,wedemonstratethatacombinatorialtargetofTLR2,TLR6,andTLR9canindeedelicitaneffectivehost-protectiveantileishmanialimmuneresponse.DisclosuresC.A.G.andT.E.arenamedasinventorsinpatentscoveringtheimmunemodulatorypropertiesofBPPcysMPEG.References1.Matsumoto,C.,T.Oda,S.Yokoyama,T.Tominari,M.Hirata,C.Miyaura,andM.Inada.2012.Toll-likereceptor2heterodimers,TLR2/6andTLR2/1induceprostaglandinEproductionbyosteoblasts,osteoclastformationandinamma-toryperiodontitis.Biochem.Biophys.Res.Commun.428:110–115.2.Takeda,K.,andS.Akira.2005.Toll-likereceptorsininnateimmunity.17:1–14.3.Sheedy,F.J.,andL.A.O’Neill.2007.TheTrollinToll:MalandTramasbridgesforTLR2andTLR4signaling.J.Leukoc.Biol.82:196–203.4.Jin,M.S.,S.E.Kim,J.Y.Heo,M.E.Lee,H.M.Kim,S.G.Paik,H.Lee,andJ.O.Lee.2007.CrystalstructureoftheTLR1-TLR2heterodimerinducedbybindingofatri-acylatedlipopeptide.130:1071–1082.5.Kang,J.Y.,X.Nan,M.S.Jin,S.J.Youn,Y.H.Ryu,S.Mah,S.H.Han,H.Lee,S.G.Paik,andJ.O.Lee.2009.RecognitionoflipopeptidepatternsbyToll-likereceptor2-Toll-likereceptor6heterodimer.31:873–884.6.Akira,S.,K.Takeda,andT.Kaisho.2001.Toll-likereceptors:criticalproteinslinkinginnateandacquiredimmunity.Nat.Immunol.2:675–680.7.Medzhitov,R.,andC.A.Janeway,Jr.1997.Innateimmunity:impactontheadaptiveimmuneresponse.Curr.Opin.Immunol.9:4–9.8.Mosser,D.M.,andJ.P.Edwards.2008.Exploringthefullspectrumofmac-rophageactivation.Nat.Rev.Immunol.8:958–969.9.Sacks,D.,andC.Anderson.2004.Re-examinationoftheimmunosuppressivemechanismsmediatingnon-cureofinfectioninmice.Immunol.Rev.201:225–238.10.Frankenburg,S.,V.Leibovici,N.Mansbach,S.J.Turco,andG.Rosen.1990.Effectofglycolipidsofparasitesonhumanmonocyteactivity:in-hibitionbylipophosphoglycan.J.Immunol.145:4284–4289.11.Srivastava,S.,S.P.Pandey,M.K.Jha,H.S.Chandel,andB.Saha.2013.LeishmaniaexpressedlipophosphoglycaninteractswithToll-likereceptor(TLR)-2todecreaseTLR-9expressionandreduceanti-leishmanialresponses.Clin.Exp.Immunol.172:403–409.12.Vargas-Inchaustegui,D.A.,W.Tai,L.Xin,A.E.Hogg,D.B.Corry,andL.Soong.2009.DistinctrolesforMyD88andToll-likereceptor2duringLeishmaniabraziliensisinfectioninmice.Infect.Immun.77:2948–2956.13.Mathur,R.K.,A.Awasthi,P.Wadhone,B.Ramanamurthy,andB.Saha.2004.ReciprocalCD40signalsthroughp38MAPKandERK-1/2inducecounteractingimmuneresponses.Nat.Med.10:540–544.14.Chandra,D.,andS.Naik.2008.Leishmaniadonovaniinfectiondown-regulatesTLR2-stimulatedIL-12p40andactivatesIL-10incellsofmacrophage/monocyticlineagebymodulatingMAPKp

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