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BlockadeofIL-10signallingclearschronicviralandbacterialinfections.ImmunizationtogetherwithblockadeofIL-10signallingorrelativelylowlevelofIL-10furtherenhancesviralandbacterialclearance.IL-10functionsthroughbindingtointerleukin10receptor(IL-10R).HereweshowedthatpeptidesP1andP2withthehydrophobicandhydrophilicpatternoftheIL10R-bindinghelixinIL-10couldbindwitheitherIL-10R1orIL-10,andinhibitinflammatorysignalswithlongdurationandnegligiblecytotoxicityinvitro.Furthermore,P2canenhanceantigenspecificCD8+TcellresponsesinmiceinducedbythevaccinebasedonalongpeptideofproteinE7inahumanpapillomavirustype16.
Editor:MassimilianoGaldiero,SecondUniversityofNaples,ITALY
Received:August10,2015;Accepted:April6,2016;Published:April21,2016
DataAvailability:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.
Competinginterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
Inthecurrentpaper,wedesignedtwopeptides(P1andP2)thatcaninhibitIL-10viastructure-basedanalysis,focusingonthehelixstructureoftheligandprotein(IL-10)withinthebindingareaofIL-10/IL-10R,aswellastheaminoacidpatternofthehelixsequence.Morespecifically,weobtainedourpeptidesbasedonphysio-chemicalpropertiesoftheIL10peptidesegmentsintheinterfaceoftheIL-10/IL-10Rcomplexstructure.InvitrotestsconfirmedtheusefulnessofthedesignedpeptidesininhibitingIL-10level;moresignificantly,theexvivoassayalsosuggestedthatonedesignedpeptidecouldenhancetheCD8+Tcellresponsesusingamousemodel.
Wepurchased6–8weeksoldadultfemaleC57BL/6(H-2b)micethatarespecificpathogenfree(SPF)fromtheAnimalResourceCentre,SunYat-SenUniversity,Guangdongprovince,ChinaandkeptthemunderSPFconditionswithirradiatedfoodandautoclavedwater,andwithcyclesoflightanddarkof12hoursatthecentre.Micewererandomlyseparatedintogroupsof3–5miceineachcage.Noanimalsbecamesickordiedpriortotheexperimentalendpoint.Themicewereeuthanizedwithcervicaldislocationaccordingtothehospital’sAECprotocol.AllexperimentswereapprovedbyandperformedincompliancewiththeguidelinesofFoshanFirstPeoplesHospitalAnimalExperimentationEthicsCommittee.
MurinemastcellMC/9celllinewaspurchasedfromATCC,USAandculturedfollowingtheprotocolsintheproductsheets.Briefly,MC/9cellswereculturedincompleteRPMI1640media(Gibco)supplementedwith10%heatinactivatedfetalcalfserum(FCS),100Uofpenicillin/mland100μgofstreptomycin/mlandwereculturedat37°Cwith5%CO2and5ngofmurineIL-4or1ngofhumanIL-10asrecommendedbyATCC,withorwithoutaddingP1,P2orP3respectively.MC/9cellproliferationwasdeterminedbyMTTassay(purchasedfromATCC,USA)followingtheinstructionofmanufacturer.
HumanmacrophagecelllineU937wasmaintainedincompleteRPMI1640media(Gibco)supplementedwith10%heatinactivatedFCS,100Uofpenicillin/mland100μgofstreptomycin/mlandwereculturedat37°Cwith5%CO2.
LongHPV16E7peptideGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR,andHPV16E7CTLepitopeRAHYNIVTF,OvaspecificCTLepitopeSIINFEKLweresynthesisedandpurifiedbyMimotopes(Melbourne,Australia).DesignedpeptidesP1,P2,P3andP4weresynthesizedbyGenicBioBiotech(Hongkong,China).Thepurityofthepeptideswasdeterminedbyreverse-phaseHPLCandwasfoundtobemorethan95%.Peptidesweredissolvedin0.5%DMSOinPBSand,ifnotusedimmediately,storedat-20°C.Lipopolysaccharide(LPS)andIncompleteFreund’sadjuvant(IFA)werepurchasedfromSigma.
RecombinantHumaninterleukin10receptoralphawaspurchasedfromCreativeBioMart,USA(Cat.NoIL10RA-212H),andwasre-suspendedinsterilizedMilliQwatertoaconcentrationof1μg/μLasstocksolution.RecombinantHumaninterleukin5receptoralphawaspurchasedfromGenscript,USA(Cat.NoZ03126-10),andwasre-suspendedinsterilizedMilliQwatertoaconcentrationof1μg/μLasstocksolution.
HumanIL10waspurchasedfromebioscience(Cat.No:BMS/346).MouseIL4waspurchasedfromebioscience(Cat.No:14–8041).
Anti-IL10receptor(1B1.3)monoclonalantibody(MAb)forexvivoimmunisationwaspurchasedfromBioXcell,USAandstoredat-80°Ctillfurtheruse.Anti-IL-10(Cat.506802),Anti-IL-10Rantibodies(Cat.308802)forinvitroexperimentswerepurchasedfromBioLedgend.PEconjugatedanti-IL10RantibodywaspurchasedfromBioLegend(Cat.308803).
InvitrostimulationofU937cells:2–5×105ofU937cellswereculturedin1mLofRPMIwith10%humanserumcontaining100Uofampicillinand100Uofstreptomycin.TheU937cellswereeitherunstimulatedorstimulatedwith4×103μMofLPS(Sigma,Cat.L3024-5MG)overnight,inthepresenceofdifferentconcentrationofP1,P2orP3oranti-IL-10(10μg/mL,~0.3μM)oranti-IL-10R(10μg/mL,~0.1μM)antibodies.Supernatantswerecollectedandstoredat-80°Ctilluse.
Isolationofperipheralbloodmononuclearcells:Heparinised(25IU/mL)peripheralbloodwastakenfrominformedindividuals,andperipheralbloodmononuclearcells(PBMCs)wereseparatedbydiscontinuousdensitygradientsofFicoll-Hypague(GEHealthcare)accordingtothemanufacturer'sprotocol.IsolatedPBMCswerewashedextensivelyusingRPMIwith10%ofhumanserum.ThisbioassaywascarriedoutstrictlyunderresearchethicsandapproachedbytheHumanEthicsCommitteeofthe1stPeople’sHospitalofFoshan,China.
InvitrostimulationofPBMCs:2–5×105ofPBMCswereculturedin1mLofRPMIwith10%humanserumcontaining100Uofampicillinand100Uofstreptomycin.ThePBMCswereeitherunstimulatedorstimulatedwith4×103μMofLPS(Sigma,Cat.L3024-5MG)overnight,inthepresenceofdifferentconcentrationofP1,P2orP3oranti-IL-10(10μg/mL,~0.3μM)oranti-IL-10R(10μg/mL,~0.1μM)antibodies.Supernatantswerecollectedandstoredat-80°Ctilluse.
7-AADassay:7-AADViabilityStainingSolutionwaspurchasedfromeBioscience(Cat.00–6993).Briefly,5μLof7-aadwereaddedto5×105cellsfor5minutes,and7-aadstainingwasmeasuredbyaBDAccuriflowcytometry.
HumanIL-10ELISAMAXDeluxe5plateswerepurchasedfromBiolegend(Cat.430604)andperformedfollowingthemanufacturer’sinstruction.HumanIL12p40andIL12p70ELISAkitswerepurchasedfromeBioscience,IL12p40orIL12p70levelsfromsupernatantscollectedfromstimulatedPBMCsweremeasuredbyELISAfollowingtheinstructionfrommanufacturer.
Groupsoffivetoeightmicewereimmunizeds.c.with50μglongHumanPapillomavirusType16(HPV16)E7peptide,or15μgofLPS,withorwithout250μgofanti-IL10RantibodiesdissolvedinPBS.InthecaseofimmunizationwithincompleteFreund'sadjuvant(IFA),thedissolvedlongHPV16E7peptidewasemulsifiedin50%(v/v)IFAbefores.c.vaccination.Thetotalinjectedvolumewas100μL/mouse.Micewerelightlyanaesthetizedwithisofluorane(Abbott,Maidenhead,U.K.)duringimmunization.
StatisticalanalysiswasperformedbythetwotailedStudent’stest.Survivalratecomparisonamongdifferentgroupswasperformedbylogranktest,byusingPrism5.0(GraphpadSoftware,SanDiego).ResultsareconsideredassignificantifPvalueislessthan0.05.
(A)MALDImassspectraofIL-10R1whenmixedwithP1,P2andP3asshown.OnlyP1andP2displayedapeakcorrespondingtothemassofthecomplexstructure.(B)AnoverlayofsensorgramsoftheSPRcompetitivebindingassayofpeptidesP1andP2.ComparedwithsensorgramswithonlyIL-10orthecorrespondingpeptideatthesameconcentration,alossintotalresponsewasobservedwhenP1orP2wereco-injectedwithIL-10.(C)IL-10Rexpressionlevelsin3×105U937withanti-humanCD210usingflowcytometry:stimulatedwithdifferentamountofLPSovernight;unstimulatedandstimulatedwithLPS(4×103μM),LPS(4×103μM)+P1(4.5μM),P2(4.1μM),P3(5.6μM)orP4(4.2μM),andLPS(4×103μM)+aIL10R1overnight,respectively;themeanfluorescenceintensity(MFI)resultofIL10-Rexpression.
(A)DifferentnumbersofmouseMC/9mastcellswereculturedinRPMI1640mediawith10%FCSand2×105μMofIL4for48hours,bluearrowindicatedthecellnumberschosenformaximalgrowthpotential.(B)1×104ofMC/9cellswereculturedeitherinmediawith2×105μMofIL4(media),orplus5×105μMofIL10(IL10only),orplus5×105μMofIL10togetherwithdifferentpeptides(P1,P2,P3andP4at10μg/mL,equalsto4.5,4.1,5.6and4.2μM,respectively)for48hoursbeforeMTTassaywasperformed.
SupernatantsweremeasuredinthepresenceofIL-10byELISA.TheconcentrationofLPSis4×103μM:(A)3×105humanU937wereeitherleftunstimulated(UN)orstimulatedwithLPS,LPS+0.3μMofanti-IL10(aIL10),LPS+0.1μMofaIL10R,LPS+P1,2,3and4at4.5,4.1,5.6,4.2μMovernight,respectively.(B)1×105ofU937cellsweretreatedwithLPSanddifferentconcentrationofP1overnight.(C)1×105ofU937cellsweretreatedwithLPSanddifferentconcentrationofP2overnight.(D)1×105ofU937cellswereunstimulatedorstimulatedwithLPS,LPS+aIL10,LPS+aIL10R,LPS+P1andLPS+P2for4,12,24and48hours.
P2increasesIL-12secretionbyLPSstimulatedPBMCs:(A)3×105humanPBMCswereeitherleftovernightunstimulated(UN)orstimulatedwithLPS(4×103μM),LPS+0.3μMofanti-IL10(LPS+aIL10),LPSandP1at9.0μM,LPSandP2at8.2μM,respectively.SupernatantsweremeasuredforthepresenceofIL-12p40byELISA.(B)1×105ofhumanPBMCswereeitherunstimulatedorstimulatedwithLPSorLPSwithP3,P4anddifferentconcentrationofP2overnight,IL-12p40fromsupernatantsweremeasuredbyELISA.(C)1×106PBMCswerestimulatedinthepresenceof100ng/mLofLPS,5μg/mLofP2orP3for72hours.Cellswerestimulatedwithstimulationcocktail(ebioscience)inthefinal6hoursandstainedwithanti-CD3,anti-CD8andintracellularlystainedwithIFNγ.FACSwereperformedandCD3+CD8+IFNγ+wereanalyzedwithFlowjo.
(A)5x105ofmousespleniccellswereeitherleftunstimulated,orstimulatedwith100ngofLPS,orsameamountofLPSandP2,P3andanti-IL-10Rantibodiesovernight.IL12p40fromsupernatantsweremeasuredbyELISAasdescribedinMaterialsandMethods.(B)FourC57BL/6micefromeachgroupwereprimedeitherwithHPV16E7peptide/MPLA/aIL10RantibodiesorwithHPV16E7/IFAwithP1,P2,P3,irrelevantpeptide(OVACTLepitope)orleftunimmunizedonday0,andthenboostimmunizedwithlongE7peptide/IFAonday7.Sixdaysafterfinalimmunization,spleenfromimmunizedmicewerecollected,singlespleencellsisolated,andculturedinthepresenceofaMHCIrestrictedHPV16E7specificpeptideRAHYNIVTFovernight,ELISPOTassayforIFNγwasperformedasdescribedinmaterialsandmethods.
Wereportedhereastructure-baseddesignfordiscoveringnewproteintopo-mimeticsthatinhibitsIL-10/IL-10Rinteraction.WedemonstratedthatdesignedpeptidesP1andP2,butnotcontrolpeptides,bindtoIL-10R1bydifferentinvitroassays.P1andP2inhibitthegrowthofIL-10dependingmurinemastcellMC/9,aswellasLPSmediatedIL-10productionbyhumanmacrophagecelllineU937,inadoseandtimedependentmanner.P2alsopromotesLPSmediatedIL-12productionbyhumanPBMCs.Moreover,P2enhancesCD8TcellresponsesinducedbyanE7peptidebasedvaccineforhumanpapillomavirus16.
Asaproofofconcept,inthiswork,weusedtheinvitroandexvivoassaystoshowthatthisparticularα-helixsegmentwith“HHPP”patterncouldbetargetedtodesignIL-10inhibitingpeptide.Thedesignedsequencesworthfurthervalidationandoptimisation.OtherpeptidesegmentsofIL-10orIL-10Rmightalsobeusedtodirectthedesignofinhibitors,however,thedeterminationofthekeystructuralcharacteristicwouldbevital.Inabroadercontext,theresultsfromthisstudyprovideideasintothedevelopmentofmuchsimplerpeptide-basedtherapeuticsthantheresource-consumingtechniquessuchphagedisplayandhumanizedmonoclonalantibodyproduction.Sincemoredisease-relatedproteinsandreceptorshavebeencharacterisedwithreliableandgenericstructuralinformation,thisdesignconceptcouldbeexpandedandutilisedinthedesignofproteinsurfacetopo-mimeticsforasimilarscenario.
OnlyP1andP2displaypeakscorrespondingtothemassofthepeptide-proteincomplexstructures(inthespectra,X=IL-10,B=P1andC=P2).
(TIF)
Nopeakcorrespondingtothemassofthepeptide-proteincomplexstructurewasfound.OnlypeaksofIL-4oligomerscanbeobserved(e.g.,21+denotestosinglychargeddimer).
Nopeakcorrespondingtothemassoftheprotein-peptidecomplexstructurewasfound(inthespectra,X,BandCdenotetoIL-10,P2andP4,respectively).P2andP4wouldoligomeriseunderthecondition,respectively.
SupernatantsweremeasuredforthepresenceofIL-10byELISA.TheamountofLPS(abbreviatedas‘L’whencoupledwithotherreagents)is4×103μM,3×105humanU937cellswereeitherleftunstimulated(UN,repeated)orstimulatedwithLPS(repeated),LPS+aIL10withdifferentconcentration,LPS+aIL10Rwithdifferentconcentrationovernight,respectively.
M2oftheXaxisisthe7aad+cells(deadcells),Yaxisrepresentsthecellnumbers.
(DOCX)
WegratefullythankDrAlunJones(InstituteforMolecularBioscience,theUniversityofQueensland)foradviceandassistancewiththeMALDIMS.ThisresearchwasundertakenwiththeassistanceofresourcesfromtheNationalComputationalInfrastructure(NCI),whichissupportedbytheAustralianGovernment.
Conceivedanddesignedtheexperiments:XLTWYZ.Performedtheexperiments:GNSCTWJZ.Analyzedthedata:XLTWYZYYZL.Contributedreagents/materials/analysistools:SFCBZKMSWMWYWYZ.Wrotethepaper:XLTWYZKMSW.
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