Suppression of Propionibacterium acnes Infection and the Associated Inflammatory Response by

18 Pages·2015·4.63 MB·English

RESEARCHARTICLE
Suppression of Propionibacterium acnes
Infection and the Associated Inflammatory
Response by the Antimicrobial Peptide P5 in
Mice
SunhyoRyu1,HyoMiHan1,PeterI.Song2,CherylA.Armstrong2☯*,YoonkyungPark1,3☯*
1 DepartmentofBiomedicalScience,ChosunUniversity,Gwangju,Korea,2 DepartmentofDermatology,
UniversityofColoradoDenverAnschutzMedicalCampus,Aurora,Colorado,UnitedStatesofAmerica,
a11111
3 ResearchCenterforProteineousMaterials,ChosunUniversity,Gwangju,Korea
☯Theseauthorscontributedequallytothiswork.
* [email protected](YP); [email protected](CA)
Abstract
OPENACCESS
Thecutaneousinflammationassociatedwithacnevulgarisiscausedbytheanaerobicbac-
Citation:RyuS,HanHM,SongPI,ArmstrongCA,
ParkY(2015)SuppressionofPropionibacterium teriumPropionibacteriumacnesthroughactivationoftheinnateimmunesystemintheskin.
acnesInfectionandtheAssociatedInflammatory Currentstandardtreatmentsforacnehavelimitationsthatincludeadverseeffectsandpoor
ResponsebytheAntimicrobialPeptideP5inMice. efficacyinmanypatients,makingdevelopmentofamoreeffectivetherapyhighlydesirable.
PLoSONE10(7):e0132619.doi:10.1371/journal.
Inthepresentstudy,wedemonstratetheprotectiveeffectsofanovelcustomizedα-helical
pone.0132619
cationicpeptide,P5,againstP.acnes-inducedinflammatoryresponsesinvitroandinvivo.
Editor:MauroPicardo,SanGallicanoDermatologic
ApplicationofP5significantlyreducedexpressionoftwoinflammatorycytokinesIL-8and
Institute,ITALY
TNF-αinP.acnes-treatedprimaryhumankeratinocytes,whereP5appearedtoactinpart
Received:March3,2015
bybindingtobacteriallipoteichoicacid,therebysuppressingTLR2-to-NF-κBsignaling.In
Accepted:June16,2015 addition,inamousemodelofacnevulgaris,P5exertedbothanti-inflammatoryandantimi-
Published:July21,2015 crobialeffectsagainstP.acnes,butexertednocytotoxiceffectsagainstskincells.These
resultsdemonstratethatP5,andperhapsothercationicantimicrobialpeptides,offerthe
Copyright:©2015Ryuetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative uniqueabilitytoreducenumbersP.acnescellsintheskinandtoinhibittheinflammation
CommonsAttributionLicense,whichpermits theytrigger.Thissuggeststhesepeptidescouldpotentiallybeusedtoeffectivelytreatacne
unrestricteduse,distribution,andreproductioninany
withoutadverselyaffectingtheskin.
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:ThisworkwassupportedbyaNational
ResearchFoundationofKorea(NRF)grantfunded Introduction
bytheKoreanGovernment(MEST;No.2011-
Acnevulgarisisamultifactorialinflammatorydisease,thesymptomsofwhichincludecome-
0017532)andGlobalResearchLaboratory(GRL)
dones,papules,pustules,nodules,cystsandpilosebaceousinflammation,oftenleadingtosig-
Grant(NRF-2014K1A1A2064460).Thefundershad
noroleinstudydesign,datacollectionandanalysis, nificantscaringanddisfigurementofthefaceanduppertrunk[1].Propionibacteriumacnes(P.
decisiontopublish,orpreparationofthemanuscript. acnes)playsakeyroleinthepathogenesisofacne.Thisubiquitousgram-positivebacteriumis
partofthenormalskinmicroflora,butispresentinabnormallyhighnumberswithinpilose-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. baceousfolliclesinpatientswithacne[2].Itiswidelyacceptedthatinflammatoryacnereflects
PLOSONE|DOI:10.1371/journal.pone.0132619 July21,2015 1/18
InflammatoryResponsebytheAntimicrobialPeptideP5
theimmuneresponseofthehosttoP.acnes,whichcanstimulatetheproductionofproinflam-
matorycytokinesandchemokines(e.g.,IL-8andTNF-α)byimmunesystemcells,therebytrig-
geringthegranulomatousreactionsofinflammatoryskindisease[3].Consistentwiththatidea,
P.acnesreleaseschemoactivefactorsthatattractneutrophils,monocytesandlymphocytes[4],
andactsviathetoll-likereceptor(TLR)2-to-NF-κBsignalingpathwaytostimulateproduction
ofpro-inflammatorycytokines,chemokinesandadhesionmolecules[5,6].
ReductionofthenumbersofP.acnesintheskinbyacnemedicationscorrelateswithclinical
improvement[2].However,treatmentofacnevulgariswithcommonlyusedantibiotics,
includingoraltetracyclineandtopicalerythromycinandclindamycin,hasincreasedtheresis-
tanceofP.acnestoantibioticsandincreasedthelikelihoodoftherapeuticfailure[7].Benzoyl
peroxide(BPO),alipophilicnon-antibioticantibacterialagent,isalsohighlyeffectiveagainst
P.acnes;however,ithasahighminimalinhibitoryconcentration(MIC:150μg/ml)[8,9].Oral
isotretinoin,apotentoralretinoidreservedforthetreatmentofsevereacne,iseffective,butits
useislimitedbyitsmanyseveresideeffects[10].Recently,a5%dapsonegel(asyntheticsul-
fone)wasshowntohavesomeefficacyinthetreatmentofacnevulgaris[11],butpotential
hematologictoxicitylimitsitsclinicalutility[12].Itisthusclearthatdevelopmentofasafe
therapeuticreagentthathasnosideeffectsbutstrongantibacterialactivitywouldhighlydesir-
ableforthetreatmentofacne.Onepromisingapproachistheuseofantimicrobialpeptides
(AMPs),whichefficientlykillmicrobialpathogensandmodulatehostimmuneresponses[13].
CAisa37-aminoacidcationicAMPisolatedfromthecaterpillarofthececropiamoth
(Hyalaphoracecropia)[14],whileMAisa23-aminoacidcationicAMPfromtheskinofthe
Africanclawedfrog(Xenopuslaevis)[15].BothCAandMAexertstrongantibacterialeffects
withlittleornocytotoxicityaffectingmammaliancells[14,15].Aprimarytargetforthesepep-
tidesisthelipidbilayerofthebacterialcytoplasmicmembrane,towhichtheybindandinduce
membranepermeabilization[16].WerecentlyreportedasyntheticCA-MAhybridanalogue,
P5,whichwasdesignedtoincreasethenetpositivechargeandhydrophobicityofthepeptide
[17,18].P5showedgreatlyincreasedantibacterialactivityagainstbothGram-positiveand
Gram-negativebacteria,actingviaamembranolyticmechanism,butnohemolyticorcytotoxic
activityinmammaliancells.Notably,P5hasstrongerantibacterialandantifungalactivities
thanCA-MAagainstavarietyofaerobicmicrobes,buthaslittleornohemolyticorcytotoxic
activityagainstnormaleukaryoticcells[17–20].Moreover,ithasnotyetbeendemonstrated
whetherP5hastheabilitytoeffectivelypreventortreatinflammatoryskindiseasessuchas
acne.Inthepresentstudy,therefore,ouraimwastoassessthepotentialutilityofP5asathera-
peuticagentforthetreatmentofinflammatoryacnevulgarisbasedonitspotentantibacterial
effectsanditsabilitytosuppressdevelopmentoftheinflammatoryresponsestriggeredbyP.
acnes.Todothat,wetestedtheeffectsofP5oninflammatorycytokineproductioninlivingP.
acnes-infectedhumankeratinocytes(HKs)invitro,andinvestigatedthemolecularmechanism
oftheanti-inflammatoryeffectsofP5inamousemodelofP.acnes-inducedinflammatory
acne.
MaterialsandMethods
EthicsStatement
ThisstudywascarriedoutinstrictaccordancewiththerecommendationsintheGuideforthe
CareandUseofLaboratoryAnimalsoftheNationalInstitutesofHealth.Theprotocolwas
approvedbytheCommitteeontheEthicsofAnimalExperimentsoftheUniversityofArkan-
sasforMedicalSciences(PermitNumber:A3063-01).Allsurgerywasperformedunder
sodiumpentobarbitalanesthesia,andalleffortsweremadetominimizesuffering.
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InflammatoryResponsebytheAntimicrobialPeptideP5
Experimentalanimalprotocol
WeutilizedfemalewildtypeInstituteofCancerResearch(ICR)mice(6–8weeks-old;Harlan,
Indianapolis,IN)inthisstudyasameansofassessingtheabilityofthenewlydesignedsyn-
theticantimicrobialpeptideP5tominimizeaPropionibacteriumacnes(P.acnes)infection,
andmodulatetheinnateinflammatoryresponseofthehostasdescribedindetailpreviously
[21].Birefly,P5(1.6μM,20μl),orPBS(20μl)wasinjectedintradermallyintotherightearsof
ICRmice24hoursafterP.acnes(1x108CFUper20μlinPBS)inoculationatthesamesite.P.
acneswasquantifiedbyplatingserialdilutionsofthehomogenateonagarplatesandincubat-
ingunderanaerobicconditionsfor48hours.Earthicknesswasmeasuredusingamicrocaliper
(Mitutoyo547-400S;MSIVikingGage,Charleston,SC)priortoinjectionandat24,48,72and
96hoursafterinjection.Forallinvivoexperiments,n=10animals/treatmentgroup/time
pointwereusedunlessotherwisespecified.Experimentalcomparisonsweremadebya
repeatedmeasuresANOVAmodelusingSASProcmixedsoftware(SASInstitute,Inc.,Cary,
NC).
Preparationofsyntheticpeptides
SyntheticAMPsCA-MA(KWKLFKKIGIGKFLHSAKKF-NH2),P5(KWKKLLKKPLLKKLLK
KL-NH2),andP4(KWKKKKKKPKFL-NH2)weresynthesizedasdescribedpreviously[17].
Thestocksolutionwasmadeataconcentrationof1mMinsterilizeddistilledwaterandfil-
teredthrougha0.22-μmporefilter.Thefilteredsolutionwasstoredat-20°Cuntiluse.
Bacterialculture
P.acnes(ATCC11828andATCC6919)(AmericanTypeCultureCollection,Manassas,VA)
wasculturedinReinforcedClostridialMedium(BD:FranklinLakes,NJ)underanaerobiccon-
ditionsusingGas-Pakat37°Casdescribedindetailpreviously[21].Staphylococcusepidermidis
(S.epidermidis)(ATCC12228)wasculturedinNutrientbroth(BD)underaerobicconditions
at37°C.Forstimulationofnormalhumankeratinocytes(HKs),bacterialcellswereharvested
bycentrifugationat3,000xgfor10minat4°C.Thebacteriawerethenwashedthreetimes
withPBSandresuspendedinstarvationmediumlackinghydrocortisoneandbovinepituitary
extract(BPE)orinPBSat1x108colony-formingunits(CFU)/ml.
Invitroantibacterialassays
Minimumbactericidalconcentrations(MBCs)ofthepeptidesagainstanaerobicandaerobic
microorganismsweredeterminedinduplicateintwoindependentexperimentsusingmicrodi-
lutionmethodswith96-wellmicrotitrecellcultureplates.Thepeptideswerefilteredthrough
0.22μmfiltersanddilutedstepwiseinappropriatebrothmediatoconcentrationsrangingfrom
100μMto0.39μM.The2-foldseriallydilutedsolutionsofeachpeptide(100μl)weremixed
with100μlofbacterialsuspensiontoadensityof2x106CFU/ml.Theplateswerethenincu-
batedfor16hat37°Cunderanaerobicoraerobicconditions.Afterincubation,thereaction
mixturesweredilutedandplatedontoappropriateagarplatesforcountingtheCFUs.The
MBCwasdefinedasthelowestpeptideconcentrationthatresultedinnovisiblemicroorganism
growthontheagarplate.
Keratinocyteculture
Humanepidermalkeratinocytes(HKs)fromforeskinwerepurchasedfromPromoCell(Hei-
delberg,Germany)andculturedinkeratinocytegrowthmedium(KGM;Clonetics,San
Diego,CA)asdescribedindetailpreviously[21].Briefly,HKcellswereculturedinKGMat
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InflammatoryResponsebytheAntimicrobialPeptideP5
37°Cinahumidifiedatmospherecontaining5%CO .Cellculturemediawaschangedevery
2
2to3daysandcellswereharvestedafterpassage3to4.HKcellswereseededataconcentra-
tionof104to105cellsperwellinto6/12/96wellcultureplatesandchamberslides.Thecells
wereallowedtogrowuptoabout70%confluence.Intherequiredexperimentalconditions,
HKcellswereculturedinKGMstarvingmedia,whichisfreeofsupplementandgrowthfac-
tors(BPE,hEGF,insulin,hydrocortisone,epinephrine,transferrinandgentamicin/ampho-
tericin-B).
Scanningelectronmicroscopy(SEM)
SEMwasperformedasdescribedpreviously[18].Allsampleswerevisualizedusingafield
emission-scanningelectronmicroscope(FE-SEM,JSM-7100F,Jeol,Japan)at20,000xmagnifi-
cation(15.0kV).
TreatmentofP.acnesand/orpeptidesinHKs
AfterculturedHKswerepre-infectedwithP.acnes(1x108CFU/ml)for24hours,asynthetic
peptide(CA-MA,P5orP4)wasaddedtoeachwelltoaconcentrationof0.8μM,1.6μMor
3.2μM.Then,thecellswereincubatedforvariousperiodsasindicatedintheresults.Cultured
HKcellsinstarvationmediumonlyservedasanegativecontrol.Thecellswerethenharvested
forRNAextraction.Thesupernatantscollectedbycentrifugationat14,000xgfor10minat
4°Cwerealiquotedandstoredat-70°CuntilusedinIL-8andTNF-αassay.
QuantitativeRT-PCR
TargetgenemRNAexpressionwasanalyzedbyreal-timeRT-PCRasdescribedinthemanu-
facturer’sprotocol(ABI7500real-timePCRsystemusingSYBRGreenmastermix;Applied
Biosystems,FosterCity,CA)asdescribedindetailpreviously[21].Theprimersequenceswere
asfollows:forIL-8,5'-GCAGTTTTGCCAAGGAGTGCT-3'(sense)and5'-TTTCTGTGTTG
GCGCAGTGTG-3'(antisense);forTNF-α,5’-ATAGCTCCCAGAAAAGCAAGC-3’(sense)
and5’-CACCCCGAAGTTCAGTAGACA-3’(antisense);forTLR2,5’-TGTCTTGTGACCGCA
ATGGT-3’(sense)and5’-GTTGGACAGGTCAAGGCTTT-3’(antisense);andfor18SrRNA,
5'-CGGCTACATCCAAGGAA-3'(sense)and5'-GCTGGAATTACCGCGGCT-3'(anti-
sense).Quantificationoftargetgeneexpressionwasnormalizedusinganinternalcontrolgene,
18SrRNA[22].Alltheexperimentswereperformedintriplicate.
Enzyme-LinkedImmunosorbentAssay(ELISA)
TheIL-8andTNF-αlevelsincollectedculturesupernatantsweredeterminedusinghuman
TNF-αandIL-8immunoassaykits(R&DSystems,Minneapolis,MN)accordingtothemanu-
facturer’sinstructions.TheopticaldensityofthewellswasmeasuredusinganELISAreaderset
to450nmwithawavelengthcorrectionsetto540nm.Alltheexperimentswereperformedin
triplicate.
ImmunofluorescentstainingforNF-κBnucleartranslocationandTLR2
cellularlocalization
ImmunofluorescenceanalysisofNF-κBandTLR2localizationwasperformedaspreviously
described[23].Briefly,HKsweretreatedwithP.acnes(1x108CFU/ml)for30minforNF-κB
or24hforTLR2inthepresenceorabsenceof1.6μMP5orP4.Thecellswerethenfirstincu-
batedwithrabbitpolyclonalanti-humanNF-κBp65antibody(RelA)orrabbitanti-human
TLR2antibody(Rockland,Gilbertsville,PA),diluted1:3000inblockingbuffer(ImmPRESS
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InflammatoryResponsebytheAntimicrobialPeptideP5
kit;VectorLaboratories,Burlingame,CA).Theywerethenincubatedfor1hwithFITC-conju-
gatedaffinity-purifiedgoatanti-rabbitIgG(1:300dilution;H+L;JacksonImmunoResearch
Laboratories,Inc.,WestGrove,GA)atroomtemperatureinthedark.Finally,thecellswere
visualizedunderamicroscope(OlympusEX51;CenterValley,PA),andimageswereacquired
usingaQICAMfast1394camera(Westmont,IL).
MTTassay
AstandardcolorimetricassayforassessingcellviabilitybasedontheactivityofMTT(yellow
tetrazoliumsalt:3-(4,5-dimethuylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)reducing
enzymeswasperformedaccordingtothemanufacturer’sinstructions(MolecularProbes,Inc.,
Eugene,OR)usingHKcells(5x103per200μlculturemedia)inthepresenceandabsenceof
P5orP4atconcentrationsrangingfrom1.6to6.4μM.Dataarepresentedasthepercentageof
viableHKcellscomparedtothepercentageofviablecellsaftertreatmentwith2%TritonX-
100,whichservedasthepositivecontrolforcellcytotoxicity.
Circulardichroism(CD)analysisofP5bindingtolipoteichoicacid(LTA)
CDspectrawererecordedat25°ConaJasco810spectropolarimeter(Jasco,Tokyo,Japan)
equippedwithatemperaturecontrolunitusingaquartzcellwitha0.1-cmpathlength.The
CDspectrafor50μMpeptidedissolvedinPBS(pH7.2)werescannedinthepresenceor
absenceof0.1%LTAdissolvedinPBS.Atleastfourscansofthe250–190nmwavelengthrange
wereconducted,afterwhichtheaverageblankspectraweresubtractedfromtheaverageofthe
samplespectra.
AnalysisofintracellularCa2+mobilizationinkeratinocytes
MobilizationofintracellularCa2+wasevaluatedtoassessthefunctionalresponseofHKstoP.
acnes-infectioninthepresenceandabsenceofsyntheticpeptidesasdescribedindetailprevi-
ously[21].Briefly,culturedHKsoncoverslipswereincubatedfor45minat37°CinPBScon-
taining2μMfura-2/AM(Invitrogen,Carlsbad,CA),themembranepermeantformofthe
fluorescentCa2+probefura-2.Afterthreewashes,thecellswerepretreatedwithorwithout
1.6μMP5orP4,afterwhichP.acneswasaddedduringtheactivemonitoringofintracellular
Ca2+mobilization.FluorescencewasmeasuredusinganInCytBasicIMFluorescenceImaging
System(IntracellularImagingInc.,Cincinnati,OH)accordingtothemanufacturer’sinstruc-
tions.TheintracellularfreeCa2+concentrationwasdeterminedbymeasuringtheratioofthe
510nmemissionselicitedbyexcitationat340and380nm.
DeterminationofP.acnesabundanceandP.acnes-induced
inflammationinvivo
P5(1.6μM,20μl)orPBS(20μl)wasintradermallyinjectedintotherightearsofICRmice
(Harlan,Indianapolis,IN)24hafterP.acnes(1x108CFUper20μlinPBS)inoculationatthe
samesite.Leftearsofthesamemicewereinjectedwith20μlofPBS.InnegativecontrolICR
mice,therightearsremaineduntreated,whiletheleftearsreceivedintradermalinjectionsof
PBS.Tenmgoftissuefrom8mmpunchbiopsiestakenfromtheears24hoursafterpeptide
injectionwerehomogenizedin250μlofsterilePBSusingatissuegrinder.P.acneswasquanti-
fiedbyplatingserialdilutionsofthehomogenateonagarplatesandincubatingthemfor48h
underanaerobicconditions.Earthicknesswasmeasuredusingamicrocaliper(Mitutoyo547-
400S;MSIVikingGage,Charleston,SC)priortopeptideinjectionandat24,48and72hours
afterinjection.
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InflammatoryResponsebytheAntimicrobialPeptideP5
Statisticalanalysis
Resultsareexpressedasmeans±SD.ANOVAwithprobabilitieswasperformedforbothover-
allsignificanceandpairwisecomparison.P<0.05wasconsideredtobestatisticallysignificant.
Results
AntibacterialeffectsofCA-MA,P5andBPOagainstskinbacteria
WecomparedtheantibacterialactivitiesofCA-MA,itsnewlydesignedanalogueP5,andBPO
againsttheskinbacteriaP.acnesATCC11828,P.acnesATCC6919andS.epidermidisATCC
12228bydeterminingtheMBCofeachstrainusingthemicrodilutionmethod.Thethree
strainswereculturedinthepresenceofvariousconcentrationseachagentfor48,72and24
hours,respectively.Bacterialgrowthwasthenevaluatedbymeasuringtheabsorbanceat600
nm,afterwhichthebacteriaweredilutedandspottedonagarplatestocounttheCFUs
(Table1).Eachsyntheticpeptide,CA-MA(parentalpeptide),P5andanegativecontrolpeptide
(P4),wastestedatconcentrationsrangingfrom0.1to12.8μMusingtwofoldserialdilutions.
BPO,whichisatraditionalcompoundusedtotreatmildtomoderateacne,wastestedatcon-
centrationsrangingfrom7.8to250μM,with5%(vv-1)DMSOservingasthecontrol.The
MBCvaluesofP5andCA-MAwere0.2and0.4μM,respectively(Table1).TheMBCforP5
was300timeslowerthanthatforBPO(62.5μM)and64timeslowerthanforP4(>12.8μM),
itsnegativecontrolpeptide.ThepotentantibacterialactivityofP5againstanaerobicP.acnesis
similartothatofclindamycin(MBC<0.2μM),acommontopicaltreatmentforacnevulgaris.
Theseresultsdemonstratethatincreasingthepositivechargeandthehydrophobicityofthe
newlydesignedAMPP5ledtoadistinctincreaseinitsbactericidalactivity.
CytotoxicityofP5againstHKs
TodeterminethecytotoxiceffectsofP5onHKs,weusedMTTassaystomeasureHKviability
inserum-freemediumwithorwithoutP5for24hat37°C.HKviabilitywasdeterminedafter
24hoftreatmentatconcentrationsrangingfrom0.5to5μM.WefoundthatHKswere100%
viableafter24hoftreatmentwithP5oritscontrol,P4(S1Table).Theseresultsdemonstrate
thatP5hasnosignificantcytotoxiceffectonHKs,evenatconcentrationsseveraltimeshigher
thannecessaryforantibacterialactivityagainstP.acnes.
P5inducesmorphologicaldisruptionofP.acnesthatsuggestsa
bactericidaleffect
CationicAMPskillbacteriaprimarilythroughmembranepermeabilizationandsubsequent
structuraldisruption.UsingSEM,wewereabletodirectlyobservecellmorphologyand
Table1. MBCsofSyntheticAMPsandConventionalAgentsagainstAcne-InducingPropionibacteriumacnes.
Antimicrobialpeptides(AMPs) MBC(μM)ofthefollowingbacteria
P.acnes(ATCC11828) P.acnes(ATCC6919) P.acnes(ATCC12228)
CA-MA 0.4 0.4 0.4
P5 0.2 0.2 0.2
P4 >12.8 >12.8 >12.8
Clindamycin <0.2 <0.2 <0.2
BPO(benzoylperoxide) 62.5 >62.5 >62.5
MBC(minimalbactericidalconcentration)wasdefinedasthelowestpeptideconcentration(μM)thatprevented100%ofbacterialgrowthonagarplates.
doi:10.1371/journal.pone.0132619.t001
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InflammatoryResponsebytheAntimicrobialPeptideP5
Fig1.MorphologicalperturbationsandblebsinducedinP.acnesbyP5.ShownareP.acnescellsafter
incubationfor20minintheabsence(A)andpresenceofCA-MA(B),P5(C)andP4(D)ataconcentrationof
1/2MBC.Arrowspointtomorphologicalperturbationsandblebs,whichwereclearlyvisiblefollowing
treatmentwithP5.
doi:10.1371/journal.pone.0132619.g001
integrityafterpeptidetreatment.WeobservedthatP5inducedmorphologicalperturbations
andblebsintheP.acnescellwallataconcentrationof0.1μM(Fig1C),whereasCA-MA(Fig
1B)andP4(Fig1D)inducedlittlemorphologicchangestothecellsurfaceatthesameconcen-
tration.TheseresultsindicatethattheantimicrobialeffectofP5againstP.acnesisconsistent
withitscationicnaturewithitsbactericidalorbacteriostaticactivity.
P5suppressesP.acnes-inducedexpressionofpro-inflammatory
cytokinesinHKs
WenextexaminedtheeffectofP5oninnateinflammatoryresponsestoP.acnesinfectionin
vitro.BecauseIL-8andTNF-αareinflammatorymediatorslikelytoparticipateintheresponse
tocutaneousinfections,weusedreal-timeRT-PCR(Fig2)andELISA(Fig3)tomeasurethe
effectsofP5(0.8or1.6μM)ontheexpressionofIL-8andTNF-α24hafterpre-infection
(1x108CFU/ml)ofHKswithP.acnes.WefoundthatrelativelevelsofbothIL-8andTNF-α
mRNA(Fig2Aand2B,respectively)andprotein(Fig3Aand3Brespectively)weresignifi-
cantlyupregulatedafterP.acnesinfection,andthoseresponsesweresignificantlyinhibitedby
P5.ParentalCA-MAhadmuchlessabilitytoinhibittheincreaseinIL-8andTNF-αexpres-
sioninducedbyP.acnesthanthemorehydrophobicP5analogue,whilethenegativecontrol
peptideP4hadnoeffectonP.acnes-inducedIL-8andTNF-αexpressioninHKs.Bycontrast,
theseAMPshadnoeffectonbasalIL-8orTNF-αexpressionwhenuninfectedHKswere
treated(Fig3Aand3B).TheseresultsindicatethatP5specificallyinhibitstheexpressionof
inflammatorycytokinesinducedbyP.acnesinfectioninHKs.
P5significantlyinhibitsP.acnes-inducedTLR2expressioninHKcells
ItisknownthatP.acnescontributestoinflammationinacnethroughactivationofthetoll-like
receptors(TLRs),especiallyTLR2[19].Therefore,tofurtherexploretheeffectofP5oninnate
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Fig2.P5inhibitsexpressionofIL-8andTNF-αmRNAinducedbyP.acnesinfectioninHKs.TotalRNAwascollectedfromP.acnes-infectedHKswith
andwithoutP5treatment.ExpressionofIL-8(A)andTNF-α(B)mRNAwasmeasuredusingquantitativeRT-PCRwithhumanIL-8-andTNF-α-specific
primers.TherelativelevelofeachmRNAwasnormalizedtotheexpressionof18SrRNA.Alldatawerecomparedtotheuntreatedcontrolvalues.Thedata
shownarerepresentativeoftriplicateexperiments.Allvaluesareexpressedasthemean±SD.*p<0.001.
doi:10.1371/journal.pone.0132619.g002
inflammatoryresponses,weusedreal-timeRT-PCRtoassessexpressionofthepatternrecog-
nitionreceptorTLR2inducedinHKsinresponsetoP.acnes(1x108CFU/ml)infectionand
thentestedtheeffectof1.6μMP5.ExpressionofTLR2mRNAwasincreasedabout2-foldin
HKs24hafterP.acnesinoculation,andthisoverexpressionwassignificantly(P<0.001)
down-regulatedbytreatmentwithP5,butnotP4(Fig4A).P5alsoinhibitedP.acnes-induced
Fig3.P.acnes-inducedsecretionofIL-8andTNF-αfromHKswasinhibitedbyP5treatment.IL-8(A)andTNF-α(B)weremeasuredinculture
supernatantsafterincubatingP.acnes-infectedHKcellsfor24hinthepresenceorabsenceof1.6μMP5,P4orCA-MA.Thedatashownarerepresentative
oftriplicateexperiments.Allvaluesareexpressedasmean±SD.*p<0.001.
doi:10.1371/journal.pone.0132619.g003
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InflammatoryResponsebytheAntimicrobialPeptideP5
Fig4.P5inhibitsexpressionofTLR2inducedbyP.acnesinHKs.(A)ExpressionofTLR2mRNAwasmeasuredusingreal-timeRT-PCRand
normalizedtotheexpressionof18SrRNA.Allvaluesareexpressedasthemean±SD.*P<0.001.(B)ImmunofluorescentlocalizationofTLR2withinHKs.P.
acnes-infectedHKswereincubatedfor24hinthepresenceorabsenceof1.6μMP5orP4,afterwhichthedistributionofTLR2wasdeterminedby
immunofluorescentlabeling.FITC-labeledTLR2isshowningreen,whiletheHoechst-stainednucleiareblue:(a)treatedwithPBS;(b)treatedwithP.acnes;
(c)treatedwithP.acnesplusP5;(d)treatedwithP.acnesplusP4;(e)treatedwithP5alone.
doi:10.1371/journal.pone.0132619.g004
expressionofTLR2proteininHKsmeasured24haftertreatment(Fig4Bc),ascomparedto
thenegativecontrolP4(Fig4Bd).Finally,treatmentwithP5alone(Fig4Be)hadnoeffecton
basalTLR2expressioninHKs.TheseresultsdemonstratethatP5specificallyinhibitsTLR2
expressionrelatedtotheinnateimmuneresponsetoP.acnes-infectioninHKcells.
P5blocksNF-κBnucleartranslocationinducedbyP.acnesinfectionin
HKcells
NF-κBisakeytranscriptionalregulatorofmultiplegenes,includingIL-8andTNF-α.TLR2
signalingleadstoactivationoftheNF-κBpathway,whichinturnstimulatesreleaseofpro-
inflammatorycytokinessuchasIL-8andTNF-α.TotestwhetherP5affectsTLRsignalingto
NF-κB,weusedimmunofluorescentstainingtoassesstheintracellulardistributionofNF-κB
p65.InuntreatedHKs,NF-κBstainingwasobservedprimarilyinthecytoplasm(Fig5A),how-
ever,nucleartranslocationofNF-κBwasrapidlyinducedbyP.acnesinfection(Fig5B).Co-
incubationofHKswithP.acnesandP5effectivelyblockedP.acnes-inducedNF-κBnuclear
translocation(Fig5C).Bycontrast,thenegativecontrolpeptideP4hadnoeffectonP.acnes-
inducedNF-κBnucleartranslocation(Fig5D).
BindingofCA-MAandP5toLTA
TheabilityofAMPstobindLTAlikelyplaysasignificantroleintheirabilitytoneutralizeLTA
shedfrombacteriaandthuspreventsinflammatoryresponses.Wethereforeanalyzedthepep-
tides’CDspectratoaskwhetheranyofthepeptidesstudiedherecouldbindtopurifiedLTAin
vitro.Whenthepeptideswerestudiedat50μMina0.1%LTAsuspension,P5adoptedamore
foldedconformationthanCA-MA(S1Fig.),whichisindicativeofitsbindingtoLTA.Thissug-
geststhatP5blockstheinductionofinflammatorycytokinesinHKcellsbybindingLTA.
PLOSONE|DOI:10.1371/journal.pone.0132619 July21,2015 9/18
InflammatoryResponsebytheAntimicrobialPeptideP5
Fig5.P5inhibitsNF-κBnucleartranslocationinP.acnes-infectedHKcells.TheintracellulardistributionofNF-κBwasdeterminedby
immunofluorescentlabelingofNF-κBp65(green),whilenucleiwereHoechststained(blue):(A)untreatedHKcells;(B)HKcellstreatedwithP.acnes;(C)HK
cellstreatedwithP.acnesplusP5;HKcellstreatedwithP.acnesplusP4(D).
doi:10.1371/journal.pone.0132619.g005
P5inhibitsP.acnes-inducedintracellularCa2+fluctuationinHKs
Ca2+signalingappearstoplayaroleatseveralstepsduringbacterialinfectionandinthecon-
trolofgeneexpression,especiallytheexpressionandsecretionofpro-inflammatorymediators
inducedbybacterialpathogens[19].WethereforeexaminedtheeffectofP5ontheintracellu-
larCa2+signalinginducedinHKsbyP.acnesinfection.AsshowninFig6,P.acnes(1x108
PLOSONE|DOI:10.1371/journal.pone.0132619 July21,2015 10/18