Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and

29 Pages·2017·7.22 MB·English

RESEARCHARTICLE
Pentoxifylline, dexamethasone and
azithromycin demonstrate distinct age-
dependent and synergistic inhibition of TLR-
and inflammasome-mediated cytokine
production in human newborn and adult
blood in vitro
a1111111111
a1111111111 EstherM.Speer1*,DavidJ.Dowling2,3,JianjinXu4,LukaszS.Ozog1,JaimeA.Mathew1,
a1111111111 AvinashChander1,DongleiYin4,OferLevy2,3,5
a1111111111
1 DepartmentofPediatrics,DivisionofNeonatology,StonyBrookUniversitySchoolofMedicine,Stony
a1111111111
Brook,NewYork,UnitedStatesofAmerica,2 DepartmentofMedicine,DivisionofInfectiousDiseases,
BostonChildren’sHospital,Boston,Massachusetts,UnitedStatesofAmerica,3 HarvardMedicalSchool,
Boston,Massachusetts,UnitedStatesofAmerica,4 DepartmentofAppliedMathematicsandStatistics,
StonyBrookUniversity,StonyBrook,NewYork,UnitedStatesofAmerica,5 PrecisionVaccineProgram,
BostonChildren’sHospital,Boston,Massachusetts,UnitedStatesofAmerica
OPENACCESS
*[email protected]
Citation:SpeerEM,DowlingDJ,XuJ,OzogLS,
MathewJA,ChanderA,etal.(2018)Pentoxifylline,
dexamethasoneandazithromycindemonstrate
Abstract
distinctage-dependentandsynergisticinhibitionof
TLR-andinflammasome-mediatedcytokine
productioninhumannewbornandadultbloodin
vitro.PLoSONE13(5):e0196352.https://doi.org/ Introduction
10.1371/journal.pone.0196352
Neonatalinflammation,mediatedinpartthroughToll-likereceptor(TLR)andinflammasome
Editor:PascalM.Lavoie,UniversityofBritish signaling,contributestoadverseoutcomesincludingorganinjury.Pentoxifylline(PTX),a
Columbia,CANADA
phosphodiesteraseinhibitorwhichpotentlysuppressescytokineproductioninnewborncord
Received:October10,2017 blood,isacandidateneonatalanti-inflammatoryagent.Wehypothesizedthatcombinations
Accepted:April11,2018 ofPTXwithotheranti-inflammatoryagents,thesteroiddexamethasone(DEX)orthemacro-
lideazithromycin(AZI),mayexertbroader,moreprofoundand/orsynergisticanti-inflamma-
Published:May1,2018
toryactivitytowardsneonatalTLR-andinflammasome-mediatedcytokineproduction.
Copyright:©2018Speeretal.Thisisanopen
accessarticledistributedunderthetermsofthe
Methods
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and WholenewbornandadultbloodwastreatedwithPTX(50–200μM),DEX(10−10–10−7M),
reproductioninanymedium,providedtheoriginal
orAZI(2.5–20μM),aloneorcombined,andculturedwithlipopolysaccharide(LPS)(TLR4
authorandsourcearecredited.
agonist),R848(TLR7/8agonist)orLPS/adenosinetriphosphate(ATP)(inflammasome
DataAvailabilityStatement:Allrelevantdataare
induction).Supernatantandintracellularcytokines,signalingmoleculesandmRNAwere
withinthepaperanditsSupportingInformation
files. measuredbymultiplexassay,flowcytometryandreal-timePCR.Druginteractionswere
assessedbasedonLoewe’sadditivity.
Funding:ThisstudyinES’slaboratorywas
financiallysupportedbytheDepartmentof
PediatricsIntramuralGrantProgram,StonyBrook Results
Children’sHospitalandStonyBrookUniversity
PTX,DEXandAZIinhibitedTLR-and/orinflammasome-mediatedcytokineproductionin
SchoolofMedicine,StonyBrook,NY.OLis
supportedbyU.S.NationalInstitutesofHealth newbornandadultblood,whetheraddedbefore,simultaneouslyorafterTLRstimulation.
PLOSONE|https://doi.org/10.1371/journal.pone.0196352 May1,2018 1/29
Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
(NIH)NationalInstituteofAllergyandInfectious PTXpreferentiallyinhibitedpro-inflammatorycytokinesespeciallyTNF.DEXinhibitedIL-10
Diseases(NIAID)InfantImmunitygrant innewborn,andTNF,IL-1β,IL-6andinterferon-αinnewbornandadultblood.AZIinhibited
R01AI100135-01anditsadministrative
R848-inducedTNF,IL-1β,IL-6andIL-10,andLPS-inducedIL-1βandIL-10.(PTX+DEX)
supplementsaswellasaNIAIDAdjuvant
DiscoveryProgramContractNo. synergisticallydecreasedLPS-andLPS/ATP-inducedTNF,IL-1β,andIL-6,andR848-
HHSN272201400052C.Thefundershadnorolein inducedIL-1βandinterferon-α,while(PTX+AZI)synergisticallydecreasedinductionof
studydesign,datacollectionandanalysis,decision
TNF,IL-1β,andIL-6.SynergisticinhibitionofTNFproductionby(PTX+DEX)wasespecially
topublish,orpreparationofthemanuscript.
pronouncedinnewbornvs.adultbloodandwasaccompaniedbyreductionofTNFmRNA
Competinginterests:Theauthorshavedeclared
andenhancementofIL10mRNA.
thatnocompetinginterestsexist.
Conclusions
Age,agent,andspecificdrug-drugcombinationsexertdistinctanti-inflammatoryeffects
towardsTLR-and/orinflammasome-mediatedcytokineproductioninhumannewbornblood
invitro.SynergisticcombinationsofPTX,DEXandAZImayofferbenefitforpreventionand/
ortreatmentofneonatalinflammatoryconditionswhilepotentiallylimitingdrugexposure
andtoxicity.
Introduction
Inflammation,driveninpartviaengagementofpatternrecognitionreceptors(PRRs)by
endogenousandexogenousagonists,contributestomultipleperinataldiseasesincludingsep-
sis,bronchopulmonarydysplasia(BPD)andbraininjury[1–3].Corticosteroidsarecurrently
themainanti-inflammatorytreatmentinneonatesbuthavesignificantshort-andlong-term
adverseeffectssuchasgastrointestinalandcardiovascularcomplications,insulinresistanceas
wellaspotentialgrowthrestrictionandneurodevelopmentaldisability,especiallywithearly
useofhigh-dosedexamethasone(DEX)duringthefirstweekoflife[4–5].Thereisthusan
unmetmedicalneedforeffectiveanti-inflammatoryalternativestocurrentlyemployedcorti-
costeroid-basedtreatments,whichmayhavelessadverseeffectsinthetreatmentofneonatal
inflammationandpreventitssubsequentsequelae.
Thexanthinederivativepentoxifylline(PTX)inhibitsarangeofphosphodiesterasesand
increasesintracellularcyclicadenosinemonophosphate(cAMP)inavarietyofcellsandtis-
sues,therebysuppressingproductionofpro-inflammatorymediatorsincludingtumornecro-
sisfactor(TNF)[6].Duetoitsanti-TNFactivity,PTXhasbeenstudiedinanimalsandhuman
adultsandnewbornstoameliorateinflammatoryconditionssuchasBPD,meconiumaspira-
tion,necrotizingenterocolitis,andhypoxicischemicencephalopathy[7–11].PTXisbeing
evaluatedasapotentialanti-inflammatoryadjuncttoantibioticsinneonatalsepsis,andseveral
smallstudiesofPTXforthisindicationraisethepossibilitythatitmaydecreaseall-causemor-
tality[12–13].Azithromycin(AZI),amacrolideantibiotic,exertsanti-inflammatoryactions
througharangeofimmunomodulatorymechanismsthatinvolveinhibitionofpro-inflamma-
torypathwaysaswellasphenotypicandfunctionalchangesofinnateimmunecells[14–16].
Combinationpharmacotherapyisanestablishedapproachtomaximizedesiredeffectswhile
limitingunwantedadverseeffectsthroughdosereductionsoftheindividualcompounds
[17–18].
Giventheneedtoreduceinflammatoryinjuryinthenewborn,westudiedage-dependent
effectsofanti-inflammatorydrugcombinationsinnewbornandadultbloodinvitro.We
hypothesizedthatPTX+DEXorAZI(DEX/AZI)mayprovidebroader,moreprofoundand/
PLOSONE|https://doi.org/10.1371/journal.pone.0196352 May1,2018 2/29
Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
orsynergisticbluntingofneonatalToll-likereceptor(TLR)-and/orinflammasome-mediated
inflammatorycytokineproduction.Asamodelforinfection-inducedneonatalinflammation,
westimulatedbloodwithTLRagonists(TLRAs)thatactivateTLR4(theendotoxinlipopoly-
saccharide(LPS)),TLR7/8(R848,animidazoquinolinethatinducesadult-levelinflammatory
cytokineproductioninnewbornmonocytes)[19],orbothTLRpathwaysandtheinflamma-
someincludingR848[19]andthecombinationofLPSandadenosinetriphosphate(ATP)
[20].Usingthesewholebloodassays,whichpreservetheage-specificimmunomodulatorysol-
ubleandcellularfactorsnaturallypresentinthenewborn[21–23],wecomparedtheanti-
inflammatoryeffectsof(PTX+DEX/AZI).Wemeasuredstimuli-mediatedcytokinemRNA
expression,intracellularcytokines,supernatantcytokineconcentrations,andmonocytesignal-
ingmoleculesinnewbornandadultblood,andcalculatedestimatesforsynergisticanti-
inflammatorydruginteractions.Wefoundthattheagentsstudiedexerteddistinctinhibitory
effectsonthedifferentPRRpathwaysstudiedthatvariedwiththeagent,agentcombination
andageofstudyparticipant.Whereas(PTX+AZI)synergisticallyinhibitedpro-inflammatory
cytokineproductioninbothnewbornandadultblood,(PTX+DEX)demonstratedmorepro-
foundsynergisticinhibitionofLPS-andR848-inducedTNFproductioninnewbornvs.adult
blood.Ourstudysuggeststhatthesubstantialdrug(includingsteroid)-sparingeffectofthese
potentanti-inflammatoryagents,especiallywhenusedincombination,mightoffernew
approachesfortherapeuticbenefitinpreventionand/ortreatmentofneonatalinflammatory
conditionsincludingsepsis.
Materialsandmethods
Subjectsandhumanbloodcollection
Placentalcordbloodwascollectedfromhealthytermnewbornsbetween38to41weeks
gestationwhodeliveredbyCesareansectionwithoutlaborandwithoutcurrentinfection,
includingdocumentedintrauterineinfection(i.e.absenceofclinicalchorioamnionitis,pro-
longedfetalmembraneruptureover12h,clinicalorlaboratorysignsofearly-onsetsepsis,or
culture-provensepsisofthemotherorthenewborn)orHIV[20].Peripheralvenousblood
wasdonatedwithconsentfromhealthyadultdonorsbetween18to55yearsofage.Allblood
collectionswereinaccordancetoprotocolsapprovedbytheInstitutionalReviewBoardof
StonyBrookUniversity,StonyBrook,NY.Writteninformedconsentwasobtainedfromstudy
participants.Cordbloodwascollectedimmediatelyafterdeliveryoftheplacentaintosterile
sodiumheparintubescontaining15units/mlheparin(BectonDickinson;FranklinLakes,NJ)
throughpunctureoftheveinsonthefetalsideoftheplacentausingsteriletechniques,asprevi-
ouslydescribed[20].
Invitrostimulationandtreatmentofbloodsamples
Bloodsampleswerekeptatroomtemperatureandprocessedwithin2hoursofcollection.
Bloodwasdiluted1:1withsterilepre-warmed(37˚C)RPMI1640(LifeTechnologies;Grand
Island,NY)andafinalvolumeof200μlwasaddedtoeachwellof96-welltissueculture-
treatedround-bottompolystyreneplates(BectonDickinson).Samplesweretreatedwithanti-
inflammatoryagentsortheirrespectivevehiclecontrol,stimulatedwithTLRAs,andcultured
fortherespectivedurationundereachexperimentat37˚Cinahumidifiedincubatorat5%
CO .Tomaximizetherelevanceofourinvitrostudy,immune-modulatorsweretestedeither
2
aloneorincombinationandwithinclinicallyrelevantconcentrationranges[24–27],including
PTX(Tocris;Minneapolis,MN),DEXandAZI(SigmaAldrich;St.Louis,MO).Ultrapure
preparationofLPSfromE.coliO111:B4(InvivoGen;SanDiego,CA)wasusedforTLR4stim-
ulation,R848(InvivoGen)forTLR7/8stimulation,orLPSfollowedby5mMATP(Sigma
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
Aldrich)for20minforinflammasomeinduction.Allimmune-modulatorsandTLRAsexcept
LPSwereverifiedendotoxin-free(<0.1endotoxinunits/ml)bytheLimulusamebocytelysate
assay(CharlesRiver;Wilmington,MA).Uponcompletion,sampleswerecentrifugedinsituat
500gfor10minatroomtemperature.~120μlofsupernatantsperwellwerecarefullycollected
withoutdisturbingthecellpelletsandstoredat-80˚Cforsubsequentanalysis,aspreviously
described[20].Duplicatetechnicalreplicateswereusedforallcultureexperimentsandimmu-
noassays,whereasreal-timePCRexperimentswereconductedintriplicate.Thenumberof
independentlyconductedexperimentsutilizingbloodsamplesfromdifferentdonorswasas
indicatedforeachexperimentaldesign.TheoptimaldurationofTLRAstimulationforthedif-
ferentexperimentalprocedureswasdeterminedthroughkineticstudies[20].
Measurementofcytokineconcentrationsinculturesupernatants
SupernatantcytokineconcentrationsweredeterminedwithBio-PlexPromagneticmultiplex
assays(Bio-Rad;Hercules,CA)andanalyzedontheBio-Plex200systemwithBio-PlexMan-
ager5.0software(Bio-Rad),whichusestheBrendanfive-parameterlogisticregressionfor
standardcurvefitting.Interferon(IFN)-αconcentrationsweremeasuredbyenzyme-linked
immunosorbentassaywiththeVeriKineHumanIFN-αMulti-SubtypeSerumELISAkit(PBL
AssayScience;Piscataway,NJ).Cytokineconcentrationswereexpressedasapercentagecom-
paredtoTLRAsalone,whichweredefinedas100%.
Real-timePCR
TotalRNAwasisolatedfromculturedwholebloodaftererythrocytelysisusingQIAampRNA
BloodMinikits(Qiagen;Valencia,CA).GenomicDNAwasremovedwithRNase-freeDNase
(Qiagen).InordertoobtainadequateRNAyieldsforourexperiments,wepooledatotalof5
wellscontaining100μlwholebloodmixedwith100μlRPMIperwellforeachPCRsample.
TheconcentrationandpurityofRNAsampleswasmeasuredwithaNanoDropND-1000spec-
trophotometer(NanoDropTechnologies;Wilmington,DE).Theaverage260/280ratiowas
2.0forcordbloodandadultbloodsamples,withmostsamplesfallingwithintherecom-
mendedrangeof1.9to2.1.Fewsampleswitha260/280ratio<1.8werefurtherpurifiedas
permanufacturerinstructions.ThetranscribedcDNAsampleswerethenusedforreal-time
PCR-basedmeasurementsofmRNAexpression,whichwereperformedintriplicates,loading
5ngcDNAper10μlreactionwell.ReversetranscriptionemployedHighCapacitycDNA
ReverseTranscriptionkits(LifeTechnologies;FosterCity,CA).Real-timePCRwasperformed
usingTaqMangeneexpressionassays(LifeTechnologies).Betaactinservedasnormalization
controlandwasmultiplexedintoallreactionwells.Inordertoassurenon-interferenceofthe
housekeepinggenewiththetargetgenes,allgeneexpressionassayswerefirstpretestedby
comparingtheiramplificationefficienciesassingle-plexassaywiththeiramplificationefficien-
cieswhenmultiplexedwiththehousekeepinggene.ReactionswererunonaStepOnePlus
Real-timePCRsystem(LifeTechnologies)usingTaqManFastAdvancedMasterMix(Life
Technologies).Datawereanalyzedusingthedelta-deltathresholdcycle(ΔΔC )method[28].
T
Flowcytometry
WholebloodsampleswerepreparedwiththeCytofix/CytopermFixation/Permeabilizationkit
(BDBiosciences)with(TNF,interleukin(IL)-6,andIL-10)orwithout(IL-1β,TLRsurface
expression)additionofBrefeldinA.Afterpretreatmentwithanti-inflammatoryagentsorvehi-
clecontrolfor2hours,sampleswerestimulatedwithTLRAsandculturedat37˚Cin5%CO
2
for6hours(intracellularcytokines)or18hours(TLRexpression).Followingsurfacestaining,
redbloodcelllysisemployedFACSLysingSolution(BD).Afterfixationandpermeabilization,
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
sampleswerestainedwithmonoclonalantibodies(PE-Cy7-conjugatedmouseanti-TNF/clone
MAb11,PE-conjugatedmouseanti-IL-6/cloneAS12,APC-conjugatedratanti-IL-10/clone
JES3-19F1,PE-conjugatedmouseanti-IL-1β/cloneAS10,PE-conjugatedmouseanti-CD284/
cloneTF901,allfromBD;PE-conjugatedmouseanti-TLR7/clone4G6(Invitrogen;Carlsbad,
CA),FITC-conjugatedmouseanti-CD288/clone44C143(Invitrogen))ortheircorresponding
isotypecontrols.SampleswereanalyzedonanLSRFortessaflowcytometer(BD).Compensa-
tionbeads(BDCompBeads,BD)wereusedassingle-stainpositiveandnegativecontrols.
MonocytesweregatedwithforwardandsidescatterasCD45+CD14+cells(PerCP-Cy5.5-con-
jugatedmouseanti-CD45/clone2D1,FITC-conjugatedmouseanti-CD14/cloneM5E2or
APC-conjugatedmouseanti-CD14/cloneMφP9(BD),respectively).Datafromatleast10,000
monocyteswereacquiredforeachconditionandanalyzedwithKaluzaversion1.3software
(BeckmanCoulter;JerseyCity,NJ).Thegeometricmeanfluorescenceintensity(MFI)ofall
monocyteswasdeterminedaftersubtractionofisotypecontrols.
Formeasurementofp38mitogenactivatedproteinkinase(MAPK)andc-JunN-terminal
kinase(JNK)phosphorylationaswellastotalinhibitorofappaB(IB)α,bloodsampleswere
pretreatedwithanti-inflammatoryagentsorvehiclecontrolfor2hoursandstimulatedwith
TLRAsfor15min,followedbyimmediateredbloodcelllysisandfixationusingLyse/Fix
Buffer(BD).AfterpermeabilizationwithPermBufferII(BD),samplesweresimultaneously
stainedwiththeappropriatesurfaceandintracellularantibodies(AF647-conjugatedmouse
anti-p38[pT180/pY182]/clone36,PE-conjugatedmouseanti-JNK[pT183/pY185]/cloneN9-
66,AF647-conjugatedmouseanti-IBα/clone25/IBα/MAD-3).
Caspase-1activation
Wholebloodsampleswerepretreatedwithanti-inflammatoryagentsorvehiclecontrolfor2
hours.SampleswerethenstimulatedwithTLRAsandculturedfor1hourat37˚Cin5%CO
2
inthepresenceofFluorescentLabelledInhibitorofCaspases(FLICA)reagent(Immuno-
ChemistryTechnologies;Bloomington,MN),acell-permeablefluorescentreagentthat
covalentlybindstoactivatedcaspase-1,andstainedformonocytesurfacemarkers.Uponcom-
pletionofcultures,sampleswereimmediatelysubjectedtoredbloodcelllysisandfixation
usingLyse/FixBuffer(BD),washedandanalyzedonanLSRFortessaflowcytometer(BD).
MFIdatafromatleast10,000monocytes,gatedwithforwardandsidescatterasCD45+CD14+
cells,wereacquiredforeachconditionasdescribedabove,andanalyzedwithKaluzaversion
1.3software(BeckmanCoulter).ThegeometricMFIofallmonocyteswasdeterminedafter
subtractionofbackgroundfluorescence(noFLICAreagent).
Statisticalanalysis
Supernatantcytokineconcentrationsofsamplestreatedwithanti-inflammatoryagentswere
expressedasapercentagecomparedtocytokineconcentrationsinducedbyTLRAsalone,
whichweredefinedas100%.Linearmixedmodels,whichtakeintoaccountthepossible
dependenceamongmeasurementsfromthesamesampleunderdifferentdrugconcentrations,
wereemployedtoanalyzedrugconcentrationresponsedataforeachcytokine,stimulation
conditionandanti-inflammatoryagentindependently.Drugconcentrationsweretreatedas
categoricalvariables.Thecovariancestructureamongdrugconcentrationswasmodeledas
compoundsymmetry,whichassumesthatalldistinctmembersofaclusterareequallycorre-
latedwitheachother,inordertomodelthecorrelationamongmeasurementsfromthe
samesample.Toensureresidualnormality,someresponsedatawerelogtransformedpriorto
enteringthemodel.Thepresenceofinteractionsforcombinedanti-inflammatorytreatment
effectswasdeterminedusingtheLoewedefinitionofadditivity[29],calculatingahierarchyof
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
interactionindicestobestfitthesynergisticprofile,asdescribedbyHarbron[30].95%confi-
denceintervalsforinteractionindiceswerereported.
FlowcytometricMFIdatawerepresentedasfoldchangesnormalizedtoTLRAstimulation
alone.Linearmixedmodelswereemployedtoanalyzeeachcombinationofstimulationand
targetanalyteindependently.Thecovariancestructureamongdifferenttreatmentconditions
withinthesamesubjectwasmodeledascompoundsymmetry.Toensureresidualnormality,
theresponsedatawerelogtransformedbeforeenteringthemodel.Real-timePCRderived
ΔΔC valueswereusedforallgeneexpressionanalyses.Linearmixedmodelswereemployed
T
toanalyzeeachcombinationofstimulationconditionandgeneindependently.Thecovariance
structureamongdifferenttimepointswithinasubjectwasmodeledascompoundsymmetry.
SincemRNAexpressionformostgenesdidnotsignificantlydifferbetweennewbornsand
adults,samplesfrombothcohortswereanalyzedcombinedunlessotherwiseindicated.For
graphicpresentationonly,relativemRNAexpressionvaluesofsamplestreatedwithanti-
inflammatoryagentswerecalculatedfromΔΔC valuesandnormalizedtoTLRAstimulation
T
alone,whichwasdefinedas100%.SAS(SASInstitute;Cary,NC)statisticalsoftwarewasused
foranalysesandGraphPadPrismVersion6.01(GraphPadSoftware;SanDiego,CA)for
graphingofresults.Duetotheexploratorynatureofthisstudy,nomultipletestingadjustment
wasimplemented[31].Allstatisticaltestsweretwo-sidedandfindingswereconsideredsignifi-
cantatp<0.05.
Results
PTX,DEX,andAZIinhibitedTLR-and/orinflammasome-mediated
cytokineproductioninhumannewbornandadultbloodinadistinctand
stimulus-dependentpattern
PTX,DEX,andAZIwereusedwithinclinicallyrelevantconcentrationrangesinnewbornand
adultwholebloodassays[24–27].AlthoughPTX,DEX,andAZI(S1Fig)exertedminoreffects
oncytokineproductioninunstimulatednewbornandadultblood,theoverallquantitative
effectsweresmallincomparisontothoseobservedinstimulatedbloodsamples.Ofnote,low
concentrationofDEXinduceda3-foldincreaseinIL-1βproductioninunstimulatedblood,
whereashighconcentrationofDEXsuppressedtheproductionofthiscytokine.IFN-αcon-
centrationswereundetectableinunstimulatednewbornandadultblood.
Asexpected,whentestedintheabsenceofanti-inflammatoryagents,LPS,R848,andLPS/
ATPinducedcytokineproductioninnewbornandadultblood(S1Table).Cytokineconcen-
trationsobtainedfromsamplesexposedtoanti-inflammatoryagentswerenormalizedtosam-
plesstimulatedwithTLRAsalone(seeS3Datafileforabsolutecytokineconcentrations).PTX
inhibitedTLR-and/orinflammasome-mediatedpro-inflammatorycytokineproductionin
newbornbloodinaconcentration-dependentmanner,withTNFproductioninhibitedmost
effectivelyandpotentlyamongthemeasuredcytokines(Fig1a).DEXinhibitedTLR-and/or
inflammasome-mediatedTNF,IL-1β,IL-6,andIFN-αinnewbornbloodinaconcentration-
dependentmannerandwithcomparableefficacy,whereasthisagentinhibitedIL-10with
diminishedefficacyinnewbornbloodandincreasedIL-10inadultblood(Fig1b,S2Table).
AZIdecreasedtheproductionofR848-inducedcytokinesexceptIFN-α,andathigherconcen-
trations(10and20μM)inhibitedLPS-inducedproductionofIL-1βandIL-10innewborn
blood,respectively(Fig1candS2Table).LPS/ATP-inducedIL-1βproductionwasincreased
innewbornbloodtreatedwithAZI,whileLPS/ATP-inducedIL-6andIL-10wereinhibitedat
thehighestconcentrationofAZI(20μM)(Fig1candS2Table).
PTX,DEX,andAZIallmodestlydecreasedtheproductionoftheanti-inflammatorycyto-
kineIL-10innewbornblood,withlowerefficacyandpotencythanthemoreprofound
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
Fig1.PTX,DEXandAZIinhibitLPS-,R848-,andLPS/ATP-inducedcytokineproductioninnewborncordblood.Humancordblood(n=10)
waspretreatedwith(a)PTX(50–200μM),(b)DEX(10−10–10−7M),(c)AZI(2.5–20μM)orvehiclecontrolfor2hours.Sampleswerestimulatedwith
10ng/mlLPS,1μg/mlR848,orLPSfollowedby5mMATPforinflammasomeinduction,andculturedfor6hoursat37˚Cin5%CO2.Supernatant
cytokineconcentrationsweremeasured,andresultsexpressedasapercentage(mean±SEM)comparedtoTLRAsalone,whichweredefinedas100%.
Thecorrespondingmeancytokineconcentrationsinpg/mlinducedbyTLRAsalonewererepresentedinbracketsnexttoeachcytokineaspartofthe
graphlegends.
https://doi.org/10.1371/journal.pone.0196352.g001
inhibitionofpro-inflammatorycytokinesTNFinPTX-,TNF,IL-1β,IFN-α,andIL-6inDEX-,
andIL-1βinAZI-treatednewbornblood(Fig1,S2Fig).Thustheoveralleffectoftheseagents
wastoshifttheratioofcytokinestowardsamoreanti-inflammatoryresponse.Incontrast,in
adultblood,PTXandDEXincreasedLPS-andLPS/ATP-inducedIL-10production(S2Fig
andS2Table).Aswehavepreviouslyreported,PTXinhibitedTLR-and/orinflammasome-
mediatedTNFaswellasR848-inducedIL-1βandIL-6withgreaterefficacyandpotencyin
newborncomparedtoadultblood[20].Similarly,PTXinhibitedR848-inducedIFN-αproduc-
tionwithgreaterefficacyinnewborncomparedtoadultblood(S2Fig).Withtheexceptionof
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
Table1. Stimulus-dependentinhibitionofcytokineproductioninnewbornbloodbyPTX,DEX,andAZI.
Cytokine Anti-inflammatory ComparisonofTLRagonist Estimatedlog-differenceinslopeofcytokineproductionresponsecurves p-valuea
agent stimulations (95%CI)
TNF PTX LPS+ATPvsR848 -2.30(-3.38,-1.21) <.0001
LPSvsR848 -2.07(-3.15,-0.99) 0.0003
DEX LPS+ATPvsR848 -7.58(-10.51,-4.64) <.0001
LPSvsR848 -7.07(-9.92,-4.23) <.0001
AZI LPS+ATPvsR848 0.04(0.03,0.05) <.0001
LPSvsR848 0.04(0.03,0.05) <.0001
IL-1β PTX LPS+ATPvsR848 3.10(2.53,3.68) <.0001
LPSvsR848 2.07(1.49,2.64) <.0001
DEX LPS+ATPvsR848 -8.24(-10.90,-5.59) <.0001
LPSvsR848 -12.14(-14.71,-9.56) <.0001
AZI LPS+ATPvsR848 0.07(0.06,0.08) <.0001
LPSvsR848 0.05(0.04,0.05) <.0001
IL-6 PTX LPS+ATPvsR848 -0.93(-1.39,-0.47) 0.0001
LPSvsR848 -0.82(-1.27,-0.36) 0.0006
DEX LPS+ATPvsR848 -9.88(-11.84,-7.91) <.0001
LPSvsR848 -8.38(-10.29,-6.48) <.0001
AZI LPS+ATPvsR848 0.01(0.00,0.01) 0.0027
LPSvsR848 0.01(0.01,0.02) <.0001
IL-10 PTX LPS+ATPvsR848 0.23(-0.09,0.54) 0.1604
LPSvsR848 -0.07(-0.39,0.24) 0.6406
DEX LPS+ATPvsR848 -7.06(-8.24,-5.88) <.0001
LPSvsR848 -6.06(-7.21,-4.92) <.0001
AZI LPS+ATPvsR848 0.02(0.01,0.02) <.0001
LPSvsR848 0.02(0.02,0.02) <.0001
Cordblood(n=10)waspretreatedwithincreasingconcentrationsofPTX(50–200μM),DEX(10−10–10−7M),AZI(2.5–20μM)orvehiclecontrolfor2hours.Samples
werestimulatedwith10ng/mlLPS,1μg/mlR848,orLPSfollowedby5mMATPforinflammasomeinduction,andculturedfor6hoursat37˚Cin5%CO2.
Supernatantcytokineconcentrationsweremeasured,andresultswerenormalizedtoTLRAsalone,definedas100%.Table1comparesthechangingrateswith
increasingdrugconcentrations(i.e.,theestimatedlogdifferenceinslope)ofLPS-orLPS/ATP-inducedcomparedtoR848-inducedcytokineproductionresponse
curvesforeachanti-inflammatoryagent.Linearmixedmodelsweredevelopedtoanalyzeeachcytokineandanti-inflammatorytreatmentindependently.Drug
concentrationswereanalyzedascontinuousvariables.Tomodelthecorrelationamongmeasurementsfromthesamesample,thecovariancestructurebetween
stimulationconditionswithinthesamesubjectwasmodeledascompoundsymmetry,whichassumesthatalldistinctmembersofaclusterareequallycorrelatedwith
eachother.Toensureresidualnormality,datawerelogtransformedbeforeenteringthemodel.Confidenceinterval(CI).
ap-valueswerebasedonlinearmixedmodelt-tests.
https://doi.org/10.1371/journal.pone.0196352.t001
inhibitionofTLR-and/orinflammasome-mediatedIL-10productioninnewbornbutnotin
adultbloodtreatedwithDEX(S2Fig),AZIandDEXdidnotexhibitanydifferencesininhibi-
tionofcytokineresponsesbetweennewbornandadultbloodsamples.
Furthermore,assummarizedinTable1,allagentstesteddemonstratedstimulus-dependent
inhibitionofcytokineproduction.WhereasDEXpreferentiallyinhibitedLPS-andLPS/ATP-
inducedcytokineproductioninnewbornblood,AZIshowedsignificantlygreaterinhibition
ofR848-inducedcytokineswithlittleeffectonLPS/ATP-inducedinflammatorycytokinepro-
duction.PTX,ontheotherhand,demonstratedenhancedinhibitionofLPS-andLPS/ATP-
inducedcomparedtoR848-inducedTNFandIL-6production,butgreaterinhibitionof
R848-inducedcomparedtoLPS-andLPS/ATP-inducedIL-1βproductioninnewborncord
blood.
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
EffectoftimingofadditionofPTX,DEX,andAZIoninhibitionofTLR-
mediatedcytokineproductioninnewbornandadultblood
Inordertoassesstheimpactofthetimingofadministrationofanti-inflammatoryagents,we
furtherdeterminedifdelayedadditionofanti-inflammatoryagentsinrelationtoTLRstimula-
tionascomparedtopretreatmentorsimultaneousadministrationwouldstillbeeffective.PTX
(50or200μM)andDEX(10−8or10−7M)inhibitedTNF,IL-1β,andIL-6productioninnew-
bornandadultbloodwhetheradded2hoursbefore,simultaneously,or2hoursafterLPS(Fig
2)andR848stimulation(Fig3),albeitwithdiminishedefficacyandpotencywithdelayed
Fig2.Timingofanti-inflammatorytreatmenteffectsextentofinhibitionofLPS-inducedinflammatorycytokineproduction.Newborncordand
adultperipheralbloodwerestimulatedwith10ng/mlLPS.SamplesweretreatedwithPTX(50or200μM),DEX(10−8or10−7M),AZI(2.5or20μM)
orvehiclecontrol,whichwereaddedeither2hoursbefore,simultaneously,or2hoursafterTLRstimulation.FollowingLPSstimulation,sampleswere
culturedforfurther6hours.SupernatantcytokineswereexpressedasapercentagecomparedtoTLRAsalone,definedas100%.Fordelayedtreatment
samples,cytokineconcentrationsmeasuredatthestartofanti-inflammatorytreatmentweresubtractedfromthecorrespondingdelayedtreatment
samples,inordernottounderestimatethedelayedtreatmenteffects.Effectsoftimingandconcentrationof(a)PTX[Fig2aadaptedfrom20,SpeerEM,
etal.PediatrRes.2017;81:806–816],(b)DEX,and(c)AZIonLPS-inducedcytokineproduction(mean±SEM)innewbornandadultbloodcombined.
Todeterminesignificantdifferencesbetweentimepoints((cid:3)p<0.05,(cid:3)(cid:3)p<0.01,and(cid:3)(cid:3)(cid:3)p<0.001),linearmixedmodelswereemployedtoanalyzeeach
cytokineandstimulationindependently.Thecovariancestructurebetweenanti-inflammatoryconcentrationswithinasubjectwasmodeledas
unstructuredvariance,andthecovariancestructureamongtimepointswithinthesameconcentrationwasmodeledascompoundsymmetry.
Significantchangesincytokineproductionofsamplestreatedwithhigh(lightgraycolumns)orlow(darkgraycolumns)concentrationsofanti-
inflammatoryagentsvs.untreatedsamples(blackcolumns),calculatedfromlogtransformedrawpg/mlvalues,wereindicatedas#.Linearmixed
modelsweredevelopedtoanalyzeeachcytokineandtimepointindependentlyasdescribedabove,withp<0.05consideredsignificant.N=5perage
group.
https://doi.org/10.1371/journal.pone.0196352.g002
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Pentoxifylline,dexamethasoneandazithromycinsynergisticallyinhibitcytokinesinhumancordblood
Fig3.Timingofanti-inflammatorytreatmenteffectsextentofinhibitionofR848-inducedinflammatorycytokineproduction.Newborncordand
adultperipheralbloodwerestimulatedwith1μg/mlR848.SamplesweretreatedwithPTX(50or200μM),DEX(10−8or10−7M),AZI(2.5or20μM)
orvehiclecontrol,whichwereaddedeither2hoursbefore,simultaneously,or2hoursafterTLRstimulation.FollowingR848stimulation,sampleswere
culturedforfurther6hours.SupernatantcytokineswereexpressedinpercentcomparedtoTLRAsalone,definedas100%.Fordelayedtreatment
samples,cytokineconcentrationsmeasuredatthestartofanti-inflammatorytreatmentweresubtractedfromthecorrespondingdelayedtreatment
samples,inordernottounderestimatethedelayedtreatmenteffects.Effectsoftimingandconcentrationof(a)PTX[Fig3aadaptedfrom20,SpeerEM,
etal.PediatrRes.2017;81:806–816],(b)DEX,and(c)AZIonR848-inducedcytokineproduction(mean±SEM)innewbornandadultbloodcombined.
Todeterminesignificantdifferencesbetweentimepoints((cid:3)p<0.05,(cid:3)(cid:3)p<0.01,and(cid:3)(cid:3)(cid:3)p<0.001),linearmixedmodelswereemployedtoanalyzeeach
cytokineandstimulationindependently.Thecovariancestructurebetweenanti-inflammatoryconcentrationswithinasubjectwasmodeledas
unstructuredvariance,andthecovariancestructureamongtimepointswithinthesameconcentrationwasmodeledascompoundsymmetry.
Significantchangesincytokineproductionofsamplestreatedwithhigh(lightgraycolumns)orlow(darkgraycolumns)concentrationsofanti-
inflammatoryagentsversusuntreatedsamples(blackcolumns),calculatedfromlogtransformedrawpg/mlvalues,wereindicatedas#.Linearmixed
modelsweredevelopedtoanalyzeeachcytokineandtimepointindependentlyasdescribedabove,withp<0.05consideredsignificant.N=5perage
group.
https://doi.org/10.1371/journal.pone.0196352.g003
treatment.AZI(2.5or20μM),ontheotherhand,inhibitedLPS-andR848-inducedIL-1βand
R848-inducedTNFandIL-6productionwhenadded2hoursbefore,simultaneously,or2
hoursafterTLRAstimulation,butdidnotdiminishLPS-inducedTNFandIL-6production
(Figs2and3).
(PTX+DEX/AZI)synergisticallyinhibitedTLR/inflammasome-mediated
cytokineproductioninnewbornandadultblood
Synergisticdrugcombinationsmayprovidecomparabletreatmenteffectsatlowerconcen-
trationsoftheindividualcompounds,thuspotentiallyminimizingadverseeffects.The
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