immunostimulatoryvivox7和oppor9哪个好

1MelatoninsuppressesTLR9-triggeredproinflammatorycytokineproductioninmacrophagesbyinhibitingERK1/2andAKTactivationXiongfeiXu1,2,GuoquanWang3,LinglingAi4,JianhuiShi1,JingZhang5&Yu-XiaChen1Toll-likereceptor(TLR)signalingplaysmajorrolesininnateimmuneresponseinmacrophages.
MelatoninregulatesTLR3-andTLR4-mediatedinnateimmuneresponsesinmacrophages.
However,itremainsunknownwhethermelatoninregulatesTLR9-mediatedinnateimmuneresponsesinmacrophages.
HerewedemonstratedthatmelatoninsuppressedTLR9ligand-inducedproinflammatorycytokinesmRNAandproteinproductioninperitonealmacrophageswithoutinterruptingtheviabilityofperitonealmacrophages.
UsingamelatoninmembranereceptorsMT1/MT2antagonistluzindole,wefoundthatMT1andMT2weredispensableformelatonin'sinhibitoryeffectsonTLR9-mediatedproinflammatorycytokinesproduction,eventhoughmelatoninupregulatedmRNAexpressionofMT1andMT2inmacrophages.
Furthermore,melatonindidnotaffectmRNAexpressionsofTLR9andMyD88butattenuatedTLR9ligand-inducedERK1/2andAKTphosphorylationwithoutaffectingp38andNF-κBp65phosphorylation.
Also,melatonininhibitedTLR9-mediatedproinflammatorycytokinesproductioninvivo.
Takentogether,ourresultsdemonstratethatmelatoninsuppressesTLR9-triggeredproinflammatorycytokinesproductioninmacrophagesviamelatoninmembranereceptor-independentmannersandprobablythroughinhibitingERK1/2andAKTactivation,whichfurtherelucidatestherolesofmelatonininregulatingTLR-mediatedinnateimmuneresponsesinmacrophages.
Toll-likereceptor(TLR),animportantpattern-recognitionreceptor(PRR),iscriticalforhostdefenceagainstinvadingpathogens1,2.
TLRsinitiateinnateimmuneresponsesthroughactivatinginnateimmunecellssuchasmacrophagesanddendriticcells(DCs)1,2.
TLRfamilymembersrecognizedifferentcomponentsofpathogens,whicharereferredtoaspathogen-associatedmolecularpatterns(PAMPs),andeachTLRhasadistinctfunc-tionintermsofPAMPrecognitionandimmuneresponses1,3.
Forinstance,TLR4,TLR3andTLR9recognizelipopolysaccharide(LPS)fromgram-negativebacteria,double-strandedRNAfromvirus,andCpGDNAfrombacteriaandviruses,respectively1,3.
LigationofTLRsbyPAMPsinducestheactivationofintracellularsigna-lingmoleculessuchasMyD88andTRIF,whichinturnleadtotheproductionofnuclearfactor-κB(NF-κB)andmitogen-activatedproteinkinases(MAPKs)-dependentpro-inflammatorycytokines,ortheproductionofinterferonregulatoryfactor(IRF)-dependenttypeIinterferons(IFNs)1–3.
WithregardtoTLR9,itundergoesproteolyticcleavageinendosomesafterligationbyCpGDNA;thecleavedTLR9thenrecruitsMyD88toactivateNF-κBandMAPKsandtoinduceproductionofpro-inflammatorycytokines,orinduceproductionoftypeIIFNbyactivatingIRF71,3.
AlthoughTLRsareessentialforhostprotectiveimmunityagainstpathogens,inappropriateTLRsactivationcanleadtoprolongedinflammationandevenautoimmuneandinflammatorydiseases1,2.
Hence,1DepartmentofPathophysiology,SecondMilitaryMedicalUniversity,Shanghai,200433,China.
2DepartmentofHepatobiliaryPancreaticSurgery,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai,200433,China.
3DepartmentofNeurology,JunkangHospital,Shanghai,200125,China.
4DepartmentofOtolaryngology,No.
455HospitalofPLA,Shanghai,200052,China.
5DepartmentofPathology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai,200433,China.
XiongfeiXu,GuoquanWangandLinglingAicontributedequally.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoX.
X.
(email:xiongfeixu@126.
com)orJ.
Z.
(email:zhangjing@smmu.
edu.
cn)Received:1June2018Accepted:8October2018Published:xxxxxxxxOPEN2signalingmoleculesorsubstancesthatfine-tuneTLRpathwayshavebeenreceivingincreasedattentioninrecentyears.
ManymoleculeshavebeenfoundtonegativelyregulateTLRsignalingbydifferentmechanisms.
Thesemoleculesincludecellmembraneorintracellularmolecules,andevenintranuclearepigeneticmodifiers2,4–7.
Inapreviousstudy,Stk38proteinkinasewasfoundtopreferentiallyinhibitTLR9-activatedinflammatoryresponsesbypromotingMEKK2ubiquitinationinmacrophages4.
Furthermore,varioussubstanceshavebeenfoundtoregulateTLRsignaling,suchasmicheliolide,vasoactiveintestinalpeptide,andmelatonin8–12.
Melatoninismainlyproducedbythepinealgland,butextrapinealtissues,suchasgastrointestinaltract,retina,spleen,liverandkidney,andendothelialcellsorimmunecellscanalsoproducemelatonin13,14.
Melatoninhasawiderangeoffunctions,whicharedividedintochronobioticandnonchronobioticones13.
Thechronobioticeffectsregulatethecircadianrhythmsandseasonalchanges,andaremediatedbymelatonininthecirculationduetonocturnalpinealsynsthesis,whereasmelatoninproducedbyothercells,suchasgastrointestinalandimmunecells,isindependentofthelight/darkcycleandexertsnonchronobioticeffects,includingantioxidant,oncostatic,antiaging,andimmunomodulatoryeffects13.
Theeffectsofmelatonininthebodyaremediatedmainlybytwopathways:melatoninreceptor-dependentpathwayandmelatoninreceptor-independentpathway14,15.
MelatoninreceptorsincludemembranereceptorsMT1/MT2,andnuclearreceptorsRORα/RZR14,15.
Increasingevidencesdemonstratethatmelatoninplaysafundamentalroleinregulatingimmuneresponse,aswellasinfection,sepsis,inflammation,andautoimmun-ity13,15–18.
Recently,moreandmorestudiesshowthatmelatoninmodulatesthefunctionsofinnateimmunecellssuchasmacrophages,monocytes,naturalkillercellsandneutrophils13,17.
Inparticular,melatoninregulatesTLR3andTLR4signalingsinmacrophages8–10,13,17.
However,itremainstobeelucidatedwhethermelatoninregulatesTLR9-mediatedinnateimmuneresponsesinmacrophages.
Inthisstudy,weobservedthatmelatoninsuppressedTLR9ligand-inducedproinflammatorycytokinesproductioninprimaryperitonealmacrophages.
Moreover,itssuppressiveeffectsonTLR9signalingdidnotdependonmelatoninmembranereceptorsMT1andMT2,butprobablythroughinhibitingERK1/2andAKTactivationwithoutaffectingmRNAexpressionsofTLR9andMyD88.
Furthermore,melatoninalsodecreasedTLR9-mediatedTNF-αandIL-6productioninmouseserum.
Overall,theresultsofourstudyclarifytheinhib-itoryeffectsofmelatoninonTLR9-mediatedinnateimmuneresponsesinmacrophages,probablythroughsup-pressingERK1/2andAKTactivation.
ResultsMelatoninsuppressesTLR9ligand-inducedproinflammatorycytokinesproductioninmac-rophages.
ToinvestigatewhethermelatonincouldregulateTLR9-mediatedinflammatoryresponsesinmacrophages,primaryperitonealmacrophageswerepre-treatedwithmelatoninandthenstimulatedwithTLR9ligandCpG-ODN.
First,weinvestigatedtheeffectsofmelatoninonmRNAexpressionsofinflammatorycytokinesinmacrophagesinducedbyCpG-ODN.
AsshowninFig.
1A–F,CpG-ODNinducedTNF-α,IL-6,IL-12p35,IL-12p40andIL-10mRNAexpressionspromptly,whileIFN-βmRNAexpressionwerenotincreasedsignif-icantlyafterCpG-ODNstimulation.
Interestingly,1mMmelatoninsignificantlyattenuatedCpG-ODN-inducedupregulationofTNF-α,IL-6,IL-12p35,IL-12p40andIL-10mRNAexpressionsbutdidnotaffectIFN-βmRNAexpression(Fig.
1A–F),demonstratingthatmelatonincouldsuppressmRNAexpressionsofCpG-ODN-inducedinflammatorycytokines.
ThenweassessedtheeffectsofmelatoninonproteinexpressionofinflammatorycytokinesinmacrophageinducedbyCpG-ODN.
AsshowninFig.
2A–D,2μg/mlCpG-ODNsignificantlyinducedtheproductionofTNF-α,IL-6,IL-12p70andIL-10inmacrophages,whereas,asexpected,itinducednoproductionofINF-β(Datanotshown).
MelatonineffectivelysuppressedtheproductionofTNF-α,IL-6andIL-12p70inducedbyCpG-ODNinadose-dependentmanner(Fig.
2A–C),whereasitexertednonotablesuppressiononanti-inflammatoryIL-10production(Fig.
2D).
Ofnote,melatoninalonecouldinhibittheconstitutiveIL-10productioninmacrophages(Fig.
2D).
TheresultsaboveindicatethatmelatonincouldmarkedlysuppressTLR9-mediatedinflammatoryresponsesinmacrophages.
Notably,melatonininhibitedIL-10mRNAexpressionbutnotIL-10proteinexpressioninCpG-ODN-stimulatedmacrophages(Figs1Eand2D),indicatingthepossibleposttranscriptionalregulationbymelatonin.
TheresultalsoshowedthatmelatoninsuppressedTLR9-mediatedproinflammatorycytokinespro-ductioninmacrophagesnotthroughupregulationofanti-inflammatoryIL-10.
Toexcludethepossibilitythatmelatoninmayaffectcellviabilityofmacrophages,weusedAnnexinV/7AADstainingtoexaminetheapoptosisofprimaryperitonealmacrophagesaftermelatoninand/orCpG-ODNtreat-ment.
AsshowninFig.
3A,B,1mMmelatonindidnotincreasetheapoptoticcellpercentageofmacrophagesirrespectiveofCpG-ODNstimulation,indicatingthatmelatoninhadnocytotoxiceffectsonmacrophagesandthesuppressionofTLR9-mediatedproinflammatorycytokinesproductioncouldnotbeattributabletoadirectcytotoxiceffectbymelatonin.
MelatoninmembranereceptorsMT1andMT2aredispensableforthesuppressiveeffectsofmelatonin.
MelatoninexertsavarietyofphysiologicalandpharmacologicaleffectsontheimmunesystemthroughactivationoftwomelatoninmembranereceptorsMT1andMT2,whichbelongtoGprotein-coupledreceptors(GPCRs)19.
WefoundthatperitonealmacrophagesexpressedmelatoninmembranereceptorsMT1andMT2,andmelatonintreatmentupregulatedmRNAexpressionsofMT1andMT2inmacrophages,whereasCpG-ODNstimulationdidnotaffectmRNAexpressionsofMT1andMT2(Fig.
4A,B).
ToinvestigatewhetherMT1andMT2wererequiredforthesuppressiveeffectsofmelatoninonTLR9-mediatedinflammatorycytokinesproduction,luzindole,anonselectiveantagonistofbothMT1andMT2,wasused.
AsshowninFig.
5A–D,routineconcentrationofluzindole(10μM)20,21didnotreversetheinhibitoryeffectsofmelatoninonCpG-ODN-inducedTNF-α,IL-6andIL-12p70production,andevenfurtherenhancedthesuppressionfunctionofmelatoninon3TLR9-mediatedTNF-αandIL-6production.
Theseresultsdemonstratedthatmelatoninmembranerecep-torsMT1andMT2weredispensableforthesuppressiveeffectsofmelatoninonTLR9-mediatedinflammatorycytokinesproductioninmacrophages.
MelatonininhibitsTLR9ligand-inducedERK1/2andAKTactivation.
TLR9agonistCpG-ODNinducesproinflammatorycytokinesproductionbyactivatingMyD88-dependentMAPK,PI-3K/AKTandNF-κBpathways1,22.
ToinvestigatethemechanismbywhichmelatoninsuppressedCpG-ODN-inducedinflammatorycytokinesproduction,wefirstdetecteditseffectsonTLR9andMyD88expression,andfoundthat1mMmel-atonintreatmentdidnotaffectmRNAexpressionofTLR9andMyD88inprimaryperitonealmacrophages,irrespectiveofCpG-ODNstimulation(Fig.
6A,B).
Wethenstudiedmelatonin'seffectsonCpG-ODN-inducedMAPK,AKTandNF-κBactivationinperitonealmacrophages.
AsshowninFig.
7A,2μg/mlCpG-ODNremark-ablyinducedERK1/2,p38,AKT,andNF-κBp65phosphorylationwhileitdidnotinduceJNKphosphorylation.
Interestingly,melatoninselectivelyattenuatedCpG-ODN-inducedERK1/2andAKTphosphorylation,buthadnoeffectsonp38andNF-κBp65phosphorylation(Fig.
7A–C).
TheseresultssuggestedthatmelatoninsuppressedCpG-ODN-inducedinflammatorycytokinesproductionprobablybyinhibitingERK1/2andAKTactivation.
MelatonininhibitsTLR9-mediatedproinflammatorycytokinesproductioninvivo.
ToexploretheroleofmelatonininregulatingTLR9signalinginmacrophagesinvivo,C57BL/6Jmicewereintraperitone-allyinjectedwithCpG-ODNinthepresenceorabsenceofmelatoninpretreatmentaftermobilizingperitonealmacrophagesthroughthioglycollate(TG)treatment,andthenthelevelsofTNF-αandIL-6inmouseserumweredetected.
AsshowninFig.
8A,B,CpG-ODNinducedhighlevelsofTNF-αandIL-6inserumandmelatoninpretreatmentsignificantlyinhibitedCpG-ODN-inducedTNF-αandIL-6productioninserum,indicatingthatmelatonininhibitedTLR9-mediatedproinflammatorycytokinesproductioninvivo.
DiscussionIncreasingevidencesshowthatmelatoninnotonlyexertsanimmunosuppressiveeffectonmacrophagesbutalsoexertsanimmunostimulatoryeffectonmacrophages,possiblydependingonthemannersofmelatoninadmin-istrationorthedoseofmelatonin13.
Forexample,ithasbeenreportedthatmelatoninstimulatedasignificantincreaseofIL-12productioninhumansynovialmacrophagesandTHP-1cells,however,asignificantreductionofIL-12productionwasobservedwhenmacrophageswerepreviouslyactivatedwithLPSandthentreatedwithmel-atonin23.
RegardingTLRssignaling,ithasbeendemonstratedthatmelatoninmainlyexertsasuppressiveeffectonTLRssignaling,includingTLR3andTLR4signalings8–10,13.
Inthepresentstudy,wedemonstratedforthefirstFigure1.
TheeffectsofmelatoninonmRNAexpressionsofcytokinesmediatedbyTLR9inperitonealmacrophages.
Peritonealmacrophageswerepre-treatedwith1mMMTorsolventcontrol(ethanol,E)for1hourandthenstimulatedwith2μg/mlCpG-ODNfor0,1.
5or3hours.
TNF-α(A),IL-6(B),IL-12p35(C)IL-12p40(D),IL-10(E)andIFN-β(F)mRNAexpressionsweredetectedbyreal-timePCR.
Dataareshownasmean±s.
d.
(n=3).
*P<0.
05,**P<0.
01(Student'st-test).
Similarresultswereobtainedinthreeindependentexperiments.
4Figure2.
MelatoninsuppressesTLR9-mediatedinflammatorycytokinesproductioninperitonealmacrophages.
Peritonealmacrophageswerepre-treatedwithvariousconcentrationsofMTorEfor1hourandthenstimulatedwith2μg/mlCpG-ODNfor24hours.
TNF-α(A),IL-6(B),IL-12p70(C)andIL-10(D)inthesupernatantswereassayedbyELISA.
Dataareshownasmean±s.
d.
(n=3).
*P<0.
05,**P<0.
01(ANOVA).
Similarresultswereobtainedinthreeindependentexperiments.
Figure3.
Melatonindoesnotinfluencetheviabilityofperitonealmacrophages.
(A)Peritonealmacrophageswerepre-treatedwith1mMMTorEfor1hourandthenstimulatedwith2μg/mlCpG-ODNfor24hours.
CellswerestainedwithannexinV/7AADandtheapoptosiswasassayedbyflowcytometry.
NumbersinplotsindicatethepercentageofannexinV7AADcells(livecells),annexinV+7AADcells(earlyapoptoticcells)andannexinV+7AAD+cells(lateapoptoticcells)inmacrophages.
(B)ThepercentageoflivecellsinAwereanalyzedstatisticallyandsummarized.
Similarresultswereobtainedinthreeindependentexperiments.
5timethatmelatoninsuppressedTLR9-mediatedinflammatoryresponsesinmacrophages,indicatingthatmela-toninwasanegativeregulatorofTLR9-mediatedinnateimmunityinmacrophages.
Althoughmelatoninupreg-ulatedmelatoninmembranereceptorsMT1andMT2mRNAexpressioninmacrophages,MT1andMT2weredispensableforthesuppressiveeffectsofmelatoninonTLR9-mediatedinflammatoryresponses.
Furthermore,Figure4.
MelatoninupregulatesmelatoninmembranereceptorsMT1andMT2expressionsinperitonealmacrophages.
Peritonealmacrophageswerepre-treatedwith1mMMTorEfor1hourandthenstimulatedwith2μg/mlCpG-ODNforindicatedtimes.
MelatoninmembranereceptorsMT1(A)andMT2(B)mRNAexpressionsweredetectedbyreal-timePCR.
Dataareshownasmean±s.
d.
(n=3).
*P<0.
05,**P<0.
01(Student'st-test).
Similarresultswereobtainedinthreeindependentexperiments.
Figure5.
MT1andMT2aredispensableforthesuppressiveeffectsofmelatoninonTLR9signaling.
Peritonealmacrophageswereincubatedwith10μMluzindole(anonselectiveantagonistofmelatoninmembranereceptorsMT1/MT2)for1hourandthentreatedwith1mMMTorEfor1hour.
Afterthat,cellswerestimulatedwith2μg/mlCpG-ODNfor24hours.
TNF-α(A),IL-6(B),IL-12p70(C)andIL-10(D)inthesupernatantswereassayedbyELISA.
Dataareshownasmean±s.
d.
(n=3).
"ns"indicatesnosignificance,*P<0.
05(ANOVA).
Similarresultswereobtainedinthreeindependentexperiments.
6melatonindidnotaffectTLR9andMyD88mRNAexpression,butpossiblyattenuatedERK1/2andAKTactiva-tiontosuppressTLR9signaling.
AfterstimulationwithTLRligandssuchasLPS,polyI:CorCpG-ODN,activatedmacrophagesproducevar-iouscytokinesincludingTNF-α,IL-6,IL-12,IL-1β,IL-10,andtype1interferonincludingIFN-αandIFN-β.
TLR9ligandCpG-ODNinducesmacrophagesanddendriticcellstosecretTNF-α,IL-6,IL-12,IL-10andIFN-α/β(mainlyproducedbyplasmacytoiddendriticcells)24,25.
ThepresentstudydemonstratedthatmelatoninsuppressedthemRNAandproteinexpressionofTNF-α,IL-6andIL-12p70inducedbyCpG-ODN,indicatingthatmelatonincouldsuppressTLR9-mediatedproinflammatorycytokinesproductioninmacrophages.
Similarly,previousstudiesshowedthatmelatoninsuppressedLPS-inducedTNF-α,IL-6,IL-1β,andIL-8expressioninRAW264.
7cells9,10.
IL-10inducedbyCpG-ODN,ananti-inflammatorycytokine,couldsuppressIL-12p70pro-ductioninmacrophages26.
OurpresentstudydemonstratedthatmelatonindidnotsuppressorpromoteIL-10proteinproductioninducedbyCpG-ODN,whichisdifferentfromanotherstudyshowingthatmelatoninsup-pressedLPS-inducedIL-10expression10.
ThisindicatesthatIL-10isnotinvolvedintheinhibitoryeffectsofmel-atoninonTLR9-mediatedinflammatoryresponsesinmacrophages.
Themolecularmechanismsofactionofmelatoninmainlyincludethefollowingfourmodels:(i)viaitsinter-actionwithmembranereceptorssuchasMT1andMT2(atlowconcentration),(ii)bybindingtoitsnuclearreceptorssuchasRORαandRZR(atlowconcentration),(iii)viaaninteractionwithcytoplasmicproteinssuchascalmodulinandhydroquinone(athighconcentration),and(iv)viadirectly,receptor-independentactionssuchasscavengingROS/RNS13,15,27.
Ithasbeenshownthattheeffectsofmelatoninonimmunesystemaremedi-atedthroughmelatoninreceptor-dependentor-independentmechanisms.
Inmultiplesclerosis(MS),melatoninblocksTh17differentiationandbooststhegenerationofIL-10-secretingTregcellsbyanMT1-dependentmech-anism28,29.
MelatonininhibitstheinfectivityofLeishmaniabymurineperitonealmacrophagesandenhancesthephagocytosisofzymosanbyhumancolostrummononuclearcells,bothofwhichareattenuatedbyluzindole(anantagonistofMT1andMT2),indicatingtheinvolvementofmelatoninmembranereceptorsintheseeffects30,31.
ThepresentstudyshowedthatluzindoledidnotreversetheinhibitoryeffectsofmelatoninonTLR9-mediatedinflammatoryresponsesinmacrophages,somelatoninexertsitsinhibitoryeffectsonTLR9signalingthroughmelatoninmembranereceptor-independentway.
Giventhatmelatoninathighconcentrationalsoexertsitseffectsthroughreceptor-independentmannersinadditiontoitsinteractionwitheithermelatoninmembraneornuclearreceptors13,15,27,inthepresentstudy,high-concentrationmelatonin(reachingthelevelofmillimole)probablydirectlyregulatedtheexpressionoractivationofTLR9signalmolecules,similartoitsmechanismsinregulatingTLR3orTLR4signalinginmac-rophages8–10.
MelatonindecreasedTLR-3-mediatedTNF-αandiNOSmRNAexpressioninrespiratorysyncy-tialvirus-infectedRAW264.
7cellsviainhibitionofNF-κBactivationwithoutinfluencingTLR3andMyD88mRNAexpression8.
InLPS-stimulatedRAW264.
7cells,melatoninattenuatedtheexpressionofMyD88andtheactivationofNF-κB,andalsoinhibitedTLR4-mediatedAKTphosphorylationandattenuatedtheelevationofIRF3,whichwasinvolvedinTLR4-mediatedTRIF-dependentsignalingpathway10.
Inanotherstudy,melatonininhibitedLPSinducedproductionofnitricoxideandIL-6inRAW264.
7cellsbysuppressingNF-κBandSTAT1activation9.
Inthepresentstudy,melatonininhibitedCpG-ODN-inducedERK1/2andAKTactivationwithoutaffectingNF-κBandp38activationinmacrophages.
Furthermore,melatonindidnotaffectTLR9andMyD88mRNAexpression.
Hence,melatoninmightsuppressTLR9-mediatedinnateimmuneresponsesthroughinhib-itingERK1/2andAKTactivation.
Thisstudy,togetherwithotherpreviousstudies,indicatethatmelatoninreg-ulatesTLRsignalinginmacrophagesthroughdifferentmechanisms,dependentondifferentTLRsubtypesandtheirligands8–10.
ItremainstobefurtherinvestigatedhowmelatoninregulatesTLR9-mediatedERK1/2andAKTactivationinmacrophages.
Inourpreviousstudy,MEKK2knockdown(specificallyinhibitingERK1/2activation)significantlysuppressedCpG-ODN-inducedTNF-αandIL-6productioninmacrophages4.
Inaddition,otherstudiesalsoshowedthatwortmannin,aPI-3Kinhibitor,inhibitedtheproductionofIL-12inducedbyCpGDNAFigure6.
TheeffectsofmelatoninonmRNAexpressionsofTLR9andMyD88inperitonealmacrophages.
Peritonealmacrophageswerepre-treatedwith1mMMTorEfor1hourandthenstimulatedwith2μg/mlCpG-ODNforindicatedtimes.
TLR9(A)andMyD88(B)mRNAexpressionsweredetectedbyreal-timePCR.
Dataareshownasmean±s.
d.
(n=3).
Similarresultswereobtainedinthreeindependentexperiments.
7inbonemarrowderiveddendriticcells32,andanotherPI-3Kinhibitor,LY294002,alsoreducedTNF-αproduc-tioninducedbyCpGDNAinmacrophages33.
ThesestudiesfurthersupportthatERK1/2andAKTactivationisimportantforTLR9-mediatedinflammatorycytokinesproductioninmacrophages.
ThereisonlyonepublishedresearchreportingontherelationshipbetweenmelatoninandTLR9signaling.
ThisstudyshowedthatJNK-enhancedTLR9signalpathwaymediatedallergicairwayinflammationthroughsup-pressingmelatoninbiosynthesis34.
However,thereisnoresearchaddressingtheregulatoryfunctionofmelatoninonTLR9signaling.
OurpresentstudyshowedforthefirsttimethatmelatoninregulatedTLR9-mediatedinnateimmuneresponsesandTLR9signalingpathways.
TLR9-mediatedinnateimmunityandinflammationplaysimportantrolesininfectiousdiseases,autoimmunediseasesandcancer25,35.
Previousstudiesshowedthatpolymicrobialsepsisinmouseexperimentalperitonitismodelinducedbycaecalligationandpuncture(CLP)wasTLR9-dependent36,37.
IthasbeendemonstratedthatmelatoninprotectsmiceagainstsepticshockorsepticorgansinjuryinCLPsepsismodelsthroughdifferentmechanisms:theseincludeantioxidativeeffects,anti-nitrosativeeffects,protectiveeffectsagainstmitochondrialdysfunctionandanti-inflammatoryeffects18,38–43.
Hence,itispossiblethatmelatoninalsoprovidesprotectionsFigure7.
MelatonininhibitsTLR9-mediatedERK1/2andAKTactivationinperitonealmacrophages.
Peritonealmacrophageswerepre-treatedwith1mMMTorEfor1hourandthenstimulatedwith2μg/mlCpG-ODNforindicatedtimes.
PhosphorylatedandtotalERK1/2,JNK,p38,AKTandNF-κBp65andβ-actinweredetectedbywesternblot.
Representativeimagesareshown(A)andrelativeexpressionlevelsofphosphorylatedERK1/2andAKTinvariousgroupsareshownbythepercentageoftheirexpressionlevelsin"E+CpG0min"group(B,C).
*P<0.
05,**P<0.
01(ANOVA,MTgroupsinvarioustimesarecomparedtoEgroupsinthesametime).
Dataarerepresentativeofthreeindependentexperimentswithsimilarresults.
8againstsepsisthroughsuppressingTLR9-mediatedinflammation,whichrequirestobefurtherinvestigated.
Furthermore,ourpresentstudydemonstratedthatinvivoadministrationofmelatoninexertednotablesuppres-siveeffectsonTLR9–mediatedinflammatorycytokinesproductioninmice,furtherraisingthispossibility.
Takentogether,ourstudydemonstratedthatmelatoninsuppressedTLR9-triggeredproinflammatoryTNF-α,IL-6andIL-12p70productioninmacrophagesnotthroughmelatoninmembranereceptors-dependentmannersbutprobablythroughinhibitingTLR9-mediatedERK1/2andAKTactivation.
Theseresultsprovedthatmela-toninwasanegativeregulatorofTLR9-mediatedinnateimmunityinmacrophages.
MaterialsandMethodsMiceandreagents.
C57BL/6Jmice(female,6–8weeksold)wereobtainedfromJointVenturesSipperBKExperimentalAnimalCompany(Shanghai,China).
AllanimalexperimentswereperformedinaccordancewiththeNationalInstitutesofHealthGuidefortheCareandUseofLaboratoryAnimals,withtheapprovaloftheScientificInvestigationBoardofSecondMilitaryMedicalUniversity(Shanghai,China).
Melatonin(M5250),7-aminoactinomycin(7AAD,A9400)andluzindole(L2407)werefromSigma-Aldrich(StLouis,MO).
Phosphorothioate-modifiedCpG-ODN1668(5′-TCCATGACGTTCCTGATGCT-3′)wassynthesizedbySangonBiotechCompany(Shanghai,China).
EndotoxinintheseODNswasremovedusingEndotoxinRemovalSolution(Sigma-Aldrich)asdescribedpreviously44,andtheendotoxinlevelwas<0.
015U/mgCpG-ODN.
AntibodiesspecificforERK1/2(9107),phosphorylatedERK1/2(9101),JNK(9252),phosphorylatedJNK(9255),p38(9212),phosphorylatedp38(9211),AKT(9272),phosphorylatedAKT(4060),p65(3034),andphosphoryl-atedp65(3033)werefromCellSignalingTechnology.
AnnexinVapoptosisdetectionkitAPCwasfromeBio-science(ThermoFisherscientific).
Antibodyspecificforβ-actin(sc-1616)andHRP-coupledsecondaryantibodywerefromSantaCruz.
Cellcultureandtreatments.
Thioglycollate-elicitedmouseperitonealmacrophageswereisolatedandcul-turedaspreviouslydescribed4.
C57BL/6Jmice(6–8weeksold)wereintraperitoneallyinjectedwith1mlof3%sodiumthioglycollate(Merck)solutionpermouse.
After3days,cellsintheabdominalcavitywerecollectedbylavagingwith10–15mlFBS-freeDMEMandcentrifugated.
CellswereculturedandmaintainedinDMEMwith10%(vol/vol)FBSfor24hours.
Afterremovingthefloatingcells,adheredperitonealmacrophagesweresubjectedforfurtherexperiments.
Thecellswerepre-treatedwithvariousconcentrationsofmelatonin(stockconcentra-tion200mM,dissolvedinethanol)for1hourandthenstimulatedwith2μg/mlCpG-ODNforindicatedtimes(SupplementaryFig.
1).
Insomeexperiments,thecellswereincubatedwith10μMluzindolefor1hourbeforepretreatmentwithmelatonin(SupplementaryFig.
1C).
Detectionofcytokineproduction.
TheconcentrationsofTNF-α,IL-6,IL-12p70andIL-10inculturesupernatantsorserumweremeasuredwithELISAkitsfromR&Dsystems(Minneapolis,MN)accordingtothemanufacturer'sinstructions.
Detectionofcellapoptosis.
Aftertreatmentwithmelatoninand/orCpG-ODN(SupplementaryFig.
1B),peritonealmacrophageswereharvestedandstainedwithAnnexinV-APCand7AADaccordingtothemanufac-turer'sinstructions.
Thentheapoptosisofmacrophageswasassayedbyflowcytometry(LSRII,BDbiosciences)andthedatawereanalyzedwithFlowJoVersion5.
7.
2software(TreeStar).
Quantitativereversetranscription-PCR.
TotalRNAwasextractedwithRNAextractionkit(RNAfast200,FastagenBiotechCo.
,China)andreversed-transcriptedwithreversetranscriptaseM-MLX(Takara)followingthemanufacturer'sinstructions.
Real-timePCRwasperformedwithaSYBRRT-PCRkit(Takara)onLightCyclersystemFigure8.
MelatonininhibitsTLR9-mediatedproinflammatorycytokinesproductioninvivo.
C57BL6/Jmicewereintraperitoneallyinjectedwiththioglycollate(TG)toelicitemacrophagesonday0andthenreceived5mg/kgmelatonin(MT)orethanol(E)onday3andday4.
Onday4,micewerefurtherstimulatedbyintraperitonealinjectionof50μgCpG-ODNtwohoursaftermelatoninpretreatment.
TNF-α(A)andIL-6(B)inserumofmiceinvariousgroups(n=5)wereassayedbyELISA2hoursafterCpG-ODNinjection.
Errorbarsrepresents.
d.
**P<0.
01(ANOVA).
9(Roche).
Datawerenormalizedbythelevelofβ–actinexpressionineachindividualsample.
2Ctmethodwasusedtocalculaterelativeexpressionchanges.
ThesequencesoftheprimersusedwerefromPrimerBankasfollows:β–actinsense,5′-GGCTGTATTCCCCTCCATCG-3′andantisense,5′-CCAGTTGGTAACAATGCCATGT-3′;MT1sense,5′-TACACTGTCAAGTCAGCGCAT-3′andantisense,5′AGCAGTAACGGTTCATAGCGA-3′;MT2sense,5′-GAACAGCTCAATCCCTAACTGC-3′andantisense,5′-ACGACTACTGTAGATAGCATGGG-3′;TNF-αsense,5′-CCCTCACACTCAGATCATCTTCT-3′andantisense,5′-GCTACGACGTGGGCTACAG-3′;IL-6sense,5′-TAGTCCTTCCTACCCCAATTTCC-3′andantisense,5′-TTGGTCCTTAGCCACTCCTTC-3′;IL-12p35sense,5′-CTGTGCCTTGGTAGCATCTATG-3′andantisense,5′-GCAGAGTCTCGCCATTATGATTC-3′;IL-12p40sense,5′-TGGTTTGCCATCGTTTTGCTG-3′andantisense,5′-ACAGGTGAGGTTCACTGTTTCT-3′;IL-10sense,5′-GCTCTTACTGACTGGCATGAG-3′andantisense,5′-CGCAGCTCTAGGAGCATGTG-3′;IFN-βsense,5′-CAGCTCCAAGAAAGGACGAAC-3′andantisense,5′-GGCAGTGTAACTCTTCTGCAT-3′;TLR9sense,5′-ATGGTTCTCCGTCGAAGGACT-3′andantisense,5′-GAGGCTTCAGCTCACAGGG-3′;MyD88sense,5′-TCATGTTCTCCATACCCTTGGT-3′andantisense,5′-AAACTGCGAGTGGGGTCAG-3′.
Westernblot.
Afterstimulatedwith2μg/mlCpG-ODNforindicatedtimes(SupplementaryFig.
1D),pri-maryperitonealmacrophageswerewashedtwicewithice-coldPBSandlysedwithM-PERProteinExtractionReagent(Pierce)supplementedwithproteaseinhibitorcocktail,andthenproteinconcentrationsoftheextractsweremeasuredwithBCAassayaccordingtothemanufacturer'sinstructions(Pierce).
EqualamountsofextractswereloadedforSDS–PAGE,transferredontonitrocellulosemembranesandthenblottedwithindicatedantibod-ies,asdescribedpreviously4.
Finally,signalintensitywasdeterminedusingtheTanon5200SChemiluminescentImagingSystem(Tanon).
Imageshadbeencroppedforpresentation.
Full-sizeimageswerepresentedinSupplementaryFig.
2.
InductionofproinflammatorycytokinesbyCpG-ODNinvivo.
C57BL/6Jmice(6–8weeksold)wereintraperitoneallyinjectedwith1mlof3%sodiumthioglycollate(Merck)solutiononday0.
Onday3andday4,micewereintraperitonealinjectedwith5mg/kgmelatonin.
Onday4,micewerestimulatedbyintraperito-nealinjectionof50μgCpG-ODN2hoursaftermelatonininjection(SupplementaryFig.
1E).
TwohoursafterCpG-ODNinjection,theserumsampleswerecollectedandusedforELSIAassaysofTNF-αandIL-6.
Statisticalanalysis.
Alldatawerepresentedasmeans±standarddeviation(S.
D.
).
Thestatisticalsignifi-cancewascalculatedbyone-wayANOVAtestortwo-tailedStudent'sttestusingPrismsoftware5(GraphPadSoftware,Inc.
,SanDiego,CA).
Pvaluelessthan0.
05wasconsideredstatisticallysignificant.
DataAvailabilityAlldatageneratedoranalyzedduringthisstudyareincludedinthismanuscript.
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AcknowledgementsWethankDrHongmeiXuforcriticalrevisionofthismanuscript.
ThisworkwassupportedbythegrantsfromtheNationalNaturalScienceFoundationofChina[No.
31100630(XuX)andNo.
81502466(ZhangJ)]andNaturalScienceFoundationofShanghai[No.
18ZR1449400(XuX)].
AuthorContributionsX.
X.
contributedtothestudydesign;X.
X.
andZ.
J.
obtainedfunding;X.
X.
,W.
G.
,A.
L.
,S.
J.
,Z.
J.
andC.
Y.
X.
performedtheexperimentsandanalyzedthedata;X.
X.
wrotethemanuscript.
Alltheauthorscriticallyrevisedthearticleandhaveconsentedthefinalversionofthemanuscript.
AdditionalInformationSupplementaryinformationaccompaniesthispaperathttps://doi.
org/10.
1038/s41598-018-34011-8.
CompetingInterests:Theauthorsdeclarenocompetinginterests.
Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
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