ContentslistsavailableatScienceDirectNeuropharmacologyjournalhomepage:www.elsevier.com/locate/neuropharmAnti-inflammatoryeffectsoffluoxetineinlipopolysaccharide(LPS)-stimulatedmicroglialcellsDexiangLiua,1,ZhenWangb,1,ShangmingLiuc,FuwuWangb,ShidouZhaob,AijunHaob,*InstituteofMedicalPsychology,ShandongUniversitySchoolofMedicine,44#,WenhuaXiRoad,Jinan,Shandong250012,PRChinaKeyLaboratoryoftheMinistryofEducationforExperimentalTeratology,DepartmentofHistologyandEmbryology,ShandongUniversitySchoolofMedicine,44#,WenhuaXiRoad,Jinan,Shandong250012,PRChinacShandongProvincialKeyLaboratoryofMentalDisorders,ShandongUniversitySchoolofMedicine,44#,WenhuaXiRoad,Jinan,Shandong250012,PRChinabaarticleinfoArticlehistory:Received2December2010Receivedinrevisedform5April2011Accepted26April2011Keywords:FluoxetineMicrogliaCytokineNitricoxideNuclearfactor-kappaBabstractRecentevidencehassuggestedthatmicroglialactivationplaysanimportantroleinthepathogenesisofdepression.Activatedmicrogliacansecretevariouspro-inflammatorycytokinesandneurotoxicmedia-tors,whichmaycontributetothedevelopmentandmaintenanceofdepression.Thus,inhibitionofmicroglialactivationmayhaveatherapeuticbenefitinthetreatmentofdepression.Inthepresentstudy,usingBV2microglialcelllineandprimarymicroglialculture,weinvestigatediffluoxetine,themostwidelyusedantidepressant,caninhibitmicrogliaactivation.Ourresultsshowedthatfluoxetinesignif-icantlyinhibitedlipopolysaccharide(LPS)-inducedproductionoftumornecrosisfactor-alpha(TNF-a),interleukin-6(IL-6)andnitricoxide(NO).ByRT-PCR,themRNAlevelofthesepro-inflammatorycyto-kinesandiNOSwasalsoattenuatedbyfluoxetine.Wefurtherinvestigatedtheintracellularsignalingmechanismregulatingtheproductionofpro-inflammatorycytokinesandNOfromLPS-activatedmicroglia.TheresultsshowedthatfluoxetineinhibitedIkB-adegradation,phosphorylationandnucleartranslocationofthep65subunitofNF-kB,andphosphorylationofp38mitogen-activatedproteinkinase(MAPK)intheLPS-stimulatedmicroglia.Takentogether,ourresultssuggestthatthetherapeuticeffectsoffluoxetinearepartiallymediatedbymodulatingmicroglialactivation.Ó2011ElsevierLtd.Allrightsreserved.1.IntroductionIncreasingevidenceindicatesthatmicroglialactivationandinflammatoryprocessesmayplayimportantrolesinthepatho-genesisofdepression(Henryetal.,2008;Steineretal.,2008;Tynanetal.,2010).Microglia,theresidentmacrophagesofthecentralnervoussystem(CNS),arenowrecognizedastheprimarycompo-nentofthebrainimmunesystem(Barron,1995).Onceactivated,microgliachangetheirmorphology,proliferateandupregulatesurfacemolecules(ZielasekandHartung,1996).Inaddition,acti-vatedmicrogliahavethecapabilityofproducingpro-inflammatorycytokinesandneurotoxicmediatorssuchasnitricoxide(NO),prostaglandin(PG)E2,andsuperoxideanion(Giulianetal.,1994;NakajimaandKohsaka,1993).Severallinesofevidencehavedemonstratedthatexcessivepro-inflammatorycytokinesareinvolvedinthepathophysiologyofdepression(LicinioandWong,*Correspondingauthor.Tel.:þ8653188382050.E-mailaddress:aijunhao@sdu.edu.cn(A.Hao).1Contributedequallytothework.0028-3908/$eseefrontmatterÓ2011ElsevierLtd.Allrightsreserved.doi:10.1016/j.neuropharm.2011.04.0331999;Yirmiyaetal.,2000).Forexample,pro-inflammatorycyto-kines,includinginterferon(IFN)-g,interleukin-6(IL-6),andtumornecrosisfactor-alpha(TNF-a),havebeenshowntoincreasetheexpressionofindoleamine-2,3-dioxygenase(IDO)enzymeinbothcentralandperipheralimmune-competentcelltypes(Connoretal.,2008;Tuetal.,2005).IDO,thefirstrate-limitingtryptophan(TRP)-degradingenzyme,couldbreakdowntryptophan,theprimaryaminoacidprecursorofserotonin,intokynurenine(KYN).ThebreakdownofTRPisbelievedtocontributetoreducedserotoninavailabilitywhichhasbeenshowntoplayanimportantroleintheinductionofdepressivesymptoms(Neumeister,2003;Widneretal.,2002).Moreover,pro-inflammatorycytokineshavebeenfoundtointeractwithmanyofthepathophysiologicaldomainsthatchar-acterizedepression,includingneurotransmittermetabolism,neuroendocrinefunction,synapticplasticityandbehavior(Milleretal.,2009;Raisonetal.,2006).Inaddition,proinflammatorymediatorslikeNO,havebeenshowntoexertanegativeeffectonbrainneurogenesisandparticipateinstress-induceddepression(Jocaetal.,2007).Thus,controllingmicroglialactivationandneu-roinflammatoryprocessesmayprovetobeatherapeuticbenefitinthetreatmentofdepression.D.Liuetal./Neuropharmacology61(2011)592e599593
Ofnote,pro-inflammatorycytokinesandtheirsignalingpath-wayscanalsocontributetopathogenesisofdepression.Mitogenactivatedproteinkinase(MAPK)pathways,includingp38andextracellularsignal-regulatedkinases(ERK)1/2,whichmediatetheeffectsofcytokinesoncellproliferation/differentiationandapoptosis,aswellasgeneexpressionofinflammatorymediators,havebeenfoundtohaveanegativeeffectonbothmonoaminesynthesisandreuptake,leadingtoreducedmonoamineavailability(Moronetal.,2003;Zhuetal.,2006).Inaddition,nuclearfactor-kappaB(NF-kB),oneofrelevantinflammatorysignalingmole-cules,alsoplaysanimportantroleindepression.NF-kB,onceactivatedinimmunecells,resultsinthereleaseofinflammatorymediatorsthatpromoteinflammation.Pro-inflammatorycyto-kines,inturn,canaccessthebrain,induceinflammatorysignalingpathwaysincludingNF-kB,andultimatelycontributetoalteredmonoaminemetabolism,increasedexcitotoxicity,anddecreasedproductionofrelevanttrophicfactors(Milleretal.,2009).Fluoxetine,aselectiveserotoninreuptakeinhibitor(SSRI),iscommonlyprescribedfortreatingmajordepressionduetoitstolerabilityandsafety(GarrisonandLevin,2000;Montgomery,1998;WildeandBenfield,1998).Itiswellknownthatthetherapeuticactionoffluoxetineisascribedtoselectiveinhibitionofpresynapticserotoninreuptake(Wongetal.,1995).Recently,ithasbeenreportedthatfluoxetinehasanti-inflammatoryeffectsinanimalmodelsofperipheralinflammation.Forexample,insepticshockandallergicasthmaanimalmodels,fluoxetinewasfoundtosuppressthenumberofinflammation-relatedcellsandTNF-areleasefrommonocytes(Roumestanetal.,2007).Inaddition,fluoxetinewasfoundtohaveanti-inflammatoryeffectsinacarrageenan-inducedpawinflam-mationmodel(Abdel-Salametal.,2004;Bianchietal.,1995).Althoughtheanti-inflammatoryeffectsoffluoxetineintheperipheraltissueshavebeenwelldocumented,itseffectsoncellsintheCNS,specificallythemicrogliahaveremainedelusive.Indeed,thedetailedmolecularmechanismsunderlyingtheinhibitoryeffectsoffluoxetineonmicroglialcellsarenotfullyunderstood.Therefore,inthisstudy,wesoughttoinvestigatethepharmacologicaleffectoffluoxetineonmicrogliaactivatedbylipopolysaccharide(LPS).Alongwiththis,wealsoexamineditsmolecularmechanismregulatingmicroglialproductionofpro-inflammatorymediatorssuchasTNF-a,IL-6andNO.Inthisconnection,twoputativepathwaysincludingMAPKandNF-kBactivationthatmaybeinvolvedintheanti-inflammatoryeffectsoffluoxetinewerealsoinvestigated.2.Materialsandmethods2.1.BV2microglialcellcultureBV2cellsweremaintainedinDulbecco’smodifiedEaglemedium(DMEM,HycloneCo.,Logan,UT)with10%fetalbovineserum(FBS,HycloneCo.),2mML-glutamine,100U/mlpenicillinand100mg/mlstreptomycin(SigmaeAldrich,StLouis,MO)ina5%CO2incubator.PlatedcellsweregrowninDMEMwith10%FBSovernight.Inallexperiments,cellsweretreatedwiththeindicatedconcentrationsoffluoxetine(SigmaeAldrich,USA)intheabsenceorpresenceofLPS(100ng/ml)inserum-freeDMEM.2.2.PrimarymicroglialcellculturePrimarymicrogliawerepreparedfromthewholebrainsof1e2daysoldBALB/cmouseasdescribedpreviously(GiulianandBaker,1986).Briefly,glialcellswereculturedfor14daysinDMEM/F12(HycloneCo.)supplementedwith10%FBS(HycloneCo.).Isolatedmicroglialcellswereplatedinto24-wellplatesatadensityof2Â105cells/well.ThepurityofmicrogliacultureswasassessedusingCD11banti-body;morethan90%ofcellswerestainedpositively.Cellswereculturedfor2daysbeforedrugtreatment.2.3.CellviabilityassayCellviabilitywasdeterminedbythetetrazoliumsalt3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT,SigmaeAldrich)assay.BV2cellswereplatedinto96-wellcultureplatesatadensityof5Â104cells/mlwith200mlculturemediumperwell.Followingtreatmentwithdifferentconcentrationsoffluoxetine(SigmaeAldrich,USA),20mlMTTsolution(5mg/ml)wasaddedtoeachwellandincubatedat37Cfor4h.Themediumwasaspiratedand200mldimethylsulfoxidewasadded.Theabsorbancevaluewasmeasuredusingamultiwellspectropho-tometer(Bio-Rad,USA)at490nm.2.4.Enzyme-linkedimmunosorbentassay(ELISA)BV2cells(1Â105cells/wellina24-wellplate)weretreatedwithdifferentconcentrationsoffluoxetine(SigmaeAldrich,USA)inthepresenceorabsenceofLPS(SigmaeAldrich,USA).ThesupernatantoftheBV2cellswascollectedat24hafterdrugstimulation.ThelevelsofcytokinesincludingTNF-aandIL-6inculturemediumweremeasuredusingcommerciallyavailableenzyme-linkedimmunosor-bentassaykits(R&DSystemsInc,Minneapolis,MN,USA)accordingtomanufac-turer’sinstructions.Briefly,serialdilutionsofproteinstandardsandsampleswereaddedto96-wellELISAplates,followedbybiotinylatedanti-TNF-aorIL-6antibody.Afterrinsingwithwashbuffer,thepreparedsolutionofavidin,horseradishperox-idase-conjugatedcomplexwasaddedfollowedbyadditionofsubstratesolution.Thereactionwasstoppedbythestoppingsolution.Theopticaldensitywasdetectedat450nminamicroplatereader(Bio-RadLabs,Hercules,CA,USA).Theconcentrationofeachsamplewascalculatedfromthelinearequationderivedfromthestandardcurveofknownconcentrationsofthecytokine.2.5.NOreleaseassayAccumulationofnitriteintheculturemediumfor24hstimulationwasmeasuredbyaGriessreaction(SigmaeAldrich).NitritewastakenasameasureofNOproduction.ThesupernatantcollectedwasmixedwithanequalvolumeofGriessreagent(0.1%N-1-naphthylethylenediaminedihydrochlorideand1%sulphanila-midein5%phosphoricacid)ina96-wellplateandincubatedatroomtemperaturefor10min.Absorbancewasmeasuredat550nminamicroplatereader(Bio-RadLabs).Sodiumnitrite,dilutedinculturemediumatconcentrationsrangingfrom10to100mM,wasusedtoprepareastandardcurve.2.6.Reversetranscription-polymerasechainreactions(RT-PCR)TotalRNAwasextractedfrominducedcellculturesusingtheTrizolreagent(Gibco,Invitrogen)accordingtothemanufacturer’sinstructions.RNAconcentrationwasdeterminedbyaspectrophotometer(Bio-Rad.lab)at260nm.IdenticalamountsofRNA(1mg)werereversetranscribedintocDNAbyusingacommercialRT-PCRkit(Fermentas,Vilnius,Lithuania)accordingtothemanufacturer’sinstructions.cDNAwassubsequentlyamplifiedbyPCRwithspecificprimers(TNF-a:forward,50-CGTCAGCCGATTTGCTATCT-30;reverse,50-CGGACTCCGCAAAGTCTAAG-30;IL-6:forward,50-CCACTTCACAAGTCGGAGGCTT-30,reverse,50-CCAGCTTATCTGTTAGGAGA-30;iNOS:forward,50-CCTCCTCCACCCTACCAAGT-30,reverse,50-CACCCAAAGTGCTTCAGTCA-30;b-actin,forward,50-GTGGGGCGCCCCAGGCACCA-30,reverse,50-CTTCCTTAATGTCACGCACGATTTC-30).PCRwasconductedbyusingthefollowingconditionsfor32cycles:denaturationat94Cfor30s,annealingat60Cfor45s,andextensionat72Cfor30s.PCRproductsseparatedona1.2%agarose/TAEgelwerevisualizedbystainingwithethidiumbromide.Thedensitometricanalysisofthedatawasnormalizedtob-actin.TheintensityofbandswasdeterminedusingtheImage-ProPlus6.0software.2.7.WesternblottinganalysisCellswererinsedwithcoldphosphatebufferedsaline(PBS)andlysedincoldlysisbuffercontaining10mMTriseHCl,pH8.0,240mMNaCl,5mMEDTA,1mMdithiothreitol,0.1mMphenylmethylsulfonylfluoride,1%TritonX-100,1mMsodiumvanadate,and1g/mlofleupeptin,pepstatin,aprotinin.Celllysateswereincubatedat4Cfor20min.Thesamplewascentrifugedat12,000rpmfor10minat4C,thenthesupernatantwascollectedandproteincontentwasassayedcolorimetrically.Tenmicrogramsoftotalproteinswasloadedontoa12%SDS-PAGEgel,electrophoreti-callytransferredtopolyvinylidenedifluoridemembrane.Theprimaryantibodiesusedwereasfollows:iNOS(rabbitpolyclonalantibody,1:1000,SantaCruz),NF-kB(p65)andphospho-NF-kB(p65),IkB-a(rabbitmonoclonalantibody,1:1000,CellSignalingTechnology,USA),thephospho-ortotalformsofERK1/2,p38MAPK,JNK(rabbitpolyclonalantibodies,1:1000,CellSignalingTechnology,USA),andb-actin(mousemonoclonalantibody,1:10,000,SigmaeAldrich,USA).Secondaryantibodieswerehorseradishperoxidaseconjugatedtoeithergoatanti-rabbitIgGoranti-mouseIgG(SigmaeAldrich,USA).Themembranesweredevelopedusinganenhancedchemiluminescence(ECL)detectionsystem(Pierce,USA).TheintensityofbandswasdeterminedusingtheImage-ProPlus6.0software.2.8.ImmunofluorescenceassayMicroglialcellswerestainedforCD11b,induciblenitricoxidesynthase(iNOS),NF-kBp65andphospho-p38MAPKproteinexpression.Briefly,thecellswereplatedonglasscoverslipsovernight.Afterdrugtreatmenttheywerefixedin4%para-formaldehydefor10min.Afterpermeabilisationwith0.3%TritonX-100inPBS,the594D.Liuetal./Neuropharmacology61(2011)592e599
cellswereblockedwith10%FBSinPBS.Then,thecellswereincubatedintheprimaryantibody(mousemonoclonalanti-CD11bantibody,Millipore;rabbitpoly-clonalanti-iNOSantibody,SantaCruzBiotechnology,SantaCruz,CA;rabbitmono-clonalanti-NF-kBp65antibody,CellSignalingTech.,Beverly,MA;rabbitpolyclonalanti-phospho-p38MAPKantibody,CellSignalingTech.,Beverly,MA)overnightat4C.Followingprimaryantibodyincubation,cellswerewashedagainandincubatedintheappropriatefluorescent-conjugatedsecondaryantibody(SigmaeAldrich)for1h.ThecellswerecounterstainedbyDAPI.ImageswerecapturedwithaNikonTE2000Umicroscope.ToanalyzeCD11bstainingintheprimarymicroglialculture,atleastthreerepresentativeimagesweretakenaccordingly.Athresholdforpositivestainingwasdeterminedforeachimagethatincludedallcellbodiesandprocesses,butexcludedthebackgroundstaining.TheimageswereanalyzedusingImage-ProPlus6.0software.2.9.Nuclearproteinextractionandelectrophoreticmobilityshiftassay(EMSA)Nuclearextractsfromtreatedmicroglialcellswerepreparedasfollows.Aftertreatment,BV2cellsweretreatedwith1mloflysisbuffer(10mMTriseHCl,pH7.9,10mMNaCl,3mMMgCl2,1%NP-40)onicefor4min.After10minofcentrifugationat3000rpm,thepelletwasresuspendedin50mlofextractionbuffer(20mMHEPES,pH7.9,20%glycerol,1.5mMMgCl2,0.2mMEDTA,1mMDTT,1mMPMSF)andincubatedonicefor30min.Aftercentrifugationat14,000rpmfor5min,thesupernatantwasharvestedasthenuclearproteinextractandstoredatÀ70C.ProteinconcentrationwasdeterminedbyusingtheBCAproteinassay(Pierce,Rockford,IL,USA).Thedouble-strandedDNAoligonucleotidescontainingtheNF-kBconsensussequenceswereend-labeledusingthedigoxigenin(DIG)gelshiftkit(RocheAppliedScience,Indianapolis,IN).FivemicrogramsofthenuclearproteinswasincubatedwithDIG-labeledNF-kBoligonucleotidesandEMSAwasperformedaccordingtomanufacturer’sinstructions(Roche).Competitionstudieswereper-formedbyincubatingsamples,beforetheadditionoftheDIG-labeledNF-kBprobe,intheabsenceandpresenceof100-foldmolarexcessofunlabeledspecific(50-AGTTGAGGGGACTTTCCCAGGC-30)andnonspecific(50-GCAGAGCATATAAGGT-GAGGTAGGA-30)double-strandedoligonucleotides.2.10.TransienttransfectionandluciferaseassayTransfectionoftheNF-kBreportergeneintoBV2cellswasperformedusingLipofectamine2000(Invitrogen)accordingtothemanufacturer’sinstructions.TheNF-kBreporterplasmidcontainedthreecopiesofthekB-bindingsequencefusedtofireflyluciferasegene(Clontech,CA,USA).BV2cells(1Â105perwellina12-wellplate)weretransfectedwith0.8mgofthereporterconstructalongwith0.04mgofRenillaluciferaseplasmid(Promega,WI,USA).After48h,cellswereharvestedandaluciferaseassaywasperformedusingtheDual-LuciferaseReporterAssaySystem(Promega)accordingtothemanufacturer’sinstructions.TodeterminetheeffectoffluoxetineonLPS-inducedNF-kBactivity,cellsweretreatedwith10mMfluoxetineintheabsenceorpresenceofLPSandincubatedfor6hpriortoharvestingcellsforluciferaseassay.LuciferaseactivitywasmeasuredusingaMonolight2010lumin-ometer(AnalyticalLuminescenceLaboratory,SanDiego,CA).Renillaluciferaseactivitywasusedasaninternalcontrol.TherelativeluciferaseactivitywasthencalculatedbynormalizingfireflyluciferaseactivitytoRenillaluciferaseactivity.2.11.StatisticalanalysisAllresultswereexpressedasmeanÆS.D.ofatleastthreeindependentexper-iments.Statisticalanalysisofdatawasdonebyone-wayANOVAusingDunnett’stestinmultiplecomparisonsofmeans.Differenceswereconsideredstatisticallysignif-icantifthepvalue<0.05.3.Results3.1.FluoxetineinhibitsLPS-inducedmicroglialactivationPriortotheinvestigationoftheeffectsoffluoxetineonmicro-glialcells,MTTassaywasperformedtodetermineitscytotoxicitytoBV2cellsafter24hincubationwiththedifferentconcentrationsoffluoxetine.Cellviabilityfollowingtreatmentwithfluoxetineat0.1mM(94.51Æ4.29%),1mM(96.00Æ12.47%),and10mM(98.90Æ21.21%)wasnotsignificantlydifferentfromthecontrol(99.99Æ4.38%).However,exposureofBV2cellstofluoxetineat50mMresultedinsignificantlyfewerviablecells(65.78Æ3.9%)ascomparedtocellsinthecontrol(Fig.1).IthasbeenpreviouslyreportedthatmicroglialactivationisassociatedwithmarkedincreaseinCD11bexpression(LingandWong,1993;Rocketal.,2004).Immuno-fluorescenceanalysisshowedthat,after24hincubationwithLPS(100ng/ml),theFig.1.EffectoffluoxetineonthecytotoxicityinBV2cells.Cellsweretreatedwithvariousconcentrationsoffluoxetinefor24h.ThecytotoxicityoffluoxetinewasmeasuredbyMTTassay.DatarepresentthemeanÆSDofthreeindependentexper-iments.***p<0.001,significantlydifferentfromcontrol.FLX,fluoxetine.
expressionofCD11bwasevidentlyincreased.Treatmentwithfluoxetine(10mM)attenuatedLPS-mediatedupregulationofCD11binprimarymicroglialcells(Fig.2).3.2.EffectofthefluoxetineoncytokineproductioninLPS-stimulatedmicroglialcellsToassesswhetherfluoxetinecouldinhibitproductionofLPS-inducedpro-inflammatorycytokinesincludingTNF-aandIL-6,BV2cellsweretreatedwithLPSintheabsenceorpresenceoffluoxetinefor24h.AsshowninFig.3,fluoxetine(10mM)alonehadnoeffectontheproductionofTNF-aandIL-6inBV2cells.Stimu-lationofBV2cellswithLPSledtoasignificantincreaseintheTNF-aandIL-6levelsafter24h.TreatmentwithfluoxetinesignificantlyinhibitedtheirproductioninBV2cellsinadose-dependentmanner.Toelucidatethemechanismresponsiblefortheinhibi-toryeffectoffluoxetineonTNF-aandIL-6production,wenextexaminedthecytokinemRNAexpressionlevelsbyRT-PCR.Consistentwiththeresultsobtainedfromthecytokineproduc-tion,theLPS-stimulatedmRNAlevelsofTNF-aandIL-6werereducedbyfluoxetine,suggestingthatfluoxetinenegativelyFig.2.EffectoffluoxetineonLPS-inducedCD11bexpressioninprimarymicroglialcells.Primarymicroglialcellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for24h.ThecellswerestainedforCD11b(green)andcounterstainedwithDAPI(blue).Cd11bimmunofluorescencewasenhancedinLPS-treatedcells.However,reducedCD11bimmunofluorescencewasevidentinLPSþFLXcells.Scalebar¼50mm.Imagesarerepresentativeoftriplicatesets.FLX,fluoxetine.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
D.Liuetal./Neuropharmacology61(2011)592e599595
A
B
Fig.3.EffectoffluoxetineonLPS-inducedproductionofproinflammatorycytokineinBV2cells.(A)BV2cellsweretreatedwithfluoxetine(0.1,1,10mM)intheabsenceorpresenceofLPS(100ng/ml)for24h.EachcytokineconcentrationwasmeasuredinculturemediumusingELISA.(B)Cellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPSfor6h.ThemRNAexpressionofeachcytokinewasevaluatedbyRT-PCR.TherelativemRNAlevelswerequantifiedbyscanningdensitometryandnormalizedtob-actinmRNA.DatarepresentthemeanÆSDofthreeindependentexperiments.*p<0.05,**p<0.01,and***p<0.001vs.LPSalone.FLX,fluoxetine.
regulatedtheproductionofTNF-aandIL-6atthetranscriptionallevelintheLPS-stimulatedmicroglialcells.3.3.FluoxetinedecreasedNOproductioninLPS-stimulatedmicroglialcellsToinvestigatetheeffectsoffluoxetineonNOproductioninLPS-stimulatedBV2cells,cellsweretreatedwithLPSaloneorwithvariousconcentrationsoffluoxetinefor24h.ThelevelsofNOintheculturemediaweredeterminedwiththeGriessassay.FluoxetinesignificantlydecreasedtheLPS-inducedproductionofNOinBV2cellsinadose-dependentmanner(Fig.4A).Next,toelucidatethemechanismresponsiblefortheinhibitoryeffectoffluoxetineonNOproduction,wedeterminedtheiNOSmRNAandproteinlevelsbyRT-PCRandWesternblotanalysis.Ataconcentrationof10mM,fluoxetineeffectivelyinhibitediNOSmRNAexpressionat6hafterdrugtreatmentinBV2cells(Fig.4B).TheproteinlevelsofiNOSwerealsorepressedbyfluoxetineat24hafterdrugtreatmentinBV2cells(Fig.4C).Toconfirmtheaboveresult,immunofluores-cenceassaywasperformedtoobserveintracellulariNOSlevelsusingprimarymicroglialcells.TheresultshowedthatiNOSimmunofluorescencewhichwasenhancedbyLPSwasalsodecreasedbyfluoxetine(10mM)at24hafterLPSstimulationinprimarymicroglialcells(Fig.4D).TheseresultsshowedthatfluoxetineinhibitedNOproductionthroughdownregulationofiNOSmRNAandproteinexpressioninLPS-stimulatedmicroglialcells.3.4.FluoxetineinhibitsLPS-inducedphosphorylationofp38MAPKWethenassessedwhethertherepressiveeffectoffluoxetineonsynthesisandreleaseofproinflammatorymediatorsoccurredviaMAPKsignalingpathway.BV2cellsweretreatedwith10mMfluoxetineinthepresenceorabsenceofLPSfor30min.AsshowninFig.5A,fluoxetinedidnotinhibitLPS-inducedphosphorylationlevelsofERK1/2andJNKMAPKs,whileLPS-inducedphosphory-lationofp38MAPKwasmarkedlyinhibitedbyfluoxetineinBV2cells.Toconfirmtheaboveresult,immunofluorescencelabelingfordetectionofintracellularphosphorylationofp38MAPKwasalsocarriedoutinprimarymicrogliacells.TheresultsshowedthatupontreatmentwithLPS,therewasanincreasedimmunofluorescenceofphospho-p38MAPK,whichwasalsosignificantlyreducedbyfluoxetineinprimarymicroglialcells(Fig.5B).Thesefindingsindicatethatfluoxetineiseffectiveintheinhibitionofp38MAPKphosphorylationinLPS-stimulatedmicroglialcells.3.5.FluoxetineinhibitsLPS-inducedNF-kBactivationinmicroglialcellsNF-kBhasbeenshowntobeoneofthemostimportantupstreammodulatorsforpro-inflammatorycytokinesandiNOSexpressioninmicroglia.Thus,wenextdeterminedwhethertherepressiveeffectoffluoxetineongeneexpressionoccurredviablockadeofNF-kBactivityinmicroglialcells.First,weexaminedtheinfluenceoffluoxetineontheIkB-aandNF-kBactivitybyWesternblotandimmunofluorescenceanalysis.AsshowninFig.6A,LPStreatmentinducedmarkedlydegradationofIkB-a,whichwasreversedbyfluoxetineinBV2cellsat30minafterdrugtreatment.Inaddition,fluoxetinesignificantlysup-pressedLPS-inducedphosphorylationlevelsofNF-kBp65inBV2cells.Theaboveresultswerefurtherconfirmedbyimmunofluo-rescenceanalysis.NF-kBp65wasmainlylocalizedinthecytoplasm,treatmentwithLPS-inducedNF-kBp65translocationfromcytoplasmtothenucleus.However,fluoxetinedecreasedthetranslocationofNF-kBp65intothenucleiofBV2cells(Fig.6B).Basedontheaboveresults,theinvolvementofNF-kBactivityinfluoxetine-inducedsuppressionofpro-inflammatorycytokinesandNOwasfurtherexaminedbyEMSAandluciferaseassay.AsshowninFig.6C,stimulationofBV2cellswithLPSresultedinstrongNF-kBbinding,whichwassignificantlyinhibitedbyfluoxetine.SpecificityofproteinbindingtotheDIG-labeledprobewasalsotested.WhenexcessunlabeledNF-kBoligonucleotideswasaddedtoproteinextractsfromLPS-stimulatedBV2cells,bindingtotheDIG-labeledoligonucleotideswasabolished.Further,additionofanunlabeled596D.Liuetal./Neuropharmacology61(2011)592e599
Fig.4.EffectoffluoxetineonLPS-inducedproductionofNOandiNOSinmicroglialcells.(A)BV2cellsweretreatedwithfluoxetine(0.1,1,10mM)intheabsenceorpresenceofLPS(100ng/ml)for24h.TheNOconcentrationwasdeterminedinculturemediumusingGriessreagent.(B)BV2cellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for6h.ThemRNAexpressionofiNOSwasevaluatedbyRT-PCRandtherelativemRNAlevelswerequantifiedbyscanningdensitometryandnormalizedtob-actinmRNA.DatarepresentthemeanÆSDofthreeindependentexperiments.*p<0.05,**p<0.01,and***p<0.001vs.LPSalone.(C)BV2cellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for24h.ProteinlevelsofiNOSwereevaluatedbyWesternblot.(D)Primarymicroglialcellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for24h.Thecellswereco-stainedforCD11b(green),iNOS(red)andimagescapturedwithafluorescencemicroscope.Arrowsindicatecellsinwhichactivationisvisiblyprominent.Scalebar¼50mm.Imagesarerepresentativeoftriplicatesets.FLX,fluoxetine.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
non-competitiveoligonucleotidestoproteinextractshadnoeffectonNF-kBbinding.Inaddition,fluoxetinealsoaffectedNF-kB-mediatedtranscrip-tioninBV2cells.TheNF-kBtranscriptionalactivitywasassayedbytransfectingtheBV2cellswithaplasmidcontainingthreeNF-kBbindingsitesandaluciferasereportergene.TheluciferaseassayshowedthatfluoxetinerepressedtheLPS-stimulatedNF-kBtran-scriptionalactivationinadose-dependentmanner(Fig.6D).Insum,theseresultsindicatedthepotentialroleofNF-kBinthepossiblemechanismoffluoxetineinsuppressingpro-inflammatorycyto-kinesandNOinactivatedmicroglia.4.DiscussionInthepresentstudy,ithasbeendemonstratedthatfluoxetineinhibitsinflammatoryactivationofmicroglia.Fluoxetinesignifi-cantlyreducedinflammatorymediatorssuchasTNF-a,IL-6andNOproductioninLPS-stimulatedmicroglialcells.TheRT-PCRanalysisFig.5.EffectoffluoxetineonLPS-inducedphosphorylationofMAPKinmicroglialcells.(A)BV2cellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for30min.Totalcellextractsweresubjectedtoimmunoblotanalysisusingantibodiesagainstphospho-ortotalformsofthreeMAPKs.(B)Primarymicroglialcellsweretreatedwithfluoxetine(10mM)intheabsenceorpresenceofLPS(100ng/ml)for1h.ThecellswerestainedforCD11b(green),phospho-p38MAPK(red)andimageswerecaptured.Arrowsindicatecellsinwhichactivationisvisiblyprominent.Scalebar¼50mm.Imagesarerepresentativefromthreeindependentexperiments.FLX,fluoxetine.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
D.Liuetal./Neuropharmacology61(2011)592e599597
Fig.6.EffectsoffluoxetineonLPS-stimulatedNF-kBactivityinBV2cells.(A)Cellsweretreatedwithfluoxetine(10mM)inthepresenceorabsenceofLPS(10ng/ml)for30min.TotalcellextractsweresubjectedtoimmunoblotanalysisusingantibodiesagainstIkB-a,phospho-ortotalformsofNF-kBp65antibody.(B)BV2cellsweretreatedwith10mMfluoxetineinthepresenceorabsenceofLPSfor1h.NF-kBp65proteinlocalizationwasdeterminedusingananti-p65antibodyandanFITC-labeledanti-rabbitIgGantibody,andcellswerevisualizedusingafluorescencemicroscope.Scalebar¼50mm.Imagesarerepresentativeoftriplicatesets.(C)BV2cellsweretreatedwithfluoxetine(10mM)inthepresenceofLPS(100ng/ml)for1h.TotalnuclearextractswereisolatedandusedinanEMSAwithDIG-labeledNF-kBoligonucleotideasaprobe.Competitionassayswereconductedintheabsenceorpresenceof100Mexcessofunlabeledspecificornonspecificdouble-strandedoligonucleotide.ThepositionsofDIG-labeledNF-kBDNA/proteinbindingcomplexesareindicatedbyarrows.NSC,nonspecificcompetitor.SC,specificcompetitor.(D)TransfectedBV2cellsweretreatedwithvariousconcentrationsoffluoxetineinthepresenceorabsenceofLPS(100ng/ml)for6h.NF-kBactivitywasexpressedasrelativeluciferaseactivity.DatarepresentthemeanÆSDofthreeindependentexperiments.***p<0.001vs.LPSalone.FLX,fluoxetine.
showedthatfluoxetinemarkedlysuppressedtheTNF-a,IL-6andiNOSgeneexpressionatthetranscriptionallevel.Furthermore,theanti-inflammatorypropertyoffluoxetinewasmediatedbytheinhibitionofp38MAPKphosphorylationandNF-kBactivationinmicrogliacells.MicroglialcellsarebrainmacrophageswhichservespecificfunctionsinthedefenseofCNSagainstmicroorganisms,theremovaloftissuedebrisinneurodegenerativediseasesorduringnormaldevelopment,andinautoimmuneinflammatorydisordersofthebrain(ZielasekandHartung,1996).SeveralstimulantssuchasIFN-g,LPSandTNF-aarewellknowntoactivatemicroglialcells(Hashiokaetal.,2007;Parketal.,2007;Ullrichetal.,2001).Assuch,inthepresentstudy,wehaveusedLPSasanexperimentalmodeltoactivatemicroglia.ItshouldbenotedthatLPS-inducedmicroglialactivationmightnotbetotallycomparablewiththatduringdepression.However,themainfocusofthepresentinvitrostudyistoinvestigatetheanti-inflammatorypropertyoffluoxetineinmicroglia.LPSwasusedasanexperimentalmodeltoinducemicroglialactivationtoproduceinflammatorymediators,mainlypro-inflammatorycytokines,whichareinvolvedinthepathogen-esisofdepression.Activatedmicrogliaproduceawiderangeofproinflammatorymediators,includingTNF-a,IL-6,IL-1b,reactiveoxygenspecies(ROS),andNO.Ofthese,TNF-aandIL-6appeartoplayacriticalroleinpathologyofdepression(Dowlatietal.,2010).Fluoxetine,exvivo,significantlyinhibitedthespontaneousproductionofTNF-aandIL-6(Sacreetal.,2010).Invitro,fluoxetinedose-dependentlyinhibitedthereleaseofTNF-afromLPS-treatedmonocytes(Roumestanetal.,2007).Inanimalexperiment,fluoxetinetreat-mentsignificantlyattenuatedLPS-inducedanorexiaandbodyweightlossinrodents.Inaddition,administrationofthetricyclicantidepressant(TCA)imipramineattenuatedorcompletelyabol-ishedthebehavioraleffectsofLPS,includingdecreasedsaccharinepreference,anorexia,bodyweightloss,reducedsocialactivity,andsuppressionoflocomotorandexploratorybehaviorintheopenfieldtest(Yirmiyaetal.,2001).Consistentwiththesestudies,wefoundthatfluoxetineinhibitsLPS-inducedproductionofpro-inflammatorycytokines,TNF-aandIL-6(Fig.3A),andtheexpres-sionoftheirmRNA(Fig.3B)inBV2cells.ThisresultindicatesthatfluoxetineisabletomodulatetheactivitiesofTNF-aandIL-6atthetranscriptionallevelinactivatedmicroglia.NOisknowntobeinvolvedintheprocessesofdepressionduetoitsabilitytosubstantiallymodifythemonoaminergicneurotrans-mission(Kiss,2000).RecentstudieshavealsoshownthatdepressedpatientsarecharacterizedbyexcessiveNOproduction(Suzukietal.,2001).Inaddition,iNOSinhibitorsareeffectiveindepressiontreatment(Harkinetal.,1999).However,theroleofNOinthepathogenesisofdepressionisstillcontroversial.Indeed,NOhasbeenreportedtopromotebothneuronalsurvivalandneuronaldeath,dependinguponthelevelsofitspresence,aswellasthespecificcellularsysteminvolved(AraujoandCarvalho,2005).Furthermore,iNOSisthekeyenzymeforNOproduction,andisquantitativelyinducedinactivatedglialcellsafterexposuretostimulatorssuchasLPSandviralinfections(Chewetal.,2006;598D.Liuetal./Neuropharmacology61(2011)592e599
McGeeretal.,1993).Therefore,agentswiththeabilitytoinhibitiNOSexpressionarepotentiallybeneficialinthetreatmentofconditionsassociatedwithanoverproductionofNO.Inthepresentstudy,fluoxetinedecreasedLPS-inducedNOproductioninadose-dependentmanner(Fig.4A),andlargelyreducedthemRNAexpressionandproteinlevelsofiNOSinmicroglialcells,indicatingthattheinhibitionofNOproductionbyfluoxetineisaresultoftheinhibitionofiNOSgeneexpression.Severalinflammatorystimuli,includingLPS,commonlyactivatetheMAPKandNF-kBsignalingpathwaysinmicroglia(Bhatetal.,1998;Jungetal.,2007;Ryuetal.,2000).AlthoughtheexactintracellularsignalpathwaysofmicroglialactivationbyLPShavenotbeenclearlydefined,LPShasbeenreportedtoinduceTNF-a,IL-6andiNOSexpressioninmicroglialcellsthroughsignalpathways,includingMAPKandNF-kB(Bhatetal.,1998;Jengetal.,2005;Wilmsetal.,2010).TheMAPKfamilyofproteinscomprisesERK,SAPK/JNK,andp38MAPK(KoistinahoandKoistinaho,2002).Amongthem,p38MAPKandERKappeartobeprimarilyinvolvedintheproductionofproinflammatorymediatorsbymicroglia.Inprimarymicrogliaandmicroglialcellline,LPShasbeenshowntodose-andtime-dependentlyincreasethephosphorylationofERKandp38,aswellasincreasetheexpressionofiNOSandTNF-a(Bhatetal.,1998;Leeetal.,1994,1993).Consistentwiththesestudies,ourresultsshowedthatfluoxetineinhibitedp38butnotERKorJNKMAPKphosphorylationinLPS-stimulatedmicroglia,suggestingthatp38MAPKplaysauniqueroleinanti-inflammatorypropertyoffluoxetineinLPS-stimulatedmicroglia.NF-kBisanimportantupstreammodulatorofcytokineandiNOSexpressioninmicroglia(Pahl,1999).ItiswellknownthatblockadeofNF-kBtranscriptionalactivityinmicrogliacanalsosuppressexpressionofiNOSandpro-inflammatorycytokines,suchasIL-1b,IL-6,andTNF-a(Baldwin,1996;Moonetal.,2007).Normally,NF-kBispresentinthecytoplasmasaheterotrimercomplexconsistingofp50,p65andIkBsubunits.Uponactivationofthecomplex,phos-phorylationanddegradationofIkBexposesnuclearlocalizationsignalsonthep50/p65complex,leadingtonucleartranslocationandbindingtospecificregulatedsequencesintheDNA,thuscontrollinggenetranscription(Moynagh,2005).Thus,inhibitionofthesesignalingpathwaysmayexplainthepotentactivityoffluoxetineasasuppressorofinflammatorymediators.Inthepresentstudy,fluoxetineinhibitedtheLPS-inducedIkB-adegrada-tionandnucleartranslocationofthep65subunitofNF-kBinmicroglia.Furthermore,NF-kBDNAbindingandtranscriptionalactivitywerealsorepressedinLPS-inducedmicroglialactivation.Thesefindingssuggestthatthetranscriptionaldownregulationoftheinflammatorymediatorsbyfluoxetineresultsfromtheinhibi-tionofNF-kBsignalpathways.Manystudieshaveexploredtherelationshipbetweenantide-pressantsandpro-inflammatorycytokines.Mostofthemfocusedontheeffectofantidepressantsonperipheralreleaseofpro-inflammatorycytokines(Diamondetal.,2006;Maesetal.,1999;Xiaetal.,1996),thoughtheresultsweresometimesconflictingduetouseofdifferentmethodology,samplesizeorheterogeneityofdepression(Janssenetal.,2010).However,todate,onlyafewstudieshaveattemptedtoexaminetheanti-inflammatoryeffectofantidepressantsthroughmicroglialinhibition.Somestudieshaveshownthatantidepressantsinhibitedtheproductionofproin-flammatorymediatorsfromactivatedmicroglia(Hashiokaetal.,2007;Horikawaetal.,2010;Limetal.,2009;Vollmaretal.,2008),thoughinconsistentresultsalsoexisted(Haetal.,2006;O’Sullivanetal.,2009).OurresultsareconsistentwithpreviousevidencebyothersthatfluoxetinecouldinhibitLPS-inducedproductionofinflammatorymediatorsinmicroglia.Tothisend,thepresentstudyhasextendedthemolecularmechanismoftheeffectofantidepressantonactivatedmicroglia.However,becauseoftheconflictingresultsmentionedabove,itisstilluncleartowhatextentourfindingsmaybegeneralizedtoallSSRIs.Onelimitationofourstudyisthelackofbehavioraldataondepressionfromanimalexperiments,thoughsomeworkhasbeendonepreviouslybyothers.Forexample,fluoxetinesignificantlyattenuatedLPS-inducedreductioninfoodconsumptionandbodyweight(Yirmiyaetal.,2001).Inaddition,minocycline,aspecificinhibitorofmicroglialactivation,reducedLPS-inducedmicroglialactivation,CNScytokineproduction,andbehavioralsymptomsofsickness(Henryetal.,2008).Thepresentstudyhasshownthatanimalexperimentisusefultobetterrevealtherelationshipbetweenmicroglialactivationanddepressionand,inthisregard,additionalbehavioralstudywouldbeanimportantscopeforfuturestudy.Insum,theeffectoffluoxetineontheinflammatoryactivationofmicrogliahasbeendetermined.Thisstudyhasshownthatfluox-etineinhibitedLPS-inducedproductionofinflammatorymediatorssuchasTNF-a,IL-6andNO,andtheirgeneexpressioninmicroglia.Inaddition,theanti-inflammatorypropertyoffluoxetinewasmediatedbytheinhibitionofp38MAPKphosphorylationandNF-kBactivationinLPS-stimulatedmicroglialcells.Thesedatasuggestthattherapeuticeffectsoffluoxetinearepartiallymediatedbytheiranti-inflammatoryactionsonmicroglialactivation.AcknowledgmentsThisresearchwassupportedbyNationalBasicResearchProgramofChina(973Program),GrantNumber:2007CB512001,2011CB966201;NationalNaturalScienceFoundationofChina,GrantNumber:No.30771142,81071057;NaturalScienceFounda-tionofShandongProvince,GrantNumber:No.Z2007C11,J20082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