TLR4 Activation Under Lipotoxic Conditions Leads to Synergistic Macrophage Cell Death through a TRIF-Dependent Pathway Joel D. Schilling, Heather M. Machkovech, Li He, Abhinav This information is current as Diwan and Jean E. Schaffer of December 18, 2018. J Immunol published online 28 December 2012 http://www.jimmunol.org/content/early/2012/12/28/jimmun ol.1202208 DD oo ww nn lolo aa dd ee W hy The JI? Submit online. d fromd from • Rapid Reviews! 30 days* from submission to initial decision h h ttpttp • No Triage! 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Published December 28, 2012, doi:10.4049/jimmunol.1202208 The JournalofImmunology TLR4 Activation Under Lipotoxic Conditions Leads to Synergistic Macrophage Cell Death through a TRIF-Dependent Pathway Joel D. Schilling,*,†,‡ Heather M. Machkovech,*,† Li He,*,† Abhinav Diwan,† and Jean E. Schaffer*,† Macrophagedysfunctioninobesityanddiabetesmaypredisposetothedevelopmentofdiabeticcomplications,suchasinfection andimpairedhealingaftertissuedamage.Saturatedfattyacids,suchaspalmitate,arepresentatelevatedconcentrationsinthe plasma of patients with metabolic disease and may contribute to the pathogenesis of diabetes and its sequelae. To examine the effectoflipidexcessonmacrophageinflammatoryfunction,wedeterminedtheinfluenceofpalmitateonLPS-mediatedresponses inperitonealmacrophages.PalmitateandLPSledtoaprofoundsynergisticcelldeathresponseinbothprimaryandRAW264.7 D macrophages. The cell death had features of apoptosis and necrosis and was not dependent on endoplasmic reticulum stress, o w ceramide generation, or reactive oxygen species production.Instead, we uncovered a macrophage death pathway that required n TLR4 signaling via TRIF but was independent of NF-kB, MAPKs, and IRF3. A significant decrease in macrophage lysosomal loa d content was observed early in the death pathway, with evidence of lysosomal membrane damage occurring later in the death ed response.OverexpressionofthetranscriptionfactorTFEB,whichinducesalysosomalbiogenicprogram,rescuedthelysosomal fro phenotypeandimprovedviabilityinpalmitate-andLPS-treatedcells.Ourfindingsprovidenewevidenceforcross-talkbetween m h lipidmetabolismandtheinnateimmuneresponsethatconvergesonthelysosome. TheJournalofImmunology,2013,190:000– ttp 000. ://w w O besity and diabetes and their related complications are is a sensor that responds to both pathogen-associated molecular w associated with significant morbidity, mortality, and in- patterns,suchasLPS,andtissuedamage–relatedsignals,suchas .jim m creasedhealthcarecosts.Impairedresponsestotissue high-mobilitygroupbox1protein(11,12).Consistentwiththis, u damageareacommonfeatureofmanydiabeticcomplications, TLR4knockout(KO)micehavereducedinflammationinresponse no leadingtoadverseoutcomesfrominfectiousandnoninfectious toGram-negativebacterialinfection,aswellastosteriletissue l.o rg injury,includingmyocardialinfarctionandsurgery(1–4).Despite injury,suchasmyocardialinfarction(13,14).TLR4isa cellsur- b/ extensive epidemiologic data, the mechanisms underlying thein- facereceptorthatactivatestwodistinctintracellularsignalingpath- y g flammatoryandwoundrepairphenotypeinpatientswithdiabetes ways via the adaptor molecules MyD88 and TRIF (15). MyD88- u e remainspoorlyunderstood.Giventhecentralroleofmacrophages dependent signaling triggers the classical inflammatory cascade, st o intheregulationofinflammationandtissuehealing,dysfunction resultinginNF-kBandMAPKactivation.Incontrast,TRIFas- n D ofthesecellsindiabeteshasbeenproposedtocontributetothe sociates withTLR4afterligand-inducedinternalizationandleads e c pathogenesis ofdiabetic complications (5–8). to activation of IRF3 and IFN-b production. TRIF-dependent em Macrophage activation in response to pathogens and/or tissue signalingcanalsomediatedelayedNF-kBandMAPKactivation be damageoccurs,inpart,throughTLRs(9,10).Inparticular,TLR4 andhasbeenimplicatedinsomecelldeath–signalingpathways r 1 8 (16). , 2 Emerging evidence shows that the metabolic perturbations in 01 *DiabeticCardiovascularDiseaseCenter,WashingtonUniversitySchoolofMedi- 8 cine,St.Louis,MO63110;†DepartmentofMedicine,WashingtonUniversitySchool obesityanddiabetesinfluencemacrophagebiology.Forexample, ofMedicine,St.Louis,MO63110;and‡DepartmentofPathologyandImmunology, lipid excess promotes macrophage recruitment to adipose tissue WashingtonUniversitySchoolofMedicine,St.Louis,MO63110 andcanshiftmacrophagepolarizationtowardamoreinflammatory ReceivedforpublicationAugust8,2012.AcceptedforpublicationNovember29, phenotype (17, 18). Long-chain saturated fatty acids, such as 2012. palmitate andstearate, are increased in theplasmaandtissues of This work was supported by grants from the National Institutes of Health (R01 obese and diabetic individuals and are thought to contribute to DK064989andAMDCCU24DK076169toJ.E.S.;K08HL098373toJ.D.S.;and P60DK020579);theBurroughsWellcomeFoundation(1005935toJ.E.S.),andthe organdysfunctionunderlyingdiabetesprogressionandthepatho- DiabetesActionFoundation(toJ.D.S.). genesis ofcomplications(19,20).Invitroexposureofendothelial Addresscorrespondenceto Dr. Jean E.Schaffer,Diabetic Cardiovascular Disease cells,fibroblasts,pancreaticbcells,hepatocytes,andmyoblaststo Center,WashingtonUniversitySchoolofMedicine,St.Louis,MO63110.E-mail palmitate triggers endoplasmic reticulum (ER) stress, ceramide address:[email protected] production, and oxidativestress,which can lead to lipotoxic cell Abbreviationsusedinthisarticle:ANX,AnnexinV–GFP;DBLKO,doubleknock- out;DCF,5-(and-6)-chloromethyl-29,79-dichlorodihydrofluoresceindiacetate,acetyl death(21).The effects ofpalmitateonprimary macrophages are ester;ER,endoplasmicreticulum;KO,knockout;LC-MS/MS,liquidchromatogra- lesswellunderstood,anditremainscontroversialwhetherlong- phy-tandemmassspectrometry;LDH,lactatedehydrogenase;LMP,lysosomalmem- chainsaturatedfattyacids,suchaspalmitate,candirectlyactivate branepermeability;MT,Mito-TEMPO;nec1,necrostatin1;PI,propidiumiodide; pMAC,peritonealmacrophage;qRT-PCR,quantitativeRT-PCR;ROS,reactiveoxy- TLR4 on these cells (22, 23). genspecies;STS,staurosporine;TFEB,transcriptionfactorEB;TMR,tetramethyl- Prematureorexcessivemacrophagecelldeathhasthepotential rhodamine;WT,wild-type. toimpairinflammatoryandtissuerepairpathways.Therefore,we Copyright(cid:1)2012byTheAmericanAssociationofImmunologists,Inc.0022-1767/12/$16.00 soughttoevaluatehowthecross-talkbetweenpalmitateandTLR4 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202208 2 TLR4ANDPALMITATETRIGGERTRIF-DEPENDENT MACROPHAGEDEATH signaling impacts macrophage survival. In this article, we dem- a Tecan Infinite M200 plate reader. Total LDH content was determined onstratethatstimulationofprimarymacrophageswithLPSunder using the lysis buffer provided by the manufacturer, according to their protocol. lipotoxic conditions produces a profound synergistic cell death SubG1DNAcontent.SubG1DNAcontentinmacrophageswasdetermined phenotypethatisdependentonTLR4.Ourstudyuncoversanovel byPIstainingandflowcytometry,asdescribedpreviously(26). TRIF-dependent cell death pathway that is associated with lyso- Mitochondrial membrane permeability. Mitochondrial membrane perme- somal dysfunction and illustrates a unique mechanism by which abilitywasassessedinstimulatedmacrophagesusingaMito-IDkit(Enzo excesslipidscanpromote macrophagedysfunction. LifeSciences),asperthemanufacturer’sinstructions,andanalyzedusing aTecanInfiniteM200platereader. Materials and Methods RNAisolationandquantitative RT-PCR Reagents TotalcellularRNAwasisolatedusingQIAGENRNeasycolumnsand SB203580,PD98059,SB600125,BAY11-7085,CA-074ME,necrostatin reverse transcribed using a high-capacity cDNA reverse-transcription kit 1,phenylbutyricacid,andstaurosporine(STS)werefromEMD-Millipore. (AppliedBiosystems).Real-timequantitativeRT-PCR(qRT-PCR)was Z-VADandMito-TEMPO(MT)werefromEnzoLifeSciences.Fumonisin performedusingSYBRGreenreagent(AppliedBiosystems)onanABI BwasfromCaymanChemicals.UltrapureEscherichiacoliLPSwasfrom 7500fastthermocycler.Relativegeneexpressionwasdeterminedusingthe InvivoGen.RecombinantmouseTNF-awasfromR&DSystems.The d-dCTmethodnormalizedto36B4expression.Mouseprimersequences anti–cathepsinDAbwasfromAbcam,andtheanti-LAMP1Abwasfrom were as follows: 36B4 (forward, 59-ATC CCT GAC GCACCG CCG SantaCruz.Butylatedhydroxyanisoleandtheanti-actin,anti-tubulin,and TGA-39,reverse,59-TGCATCTGCTTGGAGCCCACGTT-39);TNFa anti–flagM2AbswerefromSigma.ThioglycollatewasfromDifco.Fatty (forward,59-CATCTTCTCAAAATTCGAGTGACAA-39,reverse,59- acidswerefromNu-ChekPrep.UltrapureBSAwasfromSeraCareand TGGGAG TAG ACACAAGGTACAACCC-39); IFNb (forward,59- wastestedforTLRligandcontaminationpriortouse.AnnexinV–GFP GACGGAGAAGATGCAGAAGAGTT-39,reverse,59-AGTTCATCC D (ANX) was from BioVision. LysoTracker Red, tetramethylrhodamine AGGAGACGTACAAC-39);GRP78(forward,59-GCCTCATCGGAC o (TMR)-dextran(10,000m.w.),andHoechstnucleardyewerefromInvi- GCACTT-39,reverse,59-AACCACCTTGAATGGCAAGAA-39);and w n trogen;acridineorangewasfromSigma.Theproteaseinhibitor(253 CHOP (forward, 59-CAG ATT CCAGTC AGA GTT CTATGG-39, re- lo ProteaseCompletetablets)wasfromRoche. verse,59-GACCACTCTGTTTCCGTTTCCT-39). ad e d Cell culture Lysosome imaging fro m Peritoneal macrophages (pMACs) were isolated from C57BL/6 or the Aftertheindicatedstimulations,cellswerestainedwith500nMLyso- h indicatedKOmice4dafteri.p.injectionofthioglycollateandplatedata TrackerRedor1mg/mlacridineorangeintissueculturemediafor15min ttp sdeernusmity, 5o0fU5/3ml1p0e5nicceillllsin/mGl isnodDiMumE,Mancdon5t0aiUni/nmgl1s0tr%eptionmacyticviantesdulffeattael. awti3th7˚5C0.0Fmorgfl/muolrTesMceRn-tddeexxtrtarannfeoxrp2erhiminenrtesg,umlaarcrmopehdaiag,esfowlleorweeidncbuybacteeldl ://w w Stimulationswereperformedonthedayafterharvest.Forflowcytometry stimulationsfor16h.Afterstaining,cellswerewashedthreetimeswith w e(GxpreeirniemreBntiso,-Opneeri)totnoefaalcicleitlalstewceerllehcaurlvteusrte.dAoltnholuogwh-asdohmeerenstcuedipeslatrees- PteBrnSa,tihvaerlvye,smteadcraospdheasgcersibwederaebocvuelt,uarenddoannaglylazsesdcboyveflroswlipcsystotamineetrdy.wAitlh- .jim m ported up to 10–15% contamination of thioglycollate-elicited cells with LysoTrackerRed,fixedwith4%paraformaldehyde,stainedwithHoechst u nonmacrophage cell types, such as eosinophils (24), we routinely found nuclear dye, and mounted on glass slides for imaging. Immunofluores- no thatonly2–4%oftheadherentcellswereSiglecF+,F4/80+.RAW264.7 cence images were obtained on a Leica wide-field fluorescence micro- l.o cells (American Type Culture Collection) were cultured in DMEM con- scope. rg taining 10% inactivated fetal serum and streptomycin sulfate. Stably b/ transfectedHEK293cellscontainingCD14/MD2/TLR4orcontrolvector Westernblotting y g were obtained from InvivoGen and cultured according to the manu- u fpaaclmtuirteart’esorrecoolemamteecnodmatpiloenxse.dGtoroBwSthAmateadi2u:m1mwoalsarsuraptpiole,masendteesdcrwibiethd F(poHrt8o)t,aTlrcietollnulXar-1p0ro0te1i%ns,,acnedll1s3weCreomlypsleedteinPr1o5te0amseM.WNeastCelr,n1b0lomttMingTfroisr est o previously (25), and BSA-supplemented mediawas used as control. For FLAG-TFEB,LAMP1,andactinwasperformedusing25mgtotalcellular n D cellstimulations,PBSorLPS(50ng/ml)wasaddedtoBSAorpalmitate protein.ForcytoplasmiccathepsinDanalysis,followingstimulationwith ec media. BSA-PBS or palmitate-LPS, cytosolic proteins were recovered by ex- em tractionwithdigitoninbuffer(150mMNaCl,50mMHEPES[pH8],50 b Mice 1m0gK/mAldmigicitoonniUn,lt1r3a cCeonmtripfluegtealPfiroltteerasueniintshib(Mitoilrlsi)paonred).coWnceestnetrrnatebdloutsifnogr er 18 Wild-type(WT)andTLR4KOC57BL/6micewereobtainedfromOriental cathepsinDusing25mgcytoplasmicproteinwasanalyzedbySDS-PAGE , 2 Bioscience.NOX2KOmicewerefromTheJacksonLaboratory.TRIFKO, andWesternblotting.Tubulinwasusedasaloadingcontrol. 01 MYD88 KO, and TRIF/MYD88 double-KO (DBL KO) C57BL/6 mice 8 werefromMarcoColonna(WashingtonUniversity);IRF3KOmicewere TranscriptionfactorEBtransfection fromMichaelDiamond(WashingtonUniversity);andIKKbfloxedmice crossed with Lys-M Cre mice were from Michael Karin (University of AmammalianexpressionvectorforexogenousexpressionofN-terminal California,SanDiego).Miceweremaintainedinapathogen-freefacility FLAG-tagged mouse transcription factor EB (TFEB) and bicistronic ex- and were fed a standard chow diet ad libitum (6% fat). All animal ex- pressionofGFPwascreatedbycloningtherequisitecodingsequencesinto perimentswereconductedinstrictaccordancewithNationalInstitutesof the BamH1 and Xho1 sites of the backbone pAAV-CMV-IRES-hrGFP. Healthguidelinesforhumanetreatmentofanimalsandwerereviewedby HEK293-TLR4/CD14/MD2 cells were transfected using Lipofectamine the Animal Studies Committee of Washington University School of 2000(Invitrogen).After24h,transfectedcellswerestimulatedwithBSA- Medicine. PBSorpalmitate-LPSforanadditional48h.Sampleswerepreparedas aboveforPIandLysoTrackerRedstainingandflowcytometryanalysis. Assessmentofcelldeath TransfectionwasassessedbyGFPfluorescence,andcelldeathwasassessed byPIstaining. ANX–propidium iodide staining. Cells were stimulated as indicated, re- coveredbytrypsinization,andstainedwithANXandpropidiumiodide(PI), Palmitate-uptake and-oxidationassays according to the manufacturer’s instructions (BioVision). Samples were analyzedforgreen(FL1)andred (FL2)fluorescence onaBectonDick- The palmitate-uptakeassay was performedas describedpreviously(27). insonFACScanflowcytometer,withanalysisof104cells/sample.Where Palmitateoxidationwasdeterminedasdescribedpreviously(28). indicated, ANX+PI2 (apoptotic) and ANX+PI+ (necrotic) cells are both C16ceramidequantification reportedasdeadcells.DataanalysiswasperformedusingFlowJosoftware (TreeStar). Macrophages were removed from the dish by scraping in PBS and ho- Lactatedehydrogenase–releaseassay.Afterstimulationof2.53105cells, mogenized by passing through a 26G needle 15 times. The lipids were supernatants were collected at the indicated time points, and lactate de- extractedbyincubating50mlcelllysatewith450mlmethanol,withthe hydrogenase(LDH)wasquantifiedwiththeCytoTox96Non-Radioactive additionof50ngC17ceramideas a recoverystandard.Liquidchroma- CytotoxicityAssay(Promega),perthemanufacturer’sinstructions,using tography-tandemmassspectrometry(LC-MS/MS)analysiswasperformed TheJournal ofImmunology 3 asdescribed(29)onanAPI4000LC-MS/MS.Valueswerenormalizedto ANX–PIstainingandflowcytometryat24hshowedasignificant DNAconcentrationinthelysate,asdeterminedusingaNanoDropspec- reductioninviabilitywith thecombinationof LPSand palmitate trophotometer. (Fig.1A,1B).Incontrast,attheseconcentrations,LPSaloneor TNF-aELISA palmitate alone produced no or minimal cell death, respectively. WealsoobservedsynergybetweenLPSandpalmitateusingan Supernatants were harvested from macrophages 6 h after the indicated LDH-release assay as an alternate measure of cell death. Treat- stimulations. TNF-awas quantified using a DuoSet ELISA kit (R&D Systems),accordingtothemanufacturer’sinstructions. ment with palmitate and LPS led to increases in extracellular LDH, quantified as the percentage maximal cellular LDH or Reactive oxygenspeciesquantification simplyasquantityofLDHinmedia(A ),becausetotalcellular 492 Stimulatedmacrophageswerewashedandincubatedwith3mM5-(and-6)- LDH levels remained constant (Fig. 1C). Synergistic cell death chloromethyl-29,79-dichlorodihydrofluorescein diacetate, acetyl ester wasdependentonthedoseofpalmitateandwasapparent atpal- (DCF,Invitrogen)inPBSor5mMMitoSOX(Invitrogen)intissueculture mitate concentrations as low as 100 mM (Fig. 1D, left panel); mediafor15minat37˚C.Cellswereremovedfromplatesandanalyzedby flowcytometryusingtheFL1(DCF)orFL2(MitoSOX)channel(104/cells however,comparableconcentrations oftheunsaturated fattyacid collectedpersample). oleate did not induce cell death when combined with LPS (Fig. 1D,rightpanel).Becauselipotoxicresponsescanbeenhancedin Statistics the presence of high glucose (32), we compared cell death in mac- Statistical analysis was performed using GraphPad Prism software. All rophagesstimulatedinthepresenceofhigh(4.5g/l)orlow(1g/l) results are expressed as means 6 SE. Groups were compared using the concentrationsofglucose.Therewasnodifferenceinmacrophage pairedStudentttestortwo-wayANOVA,asappropriate.Avalueofp# celldeathwithpalmitate-LPStreatmentatthesedifferentglucose 0.05wasconsideredsignificant. D concentrations, indicating that the phenotype was largely driven o w Results by lipid overload (Fig. 1E). Moreover, LPS did not significantly nlo affectpalmitateuptakeoroxidationinthetimeframeofthisassay a LPSandpalmitatetriggersynergisticcelldeathin d macrophages (SFyinge.r1gFis)t.iccelldeathdoesnotrequireERstress, ceramide ed fro To investigate the effects of dietary lipids on macrophage in- m flammatory responses, we cultured primary pMACs with patho- generation,oroxidativestress http pBhSyAsioalloogniecaalsccoonncteronlt)r,aitniocnosmobfipnaaltimonitawteithco5n0jungga/tmedl LtoPBSSsAtim(ou-r TcoomfubrinthaetirocnhoafrapcatlemriizteattehaenmdaLcPrSop,whaegpeecrfeollrmdeeadthANinXdu-PceIdstbaiynitnhge ://w w lation, or PBS as control (30, 31). Analysis of both primary mac- atseveraltimepointsaftertreatment.Inthisanalysis,cellsthatstain w rophagesandtheRAW264.7macrophagecelllinebycombined withANXonly(ANX+PI2)areconsideredapoptotic,whereascells .jim m u n o l.o rg b/ y g u e s t o n D e c e m b e r 1 8 , 2 0 1 8 FIGURE1. PalmitateandLPSleadtosynergisticcelldeathinmacrophages.pMACs(A)orRAW264.7cells(B)werestimulatedwithPBSor50ng/ml LPSinthepresenceof250mMpalmitate(palm)complexedtoBSAorinthepresenceofBSAalone.CelldeathwasassessedbyANX-PIflowcytometryat 24hon104cells/sample.GraphsreportcelldeathaspercentageofthetotalcellsthatwereeitherANX+/PI2orANX+/PI+.(C)Followingstimulationof pMACsasindicated,LDHwasquantifiedinthesupernatant(sup)andisreportedasabsorbanceat492nmandaspercentageoftotalcellularLDH(left panel).TotalcellularLDHfollowinglysisisshownforeachofthetreatments(rightpanel).(D)pMACswerestimulatedwithincreasingconcentrationsof palm(leftpanel)oroleate(rightpanel)inthepresenceofPBSorLPS,andcelldeathwasassessedbyLDHrelease.(E)pMACswerestimulatedwithpalm andLPSineitherhigh-glucose(4.5g/l)orlow-glucose(1g/l)media,andcelldeathwasdeterminedbyLDHrelease.(F)Ratesof14C[palm]uptake(left panel)andoxidation(rightpanel)weredeterminedfollowingLPSstimulation.Allexperimentswereperformedaminimumofthreetimesintriplicate. Barsrepresentthemean6SE.*p#0.05,BSAversuspalm,#p#0.05,PBSversusLPS.ns,Nonsignificant. 4 TLR4ANDPALMITATETRIGGERTRIF-DEPENDENT MACROPHAGEDEATH positiveforbothANXandPI(ANX+PI+)arenecrotic.Amodest death, we incubated macrophages with the RIP1 kinase inhibitor increase in necrotic cells was observedearly, by 8 h after stimu- necrostatin1(nec1).Nec1wasabletoblocknecroptoticcelldeath lation, and both apoptotic and necrotic cells gradually increased induced by TNF–Z-VAD, but it did not affect cell death after over time (Fig. 2A). By 24 h, cells were equally distributed be- palmitate-LPS treatment (Fig. 2D). Caspases are cysteine pro- tweenapoptoticandnecroticphenotypes.Atnotimewastherea teasesthatareknowntofunctionaskeyexecutorsofapoptoticcell predominance of apoptotic cells, suggesting that postapoptotic death.Toevaluatetheirroleinpalmitate-LPS–induceddeath,we necrosis does not account for the appearance of the double- pretreated macrophages with the pan-caspase inhibitor Z-VAD. positive population. In contrast, macrophages treated with the However, this combination led to profound necrotic cell death apoptosis inducer STS were predominantly ANX single positive of macrophages (Fig. 2E). Prior studies showed that treatment (Fig.2A,farrightpanel).Asimilarprofileforapoptoticversusne- with Z-VAD in combination with LPS triggers necroptosis, a re- crotic death after palmitate-LPS stimulation was observed in sponsedependentonRIP1kinaseandinhibitedbynec1(28,29). RAW264.7cellsaswell(Fig.2B).Consistentwithourobservation Whenwetreatedmacrophageswithpalmitate-LPSinthepresence that palmitate-LPS treatment led to apoptosis, as well asnecrosis, of both Z-VAD and nec1, we observed only a modest (∼25%) palmitate-LPS–treated cells exhibited an increase in SubG1 DNA, decreaseinoveralldeathcomparedwithpalmitate-LPSalone,and anindicatorofDNAcleavagethatoccursduringapoptosis(Fig. this primarily reflected a decrease in the ANX single-positive/ 2C). apoptotic population (Fig. 2E). Consistent with the LDH-release RIP1 kinase can be activated by inflammatory signals and assay,nec1treatmentalonedidnotreducepalmitate-LPS–induced triggersaprogrammedformofnecrosisknownasnecroptosis.To cell death of macrophages (Fig. 2E). Together, our findings sug- determine the role of this kinase in palmitate-LPS–induced cell gestamodel inwhichsynergisticcelldeath upontreatment with D o w n lo a d e d fro m h ttp ://w w w .jim m u n o l.o rg b/ y g u e s t o n D e c e m b e r 1 8 , 2 0 1 8 FIGURE2. LPSandpalmitate-mediatedsynergisticcelldeathhasfeaturesofapoptosisandnecrosis.(A)pMACswerestimulatedwith50ng/mlofLPS and250mMpalmitateor80nMSTS,andcelldeathwasassessedbyANX-PIflowcytometryattheindicatedtimepoints.Representativeflowplotsare shown with the percentage of apoptotic (ANX+PI2) and necrotic (ANX+PI+) cells indicated in the lower and upper right quadrants, respectively. (B) pMACs(leftpanel)orRAW264.7cells(rightpanel)werestimulatedwithpalmitate-LPSfortheindicatedtimes;theproportionsofANX+PI2andANX+ PI+ cells are shown. (C) pMACs were stimulated with BSA-PBS or palmitate (palm)-LPS for 16 h, and SubG1 DNAwas assessed by PI staining of permeabilizedcells.Representativegraphsareshown,withthepercentageofcellscontainingcleavedDNAindicatedbythegate.(D)Macrophageswere treatedasindicatedinthepresenceofvehicle(veh)ornec1(50mM),andLDHreleasewasquantified.(E)pMACswerestimulatedwithpalm-LPSinthe presenceofvehicle,Z-VAD(20mM),and/ornec1(50mM)for24h,andcelldeathwasdeterminedbyANX-PIstaining.Thewhiteandblackportionsof thebarsrepresentthepercentageofcellsthatwereANX+/PI2andANX+/PI+,respectively.Barsrepresentmean6SE.*p#0.05,BSA-PBSversuspalm- LPS,#p#0.05,vehversusinhibitor.ns,Nonsignificant. TheJournal ofImmunology 5 palmitate and LPS involves both caspase-dependent and -inde- NOX2werenotprotectedfrompalmitate-LPS–inducedcelldeath pendentcell death mechanisms. (Fig.3D,rightpanel).Interestingly,RAW264.7macrophageshad PreviousreportsinavarietyofcelltypesimplicatedERstress, detectable ROS after palmitate-LPS treatment by DCF flow ceramideproduction,andoxidativestressinthecelldeathinduced cytometry (Fig. 3E). However, although apocynin completely by palmitate (33–35). This raised the possibility that LPS may inhibitedthisROSproduction,itfailedtosignificantlyreducecell amplify lipotoxic cell death by augmenting these cellular stress deathinthesecells(Fig.3E).TodeterminewhetherpMACsfailed responses. ER stress, as measured by CHOP and GRP78 mRNA to generate a ROS response as a consequence of maximal acti- induction,wassignificantlyupregulatedinpalmitate-LPS–treated vationduringisolation,wealsoassessedTNF-areleaseinresponse cells (Fig. 3A). However, treatment with the ER stress reliever to LPS. Using this measure of activation, pMACs were more phenylbutyric acid (36) failed to protect macrophages from cell quiescent than were the RAW cells at baseline and generated a death, despite significantly reducing the expression of ER stress robustTNF-aresponsetoLPSstimulation(Fig.3F),afindingthat markers (Fig. 3A). C16 ceramide levels were also markedly ele- argues against maximal preactivation. Together, these findings vated in palmitate-LPS–stimulated cells, but treatment with the suggestthatERstress,ceramideproduction,oroxidativestress ceramide synthase inhibitor fumonisin B at a concentration that alone is not sufficient to explain macrophage death produced by reduced C16 ceramide levels by 80% failed to protect macro- palmitate andLPS. phagesfromcelldeath(Fig.3B).Unexpectedly,wedidnotob- SynergisticcelldeathrequiresTLR4butisindependentof serveanincreaseinreactiveoxygenspecies(ROS)inpMACs NF-kBandMAPKactivation following palmitate-LPS treatment using DCF or MitoSOX staining(Fig.3C).Furthermore,treatmentofpMACswiththe Canonical LPS signaling occurs through the TLR4 receptor. To D antioxidants butylated hydroxyanisole and MT did not reduce establish the role of TLR4 signaling in the synergistic cell death o w palmitate-LPS–induced cell death (Fig. 3D, left panel), and mac- response,weusedpMACsfromWTandTLR4KOmice.Only n lo rophages from mice deficient in the NADPH oxidase subunit TLR4-expressingcellsunderwentcelldeathuponcotreatmentwith a d e d fro m h ttp ://w w w .jim m u n o l.o rg b/ y g u e s t o n D e c e m b e r 1 8 , 2 0 1 8 FIGURE3. ThecelldeathpathwayactivatedbypalmitateandLPSislargelyindependentofERstress,ceramide,andROSpathways.(A)pMACswere stimulatedwithpalmitate(palm)withorwithoutLPSinthepresenceofphenylbutyricacid(PBA)orvehicle(veh).ExpressionofERstressmarkerswas quantifiedbyqRT-PCR(at 16 h),and cell death wasdeterminedby LDH release(at 20 h).(B) C16ceramidewasquantifiedbyLC-MS/MS inpMACs pretreatedwithvehor5mMfumonisinB(FB)16hafterstimulation.CelldeathwasdeterminedbyANX-PIflowcytometry.(C)pMACswerestimulatedas indicatedfor16h,andoxidativestresswasdeterminedbyDCF(leftpanel)orMitoSOX(rightpanel)stainingandflowcytometryanalysis.Representative graphsareshown.(D)WTmacrophagesweretreatedwithpalm-LPSinthepresenceoftheantioxidantsbutylatedhydroxyanisole(BHA;100mM)orMT(500 mM)(leftpanel)orcomparedwithNOX2KOmice(rightpanel),andcelldeathwasdeterminedat20hbyLDHrelease.(E)RAW264.7cellswerestimulated withpalm-LPSinthepresenceofvehorapocynin(apo),andROS/DCF(leftpanel)orcelldeath(rightpanel)wasdeterminedbyflowcytometry.(F)pMACsor RAW264.7cellswerestimulatedwithPBSorLPS,andTNF-asecretionwasquantifiedat16h.Allexperimentswereperformedaminimumofthreetimesin triplicate.Barsrepresentmean6SE.*p#0.05,BSA-PBSversuspalm-LPS,#p#0.05,vehversusinhibitororWTversusKO.ns,Nonsignificant. 6 TLR4ANDPALMITATETRIGGERTRIF-DEPENDENT MACROPHAGEDEATH palmitate and LPS (Fig. 4A). Of note, the modest toxicity seen using well-established inhibitors of these molecules. To reduce withpalmitatetreatmentalonewasnotreducedintheabsenceof NF-kB activation we used the I-k kinase inhibitor BAY 11-7085 TLR4, indicating that the lipid-only response in these cells is andmacrophagesfrommicedeficientinIKK-b.AlthoughBAY11- TLR4 independent. To further assess the contributions of TLR4, 7085 reduced TNF-asecretion ∼75%, therewas no significant weexaminedcelldeathintheTLR4-deficientcelllineHEK293at effect on the cell death response to palmitate-LPS (Fig 4C). Con- baselineandfollowingstableoverexpressionoftheLPSreceptor sistentwith this result,IKK-b KOmacrophageshadasimilarcell complex(TLR4/MD2/CD14).Thesecellsserveasamodelsystem death phenotype comparedwith WTcells afterpalmitate-LPS forTLRsignalingthatreconstitutesdownstreamactivationofNF- treatment(Fig.4D).MAPKinhibitors(SB203580forp38,PD98059 kBandIRF3andprovideahighlytransfectablecellbackgroundin forERK,andSP600125forJNK)alsofailedtoreducesynergistic which to study TLR-mediated signaling pathways (37–40). Cell celldeathatconcentrationsthateffectivelymodulatedTNFre- death was modestly increased by palmitate alone in parental and lease(Fig.4E).Together,thesedataindicatethatTLR4isrequired TLR4/MD2/CD14-expressing cells; however, only TLR4/MD2/ forpalmitate-LPScelldeathbutthatthispathwaydoesnotrequire CD14-expressing cells showed an augmentation of death with itscanonicalinflammatory-signalingpathways. LPS (Fig. 4B), similar to findings in pMACs and RAW 264.7 TRIFisrequiredforpalmitate-LPSsynergisticcelldeath cells. Because TLR4 signaling triggers activation of NF-kB and the Among the earliest events following TLR4 ligation is its associ- MAPKsp38,JNK,andERK,weprobedthesesignalingpathways ation with the adaptor proteins MyD88 and TRIF, each of which initiates distinct signaling pathways. To address the downstream pathwayrelevanttopalmitate-LPS–mediatedsynergisticdeath, D macrophages were isolated from MyD88 KO, TRIF KO, and o w MyD88-TRIFKO(DBLKO)mice.Afterstimulationwithvehicle n lo or palmitate in the presence of PBS or LPS, cell death was de- a d termined by LDH release (Fig. 5A). Macrophages from WTand ed MyD88 KO cells demonstrated similar cell death phenotypes; fro however, TRIF KO macrophages were significantly protected m h fromsynergisticcelldeath.Macrophagesfrommicelackingboth ttp MyD88 and TRIF were completely protected from the effect of ://w LPS on lipotoxicity. We confirmed the importance of TRIF-me- w diated signaling in the cell death phenotype by ANX-PI staining w of WT versus TRIF KO macrophages challenged with LPS and .jim m palmitate (Fig. 5B). Consistent with our data from TLR4 KO u mice, TRIF–deficient macrophages were protected from syner- no gisticcelldeathbutnotthemodestbaselinetoxicityofpalmitate l.o rg (Fig.5B).Thus,TLR4-TRIFsignalingisrequiredfortheobserved b/ synergisticmacrophagecelldeathphenotypeuponchallengewith y g palmitate andLPS. u e The TRIF-signaling cascade leads to activation of the tran- st o scriptionfactorIRF3andtheinductionofIFN-b.Todetermine n D whether IRF3 activation is required downstream of TRIF in this e c cell death pathway, macrophages from IRF3 KO micewere stim- em ulatedwithpalmitate-LPS.Surprisingly,IRF3KOmacrophages be werenotprotectedfrompalmitate-LPS–mediatedcelldeath(Fig. r 1 8 5C).Asexpected,IFN-bmRNAwasnotinducedbyLPStreatment , 2 ofIRF3KOcells(Fig.5D).Thus,TLR4-mediatedTRIFsignaling 01 8 modulates synergistic cell death through an IRF3-independent mechanism. Cell deathinducedbypalmitateandTLR4activationis FIGURE4. SynergisticcelldeathisTLR4dependentbutindependentof associatedwithlysosomaldepletion anddestabilization NF-kBandMAPKactivation.(A)pMACsfromWTorTLR4KOmicewere Palmitate treatment leads to membrane remodeling, a process stimulated with the indicated concentrations of palmitate (palm) with or without LPS, and cell death was determined by LDH release at 20 h. (B) linkedtodysfunctionofintracellularorganelles(41,42).Giventhe HEK293 cells stably transfected with TLR4/CD14/MD2 or control vector evidencethatlysosomedysfunctioncaninducecelldeaththrough werestimulatedwith50ngLPSinthepresenceof750mMpalmorBSA. caspase-dependent and -independent mechanisms (43), we eval- CelldeathwasdeterminedbyANX-PIstainingat48h.(C)WTmacrophages uatedtheimpactofLPSandpalmitateonmacrophagelysosomes. werepretreatedwiththeNF-kBinhibitorBAY11-7085(BAY;3.125mM), LysoTrackerRedfluorescenceinmacrophagestreatedwitheither followedbystimulationwithBSA-PBSor250mMpalm-LPS.Celldeathwas LPS or palmitate was slightly reduced compared with control- determinedbyLDHreleaseat20h,andsecretionofTNF-awasquantifiedby treated cells when analyzed by flow cytometry. However, com- ELISA. (D) IKKb macrophage-specific KO cells were stimulated as indi- bined palmitate-LPS treatment of macrophages caused a marked cated,andcelldeathwasassessedbyLDHreleaseat20h.(E)pMACswere reductioninLysoTrackerRedsignal(Fig.6A–C).Functionofthe preincubatedwithMAPKinhibitors(20mMSB203580forp38;10mM lysosomalcompartmentwasalsoassessed usingTMR-dextranto PD98059 for ERK; 20 mM SP600125 for JNK), followed by palm-LPS treatment. Cell death and TNF-a secretion were determined at 20 h by labelthelysosomesandlateendosomes.Palmitateandpalmitate- LDH release and ELISA, respectively. All experiments were performed LPS treatment produced a significant decrease in the TMR-dex- aminimumofthreetimesintriplicate.Barsrepresentmean6SE.*p# transignal,suggestingthatthenormallysosomalcompartmentis 0.05,vehicleversusinhibitororWTversusKO.ns,Nonsignificant. impaired or depleted (Fig. 6D). The downshift in lysosomal TheJournal ofImmunology 7 D o w n lo a d e d fro m h ttp ://w w w .jim m u n o l.o rg b/ y g u e s t o n D e c e m b FIGURE5. TheinductionofsynergisticcelldeathbypalmitateandTLR4occursviaaTRIF-dependent,IRF3-independentmechanism.(A)Macro- er 1 phageswereisolatedfromWTmiceormicedeficientinMyD88,TRIF,orboth(DBLKO).CelldeathwasassessedbyLDHrelease20haftertheindicated 8 stimulations. (B) WTor TRIF KO cells were stimulated as indicated, and cell death was assessed by ANX-PI staining at 24 h. Representative flow , 2 0 cytometryplotsareshown.(C)pMACsfromWTorIRF3KOmicewerechallengedwith250mMpalmitate(palm)withorwithoutLPS,andcelldeathwas 18 assessedbyANX-PIstainingat24h.(D)mRNAwasisolatedfromWT(leftpanel)orIRF3KO(rightpanel)macrophages1hafterLPSstimulation.IFN-b expressionlevelsweredeterminedbyqRT-PCR.Allexperimentswereperformedaminimumofthreetimesintriplicate.Barsrepresentmean6SE.*p# 0.05,PBSversusLPS,#p#0.05,WTversusKO.L,LPS;ns,nonsignificant;P,PBS. fluorescence identified with LysoTracker staining was evident as rescence(FL3)withaconcomitantincreaseingreenfluorescence early as 8 h after stimulation and preceded the loss of mito- (FL1),whichisconsistentwithpalmitate-LPS–inducedLMP(Fig. chondrialmembranepotentialandincreaseinANXstaining(Fig. 6F). Furthermore, during LMP, release of cathepsins into the cy- 6E). tosol is associated with cell death (47). In macrophages treated Diminished staining with lysomotropic dyes can result from withpalmitate-LPS,cathepsinDproteinincreasedinthecytosol, lysosome depletion and/or lysosomal membrane permeabilization and this response was blunted in TRIF-deficient macrophages (LMP), both of which have been associated with lysosomal-me- (Fig.6G).Toaddressthecontributionofcathepsinreleasetocell diatedcelldeath(44,45).Weusedtwocomplementaryapproaches death from palmitate-LPS challenge, we pretreated stimulated to assess LMP in palmitate-LPS–stimulated macrophages. Acri- macrophages with the cathepsin B inhibitor, CAO-74. Inhibition dineorangeisalysomotropicdyethatfluorescesred/orange(FL3 of cathepsin B partially protected cells from palmitate-LPS–in- channel)athighconcentrations,suchasinthelysosome,andgreen duced cell death (Fig. 6H). We also assessed the lysosomal phe- (FL1 channel)when present atlow concentrations,such as inthe notype in TRIF KO macrophages after palmitate-LPS treatment. cytosol.LMPcausesareductioninredfluorescencewithacon- Although the decrease in LysoTracker Red signal in WT macro- comitant increase in green fluorescence (46). In macrophages phages was exacerbated with the combined LPS and palmitate treated with palmitate-LPS, we observed a decrease in red fluo- stimulusovereitherstimulusalone(Fig.6A,6I,blackversusblue 8 TLR4ANDPALMITATETRIGGERTRIF-DEPENDENT MACROPHAGEDEATH D o w n lo a d e d fro m h ttp ://w w w .jim m u n o l.o rg b/ y g u e s t o n D e c e m b e r 1 8 , 2 0 FIGURE6. EarlyandsustainedlossoflysosomesoccursinmacrophagestreatedwithpalmitateandLPS.pMACswerestimulatedasindicatedfor16h 1 8 andstainedwithLysoTrackerRed(lysoTred),followedbyflowcytometry(A,B)orimmunofluorescence(C).Arepresentativeplot(A),meanfluorescence intensityquantification(B),andimmunofluorescentimage(C;scalebar25mm;LysoTrackerRed,Hoechstnucleardyeblue)areshown.(D)pMACswere preloadedwithTMR-dextranfor2handthentreatedwiththeindicatedstimulifor16h,followedbyflowcytometryanalysis.Arepresentativegraphis shown.(E)QuantificationofLysoTrackerRedlowcells(LTlow),annexin-positivecells(ANXhigh),andmitochondrialmembranepotential(MMP)at8and 16hafterstimulationwithBSA-PBSorpalmitate(palm)-LPS.(F)pMACsweretreatedwithBSA-PBSorpalm-LPSfor16h,followedbystainingwith acridineorange.Orange(FL3)andgreen(FL1)meanfluorescenceintensity(MFI)wasquantifiedbyflowcytometry.(G)Cytoplasmicproteinwasisolated 16hafterstimulationwithBSA-PBSorpalm-LPSinWTorTRIFKOcells,andcathepsinD(cathD)levelswereassessedbyWesternblot.LAMP1blotting wasalsoperformedtoassessadequacyoffractionation.(H)pMACswerepretreatedwiththecathepsinBinhibitorCA-O74(10mM),followedbyBSA-PBS orpalm-LPS,andcelldeathwasdeterminedbyLDHreleaseat20h.(I)WTorTRIFKOpMACsweretreatedwithBSA-PBS,BSA-LPS,palm-PBS,or palm-LPSfor16h,followedbyLysoTrackerRedstaining.Representativeflowcytometrygraphsareshown.Allexperimentswereperformedaminimumof threetimesintriplicate.Bargraphsdisplaymean6SE.*p#0.05,BSAversuspalm,#p#0.05,PBSversusLPS. orgreenlines),palmitate-LPS–treatedTRIF-deficientmacrophages macrophagecelldeath.TFEBcontrolsagene-expressionprogram hadasimilarphenotypeascellstreatedwithpalmitate(Fig.6I, thatstimulateslysosomalbiogenesisanddegradativefunctionand black versus green lines). waspreviouslyshowntoimprovecellviabilityinthesettingof lysosomaldysfunctionand/ordepletion(45,48–50).Thus,we Overexpressionofthelysosomalbiogenesistranscriptionfactor tested whether gain-of-function of TFEB could reduce cell death TFEBattenuates celldeathinducedbypalmitateandLPS and prevent lysosomal depletion following palmitate-LPS treat- Our data suggested that inflammatory stress under lipotoxic con- ment.BecausepMACsarenotreadilytransfectable,theseexperi- ditionscauseslysosomedysfunctionand/orlossthatcontributesto mentswereperformedinHEK293-TLR4/MD2/CD14(HEK)cells, TheJournal ofImmunology 9 inwhichpalmitate-LPS stimulation induces synergisticcell death viability by PI staining alone, because the GFP marker precluded (Fig.4B).HEKcellsweretransientlytransfectedwithabicistronic concomitant FITC-annexin staining. Cells transfected with TFEB vector (IRES) encoding GFP, with and without TFEB sequences. demonstratedsignificantlyimprovedsurvivalcomparedwithcells UsingflowcytometrytodetectGFP-expressingcells,wedetermined withcontrol(GFP-only)vector(Fig.7C).InTFEB-transfected that∼29%ofthecellsexpressedGFPat72h(Fig7A).TFEBex- wells,untransfectedcells(GFP2)hadadeathphenotypesimilarto pressionattheproteinlevelwasalsoconfirmedviaWesternblotting control-transfectedcells.Thus,TFEB-mediatedprotectionfrom incellsthathadreceivedtheTFEB-containingplasmid(Fig.7B).To celldeath occurred ina cell-autonomous fashion (Fig. 7D). determinetheeffectofTFEBoverexpressiononcellviabilityin In a parallel set of samples we also assessed the lysosomal responsetopalmitate-LPS,wegatedonGFP+cellsanddetermined compartment in transfected cells using LysoTracker Red. GFP+ D o w n lo a d e d fro m h ttp ://w w w .jim m u n o l.o rg b/ y g u e s t o n D e c e m b e r 1 8 , 2 0 1 8 FIGURE7. OverexpressionofTFEBpreventslysosomalphenotypeandrescuescelldeathinducedbypalmitateandLPS.HEK293-TLR4/CD14/MD2 cellsweretransfectedcontrolGFPvector(cntrl)oraFLAG-TFEBexpressionconstructcontaininganIRES-GFPelement.Cellswerestimulatedasin- dicated24haftertransfection,andflowcytometrywasperformed48hlater.(A)Approximately29%ofthetotalcellsexpressedtheGFPconstruct.(B) TFEBproteinexpressionwasassessedbyWesternblot24haftertransfectionusinganti-FLAGAb.Actinisshownasloadingcontrol.(CandD)Twenty- fourhoursfollowingtransfection,cellswerestimulatedwithBSA-PBSorpalmitate(palm)-LPSfor48h,stainedwithPI,andanalyzedbyflowcytometry. RepresentativedotplotsfromtheGFP+cellsareshown(C),andsummeddataforGFP+(toppanel)andGFP2(bottompanel)cellsareplotted(D).(E) LysoTrackerRedfluorescencewasassessed48hafterstimulationwithBSA-PBSorpalm-LPSincontrolvectorandTFEB-transfectedcells.Representative graphs are shown. (F) Quantification of the palm-LPS–induced decrease in LysoTracker Red (lysoT red) fluorescence (MFI) in control and TFEB- transfectedcells.Allexperimentswereperformedaminimumofthreetimesintriplicate.Barsrepresentmean6SE.*p#0.05,controlvectorversus TFEBvector.ns,Nonsignificant.