through a TRIF-Dependent Pathway Leads to Synergistic Macrophage Cell Death TLR4 Activation ...

13 Pages·2012·1.35 MB·English

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
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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
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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
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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
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Stimulationswereperformedonthedayafterharvest.Forflowcytometry stimulationsfor16h.Afterstaining,cellswerewashedthreetimeswith w
e(GxpreeirniemreBntiso,-Opneeri)totnoefaalcicleitlalstewceerllehcaurlvteusrte.dAoltnholuogwh-asdohmeerenstcuedipeslatrees- PteBrnSa,tihvaerlvye,smteadcraospdheasgcersibwederaebocvuelt,uarenddoannaglylazsesdcboyveflroswlipcsystotamineetrdy.wAitlh- .jim
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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
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lation, or PBS as control (30, 31). Analysis of both primary mac- atseveraltimepointsaftertreatment.Inthisanalysis,cellsthatstain w
rophagesandtheRAW264.7macrophagecelllinebycombined withANXonly(ANX+PI2)areconsideredapoptotic,whereascells .jim
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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
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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
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palmitate-LPS–induced cell death (Fig. 3D, left panel), and mac- response,weusedpMACsfromWTandTLR4KOmice.Only n
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rophages from mice deficient in the NADPH oxidase subunit TLR4-expressingcellsunderwentcelldeathuponcotreatmentwith a
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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
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MyD88-TRIFKO(DBLKO)mice.Afterstimulationwithvehicle n
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or palmitate in the presence of PBS or LPS, cell death was de- a
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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
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The TRIF-signaling cascade leads to activation of the tran- st o
scriptionfactorIRF3andtheinductionofIFN-b.Todetermine n
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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
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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
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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+
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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.