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InternationalJournalof
MolecularSciences
Article
LXR RegulatesoxLDL-InducedTrainedImmunity
inMacrophages
*,†,†,,JannikSonnenberg,DennisSchwarz,HelenaKörner,
HolgerReineckeandYahyaSohrabi*
DepartmentofCardiologyICoronaryandPeripheralVascularDisease,HeartFailure,UniversityHospital
Münster,48149Münster,Germany;vivienne.******@(.);laura.******@(.);
Jannik.******@(.);Dennis.******@(.);
helena.******@(.);holger.******@(.)
*Correspondence:******@(.);yahya.******@(.)
†Theseauthorscontributedequallytothiswork.
Abstract:Reprogrammingofmetabolicpathwaysinmonocytesandmacrophagescaninducea
,wehaveanalyzed
theroleoftheLiverXreceptor(LXR),acrucialregulatorofmetabolismandinflammation,in
oxidizedlow-densitylipoprotein(oxLDL)-
incubatedwithLXRagonists,antagonists,,cells
wererestimulatedwiththeTLR-
,whileLXRinhibitionpreventedthe
oxLDL-,LXRinhibitionblockedthemetabolicchanges
,enrichmentof
activatinghistonemarksattheIL-
onthedifferentialexpressionoftheLXRisoforms,weinhibitedLXRaandLXRbgenesusingsiRNA
Citation:Findeisen,.;Voges,,siRNA-mediatedknock-downofLXRablockedtheoxLDL-induced
.;Braun,.;Sonnenberg,J.;inflammatoryresponse,whileknock-
Schwarz,D.;Körner,H.;Reinecke,H.;novelroleoftheLXR isoformintheregulationofoxLDL-
Sohrabi, RegulatesimportantaspectsofLXRsignalingininnateimmunitywithrelevancetoatherosclerosisformation.
oxLDL-InducedTrainedImmunityin
,Keywords:LXR;trainedinnateimmunity;oxLDL;immunometabolism;inflammation
23,:///
ijms23116166
AcademicEditor:KeikoHosohata
Received:3March2022
Accepted:26May2022Today,atherosclerosiscanbebestdescribedasalipid-drivenlow-gradechronicin-
Published:31May2022flammatorydiseaseofthevascularwall[1,2].Theaccumulationoflipoproteins,mainly
low-densitylipoproteins(LDL)inthesubendothelialspacefosteredbyendothelialdysfunc-
Publisher’sNote:MDPIstaysneutraltion,isgenerallyconsideredtheinitialstepduringthepathogenesisofatherosclerosis[2].
withregardtojurisdictionalclaimsin
publishedmapsandinstitutionalaffll-Intheintimallayer,phagocyticcellssuchasmacrophagesortransformedsmoothmus-
[2,3].Intramurallipoproteinscanbemodifled
throughinteractionswithmetalions,reactiveoxygenspecies(ROS),andenzymes,suchas
myeloperoxidase[4].Thesemodifledlipoproteins,forexample,oxidizedLDL(oxLDL),
canserveasso-calleddamage-associatedmolecularpatternmolecules(DAMPs),whichcan
Copyright:©(PRR),thecentralregulatorsoftheimmuneresponse.
LicenseeMDPI,Basel,-mediatedactivationofPRRs,suchastoll-likereceptors(TLRs),triggersproinflam-
Thisarticleisanopenaccessarticlematorysignalingpathways,therebyinducingthesecretionofvariouschemokinesor
distributedunderthetermsandcytokinessuchasIL-1 ,IL-6,orTNF [2].Theseinflammatorymediatorsfurtheractivate
conditionsoftheCreativeCommonsimmunecells,smoothmusclecellsandendothelialcellsresultinginachronicinflammatory
Attribution(CCBY)license(https://response[2,5,6].AdditionalstudieshavedemonstratedthatDAMPactivationthrough
[5–9].Trainedinnate
/).
,23,:///ijms23116166:.
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immunitydescribesalastingactivationormemory-likestatethatenhancestheimmunere-
sponseofinnateimmunecellsandevennon-immunecellstoasecondarychallenge[10,11].
Whilethetrainedinnateimmunityphenotypecanconferprotectionfrominfectiousdis-
eases,activationbynon-microbialatherogenictriggerssuchasoxLDLorLipoprotein(a)
canhaveharmfulconsequences[10,12,13].Currentevidencehasconsistentlyimplicated
metabolicandepigeneticmechanismsinregulatingtrainedinnateimmunity[10,12,14].
Trainingofmonocyteswasassociatedwithashiftfromoxidativephosphorylationto
glycolysisresultinginincreasedlactateproductioninanAkt/mTOR/Hif1 -dependent
manner[5,7,8].Furthermore,activationoffattyacidsynthesisandcholesterolmetabolism
havebeendemonstratedtocontroltrainedimmunity[14,15].Downstream,metabolic
reprogrammingcontrolsthemodulationoftheepigeneticlandscapebyimplementinga
,wehaveshownthatLiverXreceptors(LXRs),key
regulatorsofcholesterolandfattyacidmetabolism,areinvolvedintheregulationoftrained
innateimmunity[16].Usinganestablishedinvitromodeloftrainedinnateimmunityin
humanmonocytes,weshowedthatinhibitionofLXRblocksBacillusCalmette-Guerinvac-
cine(BCG)-inducedtrainedimmunity[16].Furthermore,LXRagonisttreatmentinduceda
long-terminflammatoryresponseinmonocytesindependentofanyadditionalstimuli[16].
ThisinflammatorystimulationbyLXRagonistswasaccompaniedbycharacteristicfeatures
oftrainedinnateimmunity,suchasactivatinghistonemarksoninflammatorygenepro-
motersandmetabolicreprogrammingwithincreasedHIF1 -signaling[16,17].Therole
ofLXRactivationinthedevelopmentofinnateimmunememoryhasnotbeenaddressed,
therefore,wehaveinvestigatedtheroleofLXRinoxLDL-inducedtrainedinnateimmunity
inthisstudy.
,WhereasLXRInhibitorsBlockoxLDL-InducedInflammatoryInnate
ImmuneMemory
WehavepreviouslyshownthatpharmacologicactivationofLXRduringBCGtreat-
ment(awell-studiedinduceroftrainedinnateimmunity)canstronglyenhanceinflam-
matorycytokinesynthesisuponrestimulationwiththeTLR-2agonistPam3cys,whereas
BCG-primingwasinhibitedfollowinginhibitionofLXR[16].Basedonthisobservation,
wehaveanalyzedtheroleofLXRinoxLDL-
monocyteswerepretreatedwiththeLXRagonist(T1317)andtrainedwithlow-doseoxLDL
-dayrestingperiod,,
theoxLDLeffectininducinginnateimmunememorywastestedwith10and20g/mL.
However,theexperimentswerecontinuedusing20g/mLoxLDLduetostrongerin-
flammatoryresponsesincomparisonto10g/
experiment[16],LXRactivationenhancedIL-6andTNF secretioninoxLDL-primedcells
(Figure1A,B),whileinhibitionofLXRwiththeantagonistGSK2033reducedthepriming
effectofoxLDL-training(Figure1C,D),
LXRactivationusingLXRagonistaloneenhancedinflammatoryresponsestorestimula-
tion[16,17].Withoutrestimulation,theprimedcellsdidnotproducedetectablecytokine
levels(datanotshown).Furthermore,treatmentofmonocyteswiththeLXRinverseagonist
(SR9238)signiflcantlyreducedthelevelsofproinflammatorycytokinesaswell(Figure
S1A,B).Inaddition,wedevelopedamodelofoxLDL-inducedtrainedinnateimmunity
inthehumanmonocyticcelllineTHP-,GSK2033
blockedoxLDL-traininginTHP-1cells(Figure1E,F).NeitherPMAtreatment,whichin-
ducesdifferentiation,noroxLDLtreatmentofPMA-differentiatedTHP-1cellsresultedina
:.
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(LXR)dependentregulationoftheoxidizedlow-densitylipoproteinLiverXreceptor(LXR)dependentregulationoftheoxidizedlow-densitylipoprotein
(oxLDL)-(oxLDL)--
indicatedwith20dicatedwith20µg/mLoxLDL,2g/mLoxLDL,2µMT1317(LXRagonist),(LXRagonist),µMGSK2033(LXRantagonist)orMGSK2033(LXRantagonist)
vehiclefor24h,keptfor5daysinacompletemedium,andrestimulatedwith5orvehiclefor24h,keptfor5daysinacompletemedium,andrestimulatedwith5µg/mLPam3cysforg/mLPam3cys
-6(A,C)andTNFα(B,D)-
-6(atedtomacrophagesandwereprimedasdescribedabove;thecellswererestedfor5daysandA,C)andTNF (B,D)
macrophagesandwereprimedasdescribedabove;thecellswererestedfor5daysandrestimulatedrestimulatedwith10µg/-6(E)andTNFα(F)weremeasuredinthesuper-
±SDofsixindividualsinthreedifferentexperiments.*g/-6(E)andTNF (F)<
,**p<,and***pSDofsixindividualsinthreedifferentexperiments.*<<,**p<,
and***p<.
Themetabolicshifttowardaerobicglycolysisisacrucialmechanisminestablishing
Themetabolicshifttowardaerobicglycolysisisacrucialmechanisminestablishing
trainedinnateimmunity[14].Thiscanbeseenwithmanydifferentinducersoftrained
trainedinnateimmunity[immunity,includingoxLDL[18].Enhanceda14].Thiscanbeseenwithmanydifferentinducersoftrainederobicglycolysisduringtrainedimmunity
immunity,includingoxLDL[18].Enhancedaerobicglycolysisduringtrainedimmunityis
isassociatedWITHandregulatedthroughincreasedexpressionofkeyglycolyticenzymes
associatedWITHandregulatedthroughincreasedexpressionofkeyglycolyticenzymes
suchasGLUT1,HK2,,increasedgeneexpressionwasblockedfol-
suchasGLUT1,HK2,,increasedgeneexpressionwasblocked
lowingLXRinhibition(Figure2A–C),whichwasconfirmedbyPFKFB3proteinanalysis
followingLXRinhibition(Figure2A–C),whichwasconflrmedbyPFKFB3proteinanaly-
byWesternblotting(FigureS4A–C).Accordingly,wefoundashifttowardglycolysis
sisbyWesternblotting(FigureS4A–C).Accordingly,wefoundashifttowardglycolysis
characterizedbyhigherlactateproductionandglucoseconsumption(Figure2D,E).Inter-characterizedbyhigherlactateproductionandglucoseconsumption(Figure2D,E).Inter-
estingly,theoxLDL-inducedmetabolicchangesinlactateandglucoseconsumptionwere
estingly,theoxLDL-inducedmetabolicchangesinlactateandglucoseconsumptionwere
reversedfollowingLXR-inhibitortreatment(Figure2D,E).Inaddition,LXRinverseagonistreversedfollowingLXR-inhibitortreatment(Figure2D,E).Inaddition,LXRinverseago-
SR9238inhibitedtheoxLDL-inducedexpressionofglycolyticgenes24hafterprimingnistSR9238inhibitedtheoxLDL-inducedexpressionofglycolyticgenes24hafterpriming
(FigureS2A–C).(FigureS2A–C).
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--
asindicatedwith20asindicatedwith20µg/mLoxLDL,,µMGSK2033(LXRantagonist),orvehiclefor24handeitherMGSK2033(LXRantagonist),orvehiclefor24handeither
lysedforRNAexpressionorkeptfor5daysincompletemediumforlactateassaysandglucose
lysedforRNAexpressionorkeptfor5daysincompletemediumforlactateassaysandglucose
,HK2,andPFKFB3wereanalyzedbyreal-timeqPCR(A–C).
-GLUT1,HK2,andPFKFB3wereanalyzedbyreal-timeqPCR(A–C).On
day6lactateconcentrationwasmeasuredinthecelllysateandglucoseconcentrationwasmeasureduredinthesupernatant(D,E).Graphsrepresentmeanvalues±SDofsixindividualsinthreediffer-
inthesupernatant(entexperi