An active mitochondrial biogenesis occurs during dendritic cell differentiation

and education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling or

licensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of the

article (e.g. in Word or Tex form) to their personal website or

institutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies are

encouraged to visit:

ContentslistsavailableatSciVerseScienceDirect

The

International

Journal

of

Biochemistry

&

Cell

Biology

j o u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / b i o c e l

An

active

mitochondrial

biogenesis

occurs

during

dendritic

cell

differentiation

Patrizia

Zaccagnino

_{, Maddalena}

_Saltarella

_{, Stefania}

_Maiorano

_{, Antonio}

_Gaballo

_{, Giuseppe}

_Santoro

_,

Beatrice

Nico

_,

_Michele

_Lorusso

_,

_Annalisa

_Del

_Prete

a_{DepartmentofBasicMedicalSciences,UniversityofBari,PiazzaG.Cesare11,70124Bari,Italy}

b_{InstituteofBiomembraneandBioenergetics,CNR,PiazzaG.Cesare,70124Bari,Italy}

c_{DIMO,UniversityofBari,PiazzaG.Cesare,70124Bari,Italy}

d_{HumanitasClinicalandResearchCenter,viaManzoni56,20089Rozzano(MI),Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6March2012

Receivedinrevisedform3July2012

Accepted24July2012

Available online xxx Keywords:

Dendriticcelldifferentiation

Mitochondrialrespiratorycomplexes

Mitochondrialbiogenesis

Anti-oxidantenzymes

a

b

s

t

r

a

c

t

Dendriticcells(DC)aresentinelsoftheimmunesystemderivingfromcirculatingmonocyteprecursors recruitedtositesofinflammation.Inapreviousreport(DelPreteetal.,2008)weshowedthat,after dif-ferentiation,DCexhibitedincreasednumberofcondensedmitochondriaanddynamicchangesintheir energymetabolism.AstudyispresentedhereshowingthattheDCdifferentiationprocessis character-izedbyincreasedexpressionlevelandactivityofmitochondrialrespiratorycomplexes,aswellasbyan increasedmitochondrialDNA(mtDNA)copynumber.Moreover,DCareequippedwithmoreefficient antioxidantprotectionsystems,overexpressedmostlikelytodetoxifyincreasedROSproduction,asa consequenceofthemuchhighermitochondrialactivity.Kineticanalysisofthethreemain mitochon-drialbiogenesis-associatedgenesrevealedthatthepeakinPPAR␥coactivator-1alpha(PGC-1␣)gene expressionwassuddenlyreachedfewhoursaftertheonsetofthedifferentiation.WhilePGC-1␣ expres-sionrapidlydeclines,themitochondrialtranscriptionfactorA(TFAM)andnuclearrespiratoryfactor-1 (NRF-1)expressiongraduallyincreased.Thesefindingsdemonstratethatanactivemitochondrial bio-genesisoccursduringDCdifferentiationandfurthersuggestthatanearlyinputbythemasterregulator ofmitochondrialbiogenesisPGC-1␣isneededtotriggerthesubsequentactivationofthedownstream transcriptionfactors,NRF-1andTFAMinthisprocess.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Dendriticcellsareprofessionalantigenpresentingcellsthatplay acriticalroleintheinitiationandregulationofadaptiveimmune responses(Steinman,2003).DCderivefrombonemarrow progen-itorsorfrommonocytesandtheneitherstayinthebloodstream ormigrateintoperipheraltissues,wheretheyshowanimmature phenotype.ImmatureDC,suchasLangerhanscellsintheskin,actas sentinels,continuouslytakingupantigensandundergoing activa-tion(Banchereauetal.,2000).Duringtheirmaturation,DCmigrate tosecondary lymphoid organs, where theybecome specialized immune-stimulatorycellsabletoactivatenaiveTcells(Sozzani, 2005).

Inapreviousreport,weshowedthatthedifferentiationprocess of peripheral blood monocytes to DC requires growth factor

∗ Correspondingauthor.Tel.:+390805448522;fax:+390805448538.

∗∗ Correspondingauthorat:DepartmentofBasicMedicalSciences,Universityof

Bari,PiazzaG.Cesare11,70124Bari,Italy.Tel.:+390805448522;

fax:+390805448538.

E-mailaddresses:[email protected](M.Lorusso),

[email protected](A.DelPrete).

dependent production of intracellular Reactive Oxygen Species (ROS)(DelPreteetal.,2008;seealsoSattleretal.,1999;Shengetal., 2010).As revealed by ultrastructural analysis,DC exhibited an increasednumberofcondensedmitochondriacomparedto mono-cyteprecursors.Ahigherendogenousrespiratoryactivitytogether withanincreaseinATPcontentandmitochondrialenzymecitrate synthaseactivitywasalsofound.Moreover,thepresenceof mito-chondrialComplexIinhibitorrotenoneintotheculturemedium caused inhibition of the DC differentiation process, through inhibitionofmitochondrialROSproduction(DelPreteetal.,2008). MitochondriaarebestknownfortheirroleinATPproduction throughoxidativephosphorylation,calciumhomeostasis, apopto-sis and cell signalling (Kroemer and Reed, 2000; Chan, 2006). Furthermore,mitochondriaarethemajorsourceofendogenous ROSthatplayakeyroleincellphysiologyincludingcell differenti-ationandproliferation(Kiefeletal.,2006).Mitochondriapossess theirown genomic apparatus,mtDNA, a 16.5kb closed-circular doublestrandedDNA,whichcodesforsomeoftheirproteins. Con-sequently,mitochondrialbiogenesisishighlyorchestratedbythe transcriptionalregulatorycircuitsbetweenthismtDNAandnuclear genesencodingmitochondrialproteins(GaresseandVallejo,2001; Kellyand Scarpulla,2004).Transcriptionof mtDNA requires,in addition tothemitochondrial specific DNA polymerasegamma

1357-2725/$–seefrontmatter © 2012 Elsevier Ltd. All rights reserved.

(POLG), a small number of nucleus-encoded proteins compris-ing the mitochondrial transcription factor A (TFAM) (Clayton, 1998; Scarpulla, 2002). Additional regulating elements include thetranscriptionfactorNRF-1andmembersofthePGC-1family (Scarpulla,2008).Inparticular,PGC-1␣appearsasamaster regu-latorofmitochondriabiogenesisduetoitsabilitytoactivateTFAM throughdirectinteractionwithNRF-1(GoffartandWiesner,2003; Scarpulla,2011).

Inthepresentstudytheprocessofmitochondrialbiogenesis accompanyingthedifferentiationofmonocyte precursorsinDC wasexamined. In particular, we focused onthe structural and functionalchangesinmitochondrialrespiratorycomplexesandthe expressionandactivityofanti-oxidantenzymesoccurringduring thisprocess.Moreover,adirectcomparisonoftheexpressionlevel ofmitochondrialproteinswasperformedbyusinganewtechnical approachwhichallowspuremitochondrialfractiontobeisolated frombothmonocytesandDC.Finally,themtDNAcontentandthe geneexpressionchangesoccurringduringthemonocytetoDC tran-sitionwerestudied,togetherwiththekineticsofthetranscription ofmitochondria-relatedgenesoverthe6-dayscultureperiod.

2. Materialsandmethods

2.1. Dendriticcellgenerationandcharacterization

Peripheralblood mononuclearcells(PBMC)wereisolatedby thestandardFicoll-Paque(Amersham,UK)gradientcentrifugation frombuffycoats(obtainedthroughthecourtesyofthelocalBlood Bank,PolyclinicHospital,Bari,Italy).Monocyteswerepurifiedby immunomagneticseparationusinganti-CD14conjugatedmagnetic microbeads(MiltenyiBiotec,BergischGladbach,Germany)and cul-turedfor6daysat1×106_{/mlinRPMI1640medium(BiochromAG,}

Leonorenstr,Berlin)supplementedwithheat-inactivated10%FBS (EuroClone,Milan,Italy),GM-CSF(50ng/ml)andIL-4(20ng/ml) (R&DSystems,Minneapolis,MN).Surfacephenotypeanalysiswas performedusinghumanMo-DCdifferentiationinspector, contain-ingmonoclonalantibodiesrecognizingCD14,CD83andCD209and correspondingisotypecontrols(MiltenyiBiotec).

2.2. Mitochondriaisolationwithsuperparamagneticmicrobeads MitochondriafrommonocytesandDCwerepurifiedbyusing the superparamagnetic anti-translocase of outer mitochondrial membrane 22 (-TOM22) microbeads (Miltenyi Biotec). Briefly, monocytesandDCwerewashedtwicewithphosphate-buffered saline(PBS),andlysedbyshearingthroughaneedle(29G) approx-imately40times.Aftercelldisruption,analiquotofthelysatewas examinedfortrypanblueexclusiontoensurethat80%ofthecells werelysed.Formagneticlabelling,celllysatewasincubatedwith anti-TOM22microbeadsfor60minat 4◦C, withgentleshaking. Subsequently,cellsuspensionwasloadedontoapre-equilibrated MACScolumn,andfinally,retainedmitochondriawereeluted. Fol-lowingcentrifugation at16,000×g for1min,themitochondrial pelletwasfinallyresuspendedintheappropriatestoragebuffer. 2.3. Measurementofrespiratorycomplexactivities

Monocytes and DC were suspended in a medium contain-ing 75mM sucrose, 40mM KCl, 5mM KH2PO4, 3mM MgCl2,

0.5mMEDTA and30mMTris–HCl(pH 7.4)(BufferA)and sup-plementedwith30␮g/106_{cellsdigitoninaspreviouslydescribed}

(Sgobboetal.,2007).After2min,cellsuspensionwascentrifuged and thepelletwasresuspended inhypotonic medium contain-ing25mMKH2PO4 (pH 7.2),5mMMgCl2 andtheanti-protease

cocktail tablet (Roche, Basel, CH) (BufferB). After freezingand thawingthreetimes,theproteinconcentrationwasdetermined.

EnzymeactivitiesweremeasuredusingaBeckmannDU7400 spec-trophotometer.ComplexIactivitywasassayedbyfollowingthe rotenone-sensitiveinitialrateofNADHoxidationat340–425nm (ε=6.81mM−1cm−1).200␮g/mlproteinwereaddedtoBufferB containing2.5mg/mlofBSA,2.2mMKCN,3.6␮g/mlantimycinA and71␮Mdecylubiquinone.Thereactionwasstartedbythe addi-tionof68␮MNADHfollowedby1.8␮g/mlrotenone.ComplexIV activitywasmeasuredfollowingtheoxidationofferrocytochrome cat550–540nm(ε=19.1mM−1cm−1).50␮g/mlofproteinwere addedtoBufferBsupplementedwith3.3␮g/mlantimycinAand 500␮Mdodecylmaltoside.Thereactionwasstartedbytheaddition of9␮Mreducedcytochromec,intheabsenceandinthepresence of1.6mMKCN.

2.4. MeasurementofintracellularATP

ATP concentration wasdetermined usinga bioluminescence somaticcellassaykit,followingthemanufacturer’sinstructions. 2×104_{cellswereusedforeachsample.Chemiluminescence}

sig-nals were acquired with a Victor X3 Multilabel Plate Readers (PerkinElmer,SanJosè,CA,USA).

2.5. Measurementoflactateproduction

Analysisoflactatereleasedintotheculturemediumwas car-riedoutspectrophotometricallybyfollowingNAD+ _reductionat

340nm,aspreviouslydescribed(DelPreteetal.,2008). 2.6. Anti-oxidantenzymeactivities

Superoxidedismutase(SOD)activitywasmeasuredusingthe SOD assay kit (CaymanChemical, Ann Arbor,MI), accordingto themanufacturer’sinstructions.Briefly,monocytesandDCwere centrifugedat16,000×gfor1minandthecellpelletwas homog-enizedusinga29Gneedleandcentrifugedat1500×gfor5minat 4◦C.Finally,thesupernatantwasusedforanalysisofSOD activ-ity. Theadditionof4mM KCNtotheassaywasusedtoinhibit bothCuZn-andextracellular-SOD,thusallowingthedetectionof theactivityofMnSODonly.OneunitofSODwasdefinedasthe amountofenzymecatalysing50%dismutationofthesuperoxide radical.Absorbancechangewasrecordedat450nmusingaVictor X3MultilabelPlateReaders.Catalaseactivitywasassayedby mon-itoringspectrophotometricallytherateofdecompositionofH2O2

at240nmaspreviouslydescribed(Pacellietal.,2011). 2.7. Westernblottinganalysis

Monocytes and DC orisolated mitochondriawere separated on 12% Tris–Tricine SDS-PAGE and blotted onto nitrocellulose membrane.Westernblot(WB)analysiswascarriedoutusingthe followingprimaryantibodies:Fe–Sprotein3(NDUFS3)ofComplex I(MitoSciencesInc.;Eugene,OR,USA),CoreIsubunitofComplexIII, COXIVofComplexIVandporin(VDAC)(Invitrogen,Carlsbad,CA, USA),TOM22andTFAM(GeneTex,Inc.,Irvine,CA,USA),MnSODand CuZnSOD(Millipore,Billerica,MA,USA),Catalase(Calbiochem,San Diego,CA,USA),andGAPDH(AbDSerotec,Dusseldorf,Germany), NRF-1(Abcam,Cambridge,UK)accordingtothemanufacturers’ suggestedconcentrations.Proteinsweredetectedby chemilumi-nescentLiteAblotreagent(EuroClone)andtherelativeopticalband densitieswerequantifiedbydensitometricanalysisusingQuantity One-4.4.1imagingsoftware(Bio-RadLaboratories).

2.8. mtDNAquantification

FordeterminationofthemtDNAcopynumber,totalDNAwas isolatedfrom5×106 _{monocytesandDCusingEuroGOLDTissue}

DNAMiniKit(Euroclone).ContentofmtDNApercellwasmeasured byusinghumanmitochondrialDNAqPCRDetectionkit(GeneMore s.r.l.,Mo,Italy).

2.9. Real-timequantitativereversetranscription-PCR

Transcript levels were quantified by real-time reverse transcription-PCR by extracting total RNA using Trizol (Invit-rogen)frommonocytesandDC.RNAwasthenfurtherpurifiedand DNasetreated(Roche).First-strandcDNA wassynthesizedfrom 1␮goftotalRNAusingiScriptTM_{cDNASynthesisKit(Bio-Rad)and}

Oligo dT-primers, according tothe manufacturer’s instructions. mRNA expression for genes of interest was performed by RT real-time PCR withtheiQ SYBRgreenSupermix (Bio-Rad) on a Bio-Rad iCycler iQ instrument. About 10% of each RT reaction was used to run real-time PCR reactions. The real-time PCR conditionswere: 20 at94◦C, 30 at59◦C, 45 at 72◦C for 45 cycles, followed bya melt curve cycle.Quantitative normaliza-tion of cDNA in each sample was performed using GAPDH as internal control. Samples were analysed in duplicate. Relative geneexpression levelsweredetermined bythe comparativeCt

method.ForRTreal-timePCRexperimentsthefollowingprimer setswereused: HNRF-1Fsense5 -CCAGACGACGCAAGCATCAG-3, HNRF-1 R antisense 5-GGGATCTGGACCAGGCCATT-3 (gene nrf-1);HTFAM F sense5-TGTTCACAATGGATAGGCAC-3 HTFAM Rantisense5-TCTGGGTTTTCCAAAGCAAG-3(genemt-tfa); Real-ESSS-FinEx2sense5-ACCCAGACTCCCATGGTTATG-3,Real-163-R antisense 5-GGACCACTCTTTCATCCCATCC-3 (gene ndufs11); Glic-1 F sense 5-GAAGGTGAAGGTCGGAGT-3, Glic-4 R anti-sense 5-CATGGGTGGAATCATATTGGAA-3 (gene Gapdh); Cyt SOD F sense 5-TGGTTTGCGTCGTAGTCTCCT-3, Cyt SOD R anti-sense 5-AATGCTTCCCCACACCTTCA-3 (gene sod1); MnSOD F sense 5-CTGGACAAACCTCAGCCCT-3, MnSOD R antisense 5 -CTGATTTGGACAAGCAGCAA-3 (gene sod2); Catalase F sense 5-TGGAAAGAAGACTCCCATCG-3, Catalase R antisense 5 -CCAGAAGTCCCAGACCATGT-3(genecatalase);PGC-1alphaFsense 5-AAACAGCAGCAGAGACAAATGC-3, PGC-1alpha R antisense 5-TTGGTTTGGCTTGTAAGTGTTGTG-3(genepgc-1alpha). 2.10. Electronmicroscopy

Monocytes,immatureDC(iDC)andmatureDC(mDC)werefixed in3%GTA(glutaraldehyde)in0.1Msodiumphosphatebuffer(pH 7.4)for2h,washedinthesamebufferandthenpostfixedin1% OsO4at4◦C.Afterward,cellsweredehydratedingradedethanol,

and embeddedin Epon 812.60nm ultrathinsections werecut withadiamondknifeonaLKB-Vultratome,stainedwithuranyl acetatefollowedbyleadcitrateandexaminedunderaZeissEM109 electronmicroscope(Zeiss,Oberkochen,Germany).Digitalimages wereanalysed and recorded with a cooled camera Gatan CMS (GatanGmbH,München,Germany).

2.11. Statisticalanalysis

Results are expressedas mean±SEM. Statistical significance wasdeterminedbyStudent’sttest.Differenceswereconsidered significantwhenp<0.05.

3. Resultsanddiscussion

Ultrastructuralanalysisrevealedasignificantincrementinthe number ofcondensed mitochondriain immature DC compared tomonocytes(DelPreteetal.,2008).Toinvestigatewhetherthe differentiationwas accompanied by structural as wellas func-tionalchangesinmitochondrialrespiratorychaincomplexes,we analysedtheexpressionlevel ofprotein subunitsof respiratory

complexes,aswellastheirspecificenzymaticactivities.Since load-ingofequalamountofproteininWB systemdidnotshowany majordifference betweenmonocytesand DCin theabundance ofthemitochondrialcomplexesproteinlevels(Fig.1A),proteins derivedfromanequalnumberofnucleiwereloaded.ThisWB anal-ysismethod,currentlyused byothers(Frankoetal.,2008),can indeedtakeintoaccounttheproteinsynthesisoccurringduring DCdifferentiationand demonstratetheactualincreasein mito-chondrialproteinsper nucleargene copy. Fromthis analysis,a statisticallysignificantincreaseintheproteinlevelofNDFUS3of ComplexI(1.79fold),CoreIofComplexIII(2.28fold),andCoxIV ofComplexIV(2.07fold)(Fig.1B)wasobservedinDCcompared tomonocytes.Theincreaseintheexpressionofproteinsubunitsof respiratoryenzymeswasaccompaniedbychangesoftheircatalytic function.Infact,asshowninFig.1C,theenzymaticactivityof Com-plexIin monocytes,corresponding to0.22_±0.05nmol/min/106

cells,increasedto1.36±0.20nmol/min/106_{cellsinDC(6.18fold).}

Similarly,DCshowed4.47foldincreaseintheactivityofComplexIV comparedtomonocytes(6.74±0.46vs1.42±0.18nmol/min/106

cells, respectively).In order tocomparedirectly theexpression of mitochondrial proteins between monocytes and DC, a new magneticsortingmethodwasusedtoisolatepurefunctional mito-chondriafromculturedcells,whichturnedoutsuitablealsofora smallnumberofhumanprimarycells(Hornig-Doetal.,2009).WB analysisshowedthattheexpressionoftheoutermembrane pro-teinsTOM22,VDAC,andNDUFS3inmitochondriaisolatedfrom anequalnucleusnumberwassignificantlyincreasedinDC com-paredtomonocytes(Fig.1D).Overall,thesefindingsindicatethat, duringthedifferentiationprocessofmonocytesintoDC, mitochon-drialactivityisgreatlyenhanced,asrevealedbytheincreaseinthe respiratoryenzymeactivitiesandintheirproteinexpressionlevels. IndifferentiatedDC,mitochondrialoxidativephosphorylation systemappearstoprovidemostofcellularATPproduction.Infactas showninFig.2theATPcontentindifferentiatedDCincreased pro-gressivelyalongwiththedifferentiationprocess.Treatmentwith therespiratorychaininhibitorrotenonereducedmarkedlytheATP content,alsocausingaverylargeincreaseoflactateproduction. Conversely,intheabsenceofrotenone,lactatereleaseintheculture mediumwasmuchlower.Thisstronglysuggeststhattheincreased electron transfer capacity upondifferentiation impliesa higher utilizationofmitochondriaforATPsynthesis.Interestingly,the acti-vationprocessofTlymphocytesthroughCD28signallinghasbeen describedtobeassociatedmainlytoenhancedglycolysis,withonly asmallproportionofglucoseoxidizedinmitochondria(Frauwirth etal.,2002).Fromtheseresultsitcanbearguedthatcell differenti-ationandactivation,whicharedefinitelydistinctprocesses,might exhibitdifferentmetabolicstate.

WehavepreviouslyshownthatapeakofROSproduction, last-ingalmost1h,tookplaceaftertheadditionofgrowthfactorsto monocytesuspension.Thepresenceofrotenoneinthemonocyte culture,aswellasthatoftheclassicalantioxidantCatalase,caused asimilardropinROSproductionandasuppressionoftheDC differ-entiationasrevealedbyasubstantialreductioninCD1aexpression. Undertheseconditionsoxygenconsumptionactivitymeasuredat day+6resultedtobestronglyinhibited(DelPreteet al.,2008). Theseexperimentsprovideevidencefora causalityrelationship betweenROSand electrontransferactivityaccompanying mito-chondrialbiogenesis.

Furthermore,sincetheprogressivelyincreaseofmitochondrial activityofDC,comparedtomonocytes,likelyrepresentsahigher potentialofROSproduction,itseemsplausiblethatDCmightbe equipped withmore efficient antioxidant enzymatic protection systems in order to detoxify ROS. To test this hypothesis, we analysed the activity and the expression of main anti-oxidant enzymes.Fig.3reportsmeasurementsoftheactivityofCatalase (A)andofthecytosolic(CuZn)andmitochondrial(Mn)superoxide

Fig.1. MitochondrialrespiratorycomplexesduringDCdifferentiation.(A)WBanalysisofmitochondrialrespiratorycomplexproteinsinmonocytesandDC.Equalamount

ofproteins(50␮g,upperpart)orproteinscorrespondingtoequalnumberofcells(2×106_{,lowerpart)fromarepresentativedonorsamplewereloadedandthemembranes}

wereprobedfortheindicatedproteins.(B)Quantitativedataderivedfromthedensitometricanalysisofthebandsisreportedin(A).Valuesarereportedasmean±SEM

(n=4).(C)EnzymaticactivitiesofComplexIandComplexIVasnmol/min/106_{monocytesandDC.Valuesarereportedasmean±SEM(n=4).(D)WBanalysisofTOM22,}

VDACandNDUFS3frommitochondrialfractionisolatedfrom6×106_{monocytesandDC(DC/monocytes:TOM22,6.6;VDAC,8.7;NDUFS3,6.9fold).Oneexperimentoutof}

threeisrepresented(*p<0.05,**p<0.01,***p<0.001,DCvsmonocytes).

0

2

4

6

8

10

12

14

16

0 3 6

Control

Rotenone

0

200

400

600

800

0 3 6

ATP con

tent

Lact

ate produc

tion

nmol ATP/10

cells

Lactate (

μ

M)

Days Da

ys

*

**

**

***

Fig.2. ATPcontentandlactateproductionevaluatedintheabsenceandinthepresenceoftherespiratorychaininhibitorrotenone.ATPlevelsexpressedasnmolATP/106

cellsandlactatereleasedintheculturemedium(␮M)weremeasuredinmonocytes(day0),atintermediatetimepoint(day3)andinDC(day6),intheabsenceandinthe

Fig.3.Anti-oxidantenzymeactivityandexpressionduringDCdifferentiation.ActivityofCatalase(A)andMnSODandCuZnSOD(B)inmonocytesandDC.Resultsare

indicatedasmean±SEM(n=4).(C)WBanalysisofproteinsfromanequalnumberofmonocytesandDC(oneexperimentoutofthreeisrepresented).(D)Geneexpressionof

Catalase,MnSODandCuZnSODbyreal-timeqPCRexperiments.Resultsareexpressedasfoldincrease(DC/monocytes).Valuesarereportedasmean±SEM(n=4)(*p<0.05,

**p<0.01,DCvsmonocytes).

dismutase (B).The enzymatic activity of Catalase in monocyte precursors(approximately3nmol/min/106_{cells)increasedabout}

twofoldupondifferentiation.Theanalysisoftheenzymatic activ-ityofSODrevealedthatthemitochondrialMnSODformincreased muchmore(4.11±0.39vs57.78±5.3U/106 _{cells,monocytesvs}

DC)thanthecytosolicform(14.88±0.88vs94.2±15.9U/106_cells,

monocytesvs DC),suggesting that a moreintense anti-oxidant activity occurs in the mitochondrial compartment of DC. WB analysisconfirmedtheabovedata.Infact,densitometricanalysis showeda relevant increase (30.4±6.9 fold) in MnSODprotein expression,whilethecytosolicenzymes,CatalaseandCuZnSOD, resultedonlyslightlyincreaseduponDCdifferentiation(2.8±0.7 foldand4.7±0.1fold,respectively)(Fig.3C).

Changesinanti-oxidantenzymegeneexpressionoccurring dur-ing DC differentiation is shown in Fig. 3D. While the relative geneexpressionofthecytosolicenzymesCatalaseandCuZnSOD revealedasignificantincreaseinDCcomparedtomonocytes(12.7 and6fold,respectively),noinductionwasobservedintheRNAlevel ofMnSOD(0.21fold).Thisapparentlycontrastingobservationthat amajorinductionofMnSODisseenattheproteinlevelthanat theRNAlevelhasbeenalsoreportedpreviously(Angenieuxetal., 2001)andexplainedconsideringthatthelocalizationintothe mito-chondrialmatrixwouldprotectthematuremitochondrialprotein fromproteolyticdegradation.Consequently,itcanbededucedthat duringDCdifferentiationprocess,takingplacein6-daysculture,

largeincreaseintheconcentrationofmitochondrialproteins,such asMnSOD,canoccurfrommarginalincreaseofthecorresponding mRNAlevels(Angenieuxetal.,2001).Overall,thesefindings con-firmedpreviousobservations(Rivollieretal.,2006)showingthat DCareequippedwitha veryefficientanti-oxidantsystemsthat mayassurehigherresistancetooxidativeenvironments.

Thehigherefficiencyofantioxidantapparatuswefoundin mito-chondriaascompared tothecytosoliccompartmentofDC may reflectaconditionwheremostofsuperoxideisproducedinthe matrix.IthasinfactbeenreportedthatComplexIgenerated super-oxideisreleasedexclusivelyintothematrix,togetherwith50%of thesuperoxideproducedbyComplexIII(Mulleretal.,2004).On theotherhand,thekeyroleofthemitochondrialSODisillustrated by thelethalphenotypeof micelacking thematrix superoxide dismutase(Sod2)gene (Li etal.,1995).Finally,as describedfor osteogenicdifferentiationofhumanmesenchymalstemcells(Chen etal.,2008),ourobservationssuggestthatduringDCdifferentiation a coordinatedregulationbetweenmitochondrialbiogenesisand anti-oxidantdefencesystemsneedstobeorchestratedinorderto letROSproductiontriggerdifferentiationbut,atthesametime, pre-ventaccumulationofROSwhenaerobicmitochondrialmetabolism becomesdominant.

Toclarifythemechanismsunderlyingtheregulationof mito-chondrial biogenesis occurring during DC differentiation, we investigatethemtDNA copy numberand themRNAexpression

0 100 200 300 400 500

Monocytes

DC

mtDNA/cell

A B

C

-1 0 1 2 3 4 5 6 7

PGC-1

NRF-1

TFAM

α

Gene expression (Fold)

0 2 4 6 8

NRF-1 TFAM

PGC-1

Gene expression (Fold)

Monocytes

DC

α

Days

Fig.4.MitochondrialbiogenesisoccurringduringDCdifferentiation.(A)mtDNAcopynumberpercell.Valuesarereportedasmean±SEM(n=4).(B)mRNAlevelsofNRF-1,

TFAM,andPGC-1␣.mRNAlevelsweredeterminedbyRTqPCRoftotalRNAnormalizedtoGAPDH.Resultsareexpressedasfoldincrease(DC/monocytes).Dataaremeans±SEM

ofindependentdeterminations(n=4).(C)Kineticstudy(day0,+1and+6)ofmRNAlevelsofthethreegenes.Resultsfromarepresentativeexperimentareexpressedasfold

increase.

levels of the main mitochondrial biogenesis-associated genes, includingPGC-1␣,TFAM,and NRF-1, bothin monocyte precur-sorsandinDC.Real-timeqPCRexperimentsrevealedthatmtDNA copieswereapproximatelyincreasedby3.5foldinDCcompared tomonocytes(Fig.4A).TheincreaseofmtDNAwasassociatedwith thetriggeringofthemitochondrialbiogenesisactivationpathway. Infact,thegeneexpressionofNRF-1andTFAM(Fig.4B)was sig-nificantincreasedinDCcomparedtomonocytes(NRF-1,4.84and TFAM,3.78fold).Unexpectedly,attheendofthedifferentiation process,DCshowedverylowlevelofPGC-1␣geneexpression,even lowerthanmonocyte precursors(0.21fold).However,akinetic studyofthethreemainmitochondrialbiogenesis-associatedgenes revealedthatthepeakinPGC-1␣geneexpressionwassuddenly reached3haftertheadditionofspecificDCdifferentiationsoluble factors(GM-CSFandIL-4)inmonocyteculturemedium.PGC-1␣ expressionrapidlydeclinedafter24hculture,whileNRF-1and TFAMshowedagradualincreaseintheirexpressionculminatingat theendofthedifferentiationprocess(Fig.4C).Thesefindings sug-gestthatanearlyinputbythemasterregulatorofmitochondrial biogenesisPGC-1␣isneededtotriggerthesubsequentactivation ofthedownstreamtranscriptionfactors,NRF-1andTFAM,during DCdifferentiation.

Ultrastructural features of mature DC (mDC) compared to immatureDC(iDC)andmonocytes,togetherwiththeexpression levelsofbothmitochondrial biogenesisproteinsandrespiratory enzymeswereanalysed.Nosubstantialdifferenceswereobserved

betweeniDCandmDContheultrathinsections(Fig.5BandC). DCappearedaselongatedorflattenedcellswiththin plasmamem-branes filopodia and eucromatic nuclei, containing numerous rounded or rod-like mitochondria, in a typical condensed form (Fig.5EandF).Monocytesshowedbothcondensedandorthodox mitochondria,theselatterhavingathininter-membranespaceand lamellarcristae(Fig.5AandD).Thequantitativeanalysisof mito-chondriaof thedifferentcelltypesisreportedin Fig.5G.From morphometricanalysisnosignificantdifferencesinthecondensed mitochondrianumberwereobservedbetweeniDCandmDC,which were,ontheotherhand,significantlyincreasedcomparedto mono-cytes. Thusthe final DC maturation step does not seem to be accompaniedbymoreactivemitochondrialbiogenesis.

Inconclusion,whatisemergingfromtheseandthepreviously reporteddata(DelPreteetal.,2008)isthatthesuddenROS pro-ductionuponadditionofthedifferentiationfactorstothemonocyte precursorsuspensioninducesarapidincreaseofPGC-1␣,aprocess lastinglessthanoneoutthesixdaysrequiredfortheir differenti-ationintoDC.Sincetheincreaseofmitochondrialnumber/activity induced by PGC-1␣may cause in principle an increase of ROS production,thentheproteinplaysaroleintheregulationofthe ROSmetabolicprogram,leadingtoover-expressionofROS detox-ifyingenzymes,inparticularMnSOD(St-Pierreetal.,2006).ROS levelinthematrix,wheresuperoxideismostlyreleased,isthus maintaineddefinitelylow tohavecells functioningnormally.In summary,thisstudydemonstrates,forthefirsttime,thatanactive

Fig.5.TEManalysis,respiratorychainenzymeexpressionandmitochondrialbiogenesisproteinsinmDC,iDCandmonocytes.Ultrastructuralanalysisofmonocytes(A),

immature(B)matureDC(C).DC(B,C)linedbytheplasmamembranewithfillipodiesandpseudopodesshowthecytoplasmwithnumerouscondensedmitochondria(E,F).

Monocytesshowmitochondriawithorthodoxic(asterisk)andcondensed(arrow)conformation(A,D).Scalebars(originalmagnification):A–C,1.42␮m;D–F,0.25␮m.(G)

Forthemorphometricanalysis,mitochondriaof10cellsforeachsamplewerecountedontheelectronmicrographsatfinalmagnificationof12,000×bycomputer-aided

analysis.Themeanvalueforallthemicrographs±SEMareindicated(*p<0.05,iDCandmDCvsmonocytes).(H)WBanalysisofmitochondriabiogenesisandrespiratory

complexproteinsubunitsinmonocytes,iDCandmDCfromarepresentativedonorsampleoutofthreeexperiments.

mitochondrialbiogenesisoccursduringthedifferentiationprocess ofmonocytesintoDC.Moreover,thefineregulationbetween PGC-1␣andtheexpressionofmitochondrialROS-detoxifyingenzymes mightrepresentapotentialtargetinthosepathologicalconditions whereDCfunctionsandmetabolicalterationsaresimultaneously involved.

Disclosures

Noconflictsofinterestaredeclaredbytheauthors.

Acknowledgements

This work was financially supported by National Research Projects(PRIN)fromMIUR(Ministerodell’IstruzioneUniversitàe Ricerca),andItalianMinistryofHealth(ProgettoGiovani Ricerca-tori2007).WethankDr.SusannaD’OriaandDr.AnnaAbbresciafor technicalassistanceandDr.ElisabettaCrollofromthelocalblood bank.

References

AngenieuxC,FrickerD,StrubJM,LucheS,BausingerH,CazenaveJP,etal.Gene

inductionduringdifferentiationofhumanmonocytesintodendriticcells:an

integratedstudyattheRNAandproteinlevels.FunctionalandIntegrative

Genomics2001;1:323–9.

BanchereauJ,BriereF,CauxC,DavoustJ,LebecqueS,LiuYJ,etal.Immunobiology

ofdendriticcells.AnnualReviewofImmunology2000;18:767–811.

ChanDC.Mitochondria:dynamicorganellesindisease,aging,anddevelopment.Cell

2006;125:1241–52.

ChenCT,ShihYR,KuoTK,LeeOK,WeiYH.Coordinatedchangesofmitochondrial

biogenesisandantioxidantenzymesduringosteogenicdifferentiationofhuman

mesenchymalstemcells.StemCells2008;26:960–8.

Clayton DA. Nuclear-mitochondrial intergenomic communication. Biofactors

1998;7:203–5.

DelPreteA,ZaccagninoP,DiPaolaM,SaltarellaM,OliverosCelisC,NicoB,etal.Role

ofmitochondriaandreactiveoxygenspeciesindendriticcelldifferentiationand

functions.FreeRadicalBiologyandMedicine2008;44:1443–51.

FrankoA,MayerS,ThielG,MercyL,ArnouldT,Horning-DoHT,etal.CREB-1alpha

isrecruitedtoandmediatesupregulationofthecytochromecpromoterduring

enhancedmitochondrialbiogenesisaccompanyingskeletalmuscle

differentia-tion.MolecularandCellularBiology2008;28:2446–59.

FrauwirthKA,RileyJL,HarrisMH,ParryRV,RathmellJC,PlasDR,etal.TheCD28

signalingpathwayregulatesglucosemetabolism.Immunity2002;16:769–77.

GaresseR,VallejoCG.Animalmitochondrialbiogenesisandfunction:aregulatory

GoffartS,WiesnerRJ.Regulationandco-ordinationofnucleargeneexpression

dur-ingmitochondrialbiogenesis.ExperimentalPhysiology2003;88:33–40.

Hornig-DoHT,GuntherG,BustM,LehnartzP,BosioA,WiesnerRJ.Isolationof

functionalpuremitochondriabysuperparamagneticmicrobeads.Analytical

Biochemistry2009;389:1–5.

KellyDP,ScarpullaRC.Transcriptionalregulatorycircuitscontrollingmitochondrial

biogenesisandfunction.GenesandDevelopment2004;18:357–68.

KiefelBR,GilsonPR,BeechPL.Cellbiologyofmitochondrialdynamics.International

ReviewofCytology2006;254:151–213.

Kroemer G, Reed JC. Mitochondrial control of cell death. Nature Medicine

2000;6:513–9.

LiY,HuangTT,CarlsonEJ,MelovS,UrsellPC,OlsonJL,etal.Dilatedcardiomyopathy

andneonatallethalityinmutantmicelackingmanganesesuperoxidedismutase.

NatureGenetics1995;11:376–81.

Muller FL, Liu Y, Van Remmen H. Complex III releases superoxide to both

sidesoftheinnermitochondrialmembrane.JournalofBiologicalChemistry

2004;279:49064–73.

PacelliC,DeRasmoD,SignorileA,GrattaglianoI,diTullioG,etal.Mitochondrial

defectandPGC-1alphadysfunctioninparkin-associatedfamilialParkinson’s

disease.BiochimicaetBiophysicaActa2011;1812:1041–53.

Rivollier A, Perrin-Cocon L, Luche S, Diemer H, Strub JM, Hanau D, et al.

Highexpressionofantioxidantproteins indendriticcells:possible

impli-cations in atherosclerosis. Molecular and Cellular Proteomics 2006;5:

726–36.

SattlerM,WinklerT,VermaS,ByrneCH,ShrikhandeG,SalgiaR,etal.Hematopoietic

growthfactorssignalthroughtheformationofreactiveoxygenspecies.Blood

1999;93:2928–35.

ScarpullaRC.MetaboliccontrolofmitochondrialbiogenesisthroughthePGC-1

fam-ilyregulatorynetwork.BiochimicaetBiophysicaActa2011;1813:1269–78.

ScarpullaRC.Nuclearactivatorsandcoactivatorsinmammalianmitochondrial

bio-genesis.BiochimicaetBiophysicaActa2002;1576:1–14.

ScarpullaRC.Nuclearcontrolofrespiratorychainexpressionbynuclear

respira-toryfactorsandPGC-1-relatedcoactivator.AnnalsoftheNewYorkAcademyof

Sciences2008;1147:321–34.

SgobboP,PacelliC,GrattaglianoI,VillaniG,CoccoT.Carvedilolinhibits

mitochon-drialcomplexIandinducesresistancetoH2O2-mediatedoxidativeinsultin

H9C2myocardialcells.BiochimicaetBiophysicaActa2007;1767:222–32.

ShengKC,PieterszGA,TangCK,RamslandPA,ApostolopoulosV.Reactive

oxy-genspeciesleveldefinestwofunctionallydistinctivestagesofinflammatory

dendriticcelldevelopmentfrommousebonemarrow.JournalofImmunology

2010;184:2863–72.

SozzaniS.Dendriticcelltrafficking:morethanjustchemokines.Cytokineand

GrowthFactorReviews2005;16:581–92.

St-PierreJ,DroriS,UldryM,SilvaggiJM,RheeJ,JagerS,etal.Suppressionofreactive

oxygenspeciesandneurodegenerationbythePGC-1transcriptional

coactiva-tors.Cell2006;127:397–408.

Steinman RM. Some interfaces of dendritic cell biology. APMIS