Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune ...

15 Pages·2017·2.4 MB·English

Hindawi Publishing Corporation
Stem Cells International
Volume 2017, Article ID 2389753, 15 pages
http://dx.doi.org/10.1155/2017/2389753
Research Article
Allogeneic Adipose-Derived Mesenchymal Stromal
Cells Ameliorate Experimental Autoimmune
Encephalomyelitis by Regulating Self-Reactive T Cell
Responses and Dendritic Cell Function
PerAnderson,1,2ElenaGonzalez-Rey,1FranciscoO’Valle,3FranciscoMartin,2
F.JavierOliver,1andMarioDelgado1
1InstituteofParasitologyandBiomedicine“Lo´pez-Neyra”,CSIC,PTS,Granada,Spain
2GENYO,CentreofGenomicsandOncologicalResearch,Pfizer/UniversityofGranada/AndalusianRegionalGovernment,
PTS,Granada,Spain
3SchoolofMedicine,DepartmentofPathology,IBIMER,CIBM,UniversityofGranada,Granada,Spain
CorrespondenceshouldbeaddressedtoPerAnderson;[email protected];[email protected]
Received17October2016;Accepted18December2016;Published30January2017
AcademicEditor:MarcellaFranquesa
Copyright©2017PerAndersonetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Multipotentmesenchymalstromalcells(MSCs)haveemergedasapromisingtherapyforautoimmunediseases,includingmultiple
sclerosis(MS).AdministrationofMSCstoMSpatientshasprovensafewithsignsofimmunomodulationbuttheirtherapeutic
efficacyremainslow.Theaimofthecurrentstudyhasbeentofurthercharacterizetheimmunomodulatorymechanismsofadipose
tissue-derivedMSCs(ASCs)invitroandinvivousingtheEAEmodelofchronicbraininflammationinmice.Wefoundthatmurine
ASCs(mASCs)suppressTcellproliferationinvitroviainduciblenitricoxidesynthase(iNOS)andcyclooxygenase-(COX-)1/2
activities.mASCsalsopreventedthelipopolysaccharide-(LPS-)inducedmaturationofdendriticcells(DCs)invitro.Theaddition
oftheCOX-1/2inhibitorindomethacin,butnottheiNOSinhibitorL-NAME,reversedtheblockinDCmaturationimplicating
prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin
oligodendrocyteprotein-(MOG35−55-)inducedEAEinC57Bl/6mice.MechanisticstudiesshowedthatmASCssuppressedthe
functionofautoantigen-specificTcellsandalsodecreasedthefrequencyofactivated(CD11c+CD40highandCD11c+TNF-𝛼+)DCs
indraininglymphnodes(DLNs).Insummary,thesedatasuggestthatmASCsreduceEAEseverity,inpart,throughtheimpairment
ofDCandTcellfunction.
1.Introduction clearly shown that in vitro-expanded MSCs possess potent
immunomodulatoryproperties.Firstly,MSCscanmodulate
Multipotent mesenchymal stromal cells (MSCs) are non- theactivationofcellsofboththeinnateandadaptiveimmune
hematopoietic, perivascular cells which support hemato- system in vitro. Depending on species and immune cell
poiesisandarethoughttoparticipateintissuerepairinvivo studied,thisimmunomodulatoryeffectisachievedthrough
[1–3]. MSCs can be obtained from virtually all tissues and a combination of mechanisms including cytokines (IL-10,
organsofthebodyandhavebeendefinedinvitroasplastic TGF-𝛽1, and IL-6), intracellular enzymes (inducible nitric
adherent cells that express CD73, CD90, and CD105 while oxidesynthase(iNOS)inmurineMSCsandindoleamine2,3-
lacking expression of CD45, CD34, and CD14, which can dioxygenase(IDO)inhumanMSCs),growthfactors(hepato-
differentiate into osteoblasts, adipocytes, and chondroblasts cytegrowthfactor,vascularendothelialgrowthfactor),mem-
invitro[4].Althoughtheproportionofbonafidestemcells braneboundmolecules(programmeddeathligand-1,FASL),
withinMSCpreparationsislikelytobelow[5],ithasbeen and prostaglandin E2 (PGE2) [6–8]. Secondly, injection of
2 StemCellsInternational
MSCpreparationshasshowndelayallograftrejection[9,10] ethical committee at the Institute for Parasitology and Bio-
andamelioratingdiseaseinseveralanimalmodelsofinflam- medicine“Lo´pez-Neyra”andthecentralethicalcommitteeat
mation/autoimmunitybyinhibitingthedeleteriousimmune theConsejoSuperiordeInvestigacionesCientificas(CSIC).
responsesagainstself-antigens[11–14].Basedontheirsuccess Micewereanesthetizedbeforeimmunizationusingintraperi-
in experimental models, the clinical potential of human tonealinjectionsofketamine-HCl(doseat100mg/kg)mixed
autologousandallogeneicMSCsforthetreatmentofautoim- with xylazine-HCl (10mg/kg). Mice were sacrificed using
mune/inflammatory diseases has been assessed in various CO2whenextractingthespinalcordforhistologicalanalyses.
phaseI/IIandIIIclinicaltrials[15–19]. Inallothercases,miceweresacrificedusingcervicaldisloca-
tion.
Multiplesclerosis(MS)isachronicdiseaseofthecentral
nervoussystem(CNS)whereautoreactiveTcellsandmacro-
phagesattackanddisruptthecommunicationbetweenneu- 2.2. Isolation and Expansion of mASCs. Mesenchymal stro-
rons which result in a multitude of neurological symptoms mal cells were isolated from adipose tissue as previously
[20].SeveralclinicaltrialshaveutilizedMSCsforthetreat- described[28].CellswereresuspendedinMesenCult(Stem
ment of MS and a few of these studies have analyzed the Cell,Grenoble,France)containing20%mousemesenchymal
effects of injected MSCs on the immune status of patients supplements(StemCell)andpenicillin/streptomycin(Invit-
withMS.Inaphase1/2open-safetyclinicaltrialforMS,injec- rogen,Carlsbad,CA),platedatadensityof2-3×104cells/cm2
∘
tionsofautologousMSCsresultedinarapidinhibitionofthe andculturedat37 Cinhypoxiaat5%O2,5%CO2.Nonadher-
immunesystem[17]whileanotherstudyobservedincreased entcellswereremovedafter24hoursinculture.Subsequent
4 2
foxp3transcriptlevelsinperipheralbloodmononuclearcells passages were plated at 10 cells/cm and maintained at
in MSC-treated patients [21]. These data suggest that the 5% O2, 5% CO2. mASCs were used at passages 3–9. The
phenotypiccharacterizationanddifferentiationcapacityinto
therapeuticeffectofMSCsinMSdepends,atleastpartially,
adipocytes,osteocytes,andchondrocyteswereperformedas
upontheirimmunomodulatorycapacity.However,whilethe
previously described ([28]; data not shown). Human ASCs
administrationofMSCshasbeenprovensafe,theircapacity
were obtained from Cellerix SA, Tres Cantos (Madrid),
toameliorateMSremainspoor[16,17,19,22].Therefore,it
Spain,andculturedinadvancedDMEMsupplementedwith
is important to further study how MSCs can modulate the
10%FCS(Invitrogen),Glutamax(GIBCO,LifeTechnologies,
immunesystembothinvitroandinvivoinordertoimprove
CA), and 100U/mL penicillin/streptomycin (GIBCO, Life
MSC-basedtherapiesforMS[23].
Technologies)aspreviouslydescribed[14].
Thus,theobjectiveofthecurrentstudyhasbeentochar-
acterize the immunomodulatory properties of murine adi-
2.3. Assessment of mASC-Mediated Inhibition of T Cell Pro-
posetissue-derivedMSCs(mASC)invitroandinvivousing
liferation In Vitro. mASCs were treated with mitomycin C
the experimental autoimmune encephalomyelitis (EAE)
(50𝜇g/mL,SigmaAldrich,St.Louis,MO)for20minutesat
modelofCNSautoimmunity.EAEisawell-acceptedanimal
∘
37 Candwashed3timeswithcompleteRPMI1640(2mML-
model of MS sharing both its immunological and patho-
glutamine,100U/mLpenicillin/streptomycin,50𝜇M2-mer-
logical features [24]. Injection of MSCs at distinct stages of
captoethanol,and10%heat-inactivatedFCS,allfromInvitro-
diseasehasbeenshowntoinhibitdiseaseseverityinvarious
gen).MitomycinC-treatedmASCswereplatedinflatbotto-
modelsofEAE,includingrelapsing/remittingEAE,chronic
med96-wellplatesandallowedtoadherefor3-4hours.Mito-
EAE,anddiseaseinducedbyadoptivetransferofencephal-
mycinCtreatmentofmASCsdidnotimpactontheirability
itogenic T cells [11, 25–27]. We show that mASC inhibited
toinhibitTcellproliferationinvitro(seeFigureS1inSupple-
TcellproliferationinvitroviaiNOSandCOX-1/2activities.
mentary Material available online at https://doi.org/10.1155/
Bothallogeneicandxenogeneic(human)ASCsameliorated
2017/2389753).SpleensfromfemaleBalb/cmicewerehomo-
MOG35−55-induced chronic EAE in C57Bl/6 mice. Murine
genized and erythrocytes lysed using ACK buffer (0.15M
ASCs reduced EAE severity through the inhibition of the
autoimmuneTcellresponsewithnoincreaseinfoxp3Tregs. N2H×41C0l5,1c0elmlsMwKerHeCadOd3e,dantdo0w.1emllsMwNitah2EoDrTwAithatopuHt m7.4A)SaCnsd.
Importantly, mASC inhibited the maturation of dendritic
Concanavalin A (ConA, 2.5𝜇g/mL, Sigma Aldrich) was
cells (DCs) in vitro via COX-1/2 activity and reduced the
addedtoculturesasamitogenicstimulusofTcells.L-NAME
percentageofactivatedDCsinthedrainingLNsofEAEmice.
(1mM), indomethacin (20𝜇M), and L-norvaline (10mM)
Our data suggests that MSC, through their modulation of
(allfromSigmaAldrich)wereaddedattheinitiationofthe
TcellandDCfunction,canameliorateinflammatory/auto-
cocultures. After 3 days, cells were pulsed with 0.5𝜇Ci/well
immuneCNSdisease.
3
[ H]-thymidine (Perkin Elmer, Waltham, MA) for 6 hours
and harvested onto glass fiber filters using a FilterMate 96
2.MaterialsandMethods
3
well-harvester(PerkinElmer).Uptakeof[ H]-thymidinewas
2.1.Animals. MaleBalb/c(6–10weeks)andfemaleC57Bl/6 measured on a 1450 Microbeta Trilux scintillation counter
(6–8 weeks) (Charles River, Barcelona, Spain) were used to (WallacOy,Turku,Finland).
initiate cultures of mASCs and for the induction of EAE,
respectively.Experimentalprotocolswereperformedaccord- 2.4. Measurement of Nitrite Production. To assess iNOS
ing to the National/EU Guidelines for the Care and Use of activity in mASCs, supernatants from control/stimulated
LaboratoryAnimalsinResearchwiththeapprovalofthelocal mASCs and mASC: DC cocultures were assayed for nitrite
StemCellsInternational 3
contents using the Griess assay. In brief, 100𝜇L of Griess processed forparaffin inclusionandsectioning.Transversal
reagent(a1/1mixtureof1%p-aminobenzene-sulfonamidein sections(4𝜇mthickness)werestainedwithLuxolfastblue,
5%H3PO4and0.1%naphthylethylenediaminedihydrochlo- cresylviolet,andhematoxylinfollowingtheKlu¨ver-Barrera
ride in distilled H2O; Sigma Aldrich) was added to 100𝜇L technique[31]andwereanalyzedforthepresenceofareasof
culturesupernatantsandstandard(NaNO2)in96-wellplates. demyelination and cell infiltration using a light microscope
Plates were incubated at room temperature for 10min, and (Olympus,Tokyo,Japan).Forimmunohistochemistry,spinal
absorbancewasmeasuredat550nm. cord sections were obtained as described for paraffin pro-
cessingfollowedbyblockingstepswithperoxidaseblocking
∘
reagents, heat-treated in 1mM EDTA buffer pH 8 at 95 C
2.5.MeasurementofArginaseActivity. Arginaseactivitywas
during 20min for antigenic unmasking, and incubated for
measured as previously described [29]. Urea was used for
30min at room temperature with polyclonal anti-myelin
thestandardcurve.Theproteinconcentrationsofcelllysates
basicproteinAb(MasterDiagnostica,Granada,Spain).The
were measured using the bicinchoninic acid (BCA) assay
immunohistochemical study was done on an Autostainer
[30]. One unit of enzyme activity is defined as the amount
480 (Thermo Fisher Scientific Inc., Waltham, MA) using
ofenzymethatcatalyzestheformationof1mmolofureaper
the polymer-peroxidase-based method and developed with
min.
diaminobenzidine.
2.6. Induction of EAE. Female C57Bl/6 mice (6–8 weeks-
2.8.AssessmentofAutoreactiveTCellResponsesinEAEMice.
old) were immunized s.c. in the flanks with 150𝜇L of an
Spleenanddraininglymphnode(DLN)cellsfromEAEmice
emulsion (75𝜇L/flank) containing 150𝜇g MOG35−55 (Gen- treatedi.p.withPBS(control,𝑛=4)orallogeneicmASC(𝑛=
script, Hong Kong, China) in PBS mixed with an equal 4) at the onset of disease (clinical scores: 0-1) were isolated
volumeofcompleteFreund’sadjuvant(CFA)supplemented
7daysaftermASCinjectionandstimulatedwithMOG35−55
with 4mg/mL Mycobacterium tuberculosis H37Ra (Difco, (50𝜇g/mL)oranti-CD3(1𝜇g/mL;145-2C11;BDPharmingen,
Detroit, MI). Mice were injected i.p. with 200ng pertussis San Diego, CA) at 1.5 × 106 cells/mL in flat bottomed 96-
toxin(SigmaAldrich)inPBSonthedayofimmunizationand
6
wellplatesforproliferationandat10 cells/mLin24-wellfor
2dayslater.Immunizedmicewererandomlydistributedin
differentgroups.Group1:controlmice(𝑛=8)wereinjected cytokinedetermination.Supernatantswerecollectedafter48
hoursandassayedforcytokinelevelsbyELISA.After3days,
i.p. with PBS at the onset of disease (clinical scores: 0-1).
3
Group2:controlmice(𝑛 = 13)wereinjectedi.p.withPBS cellproliferationwasdeterminedby[ H]-thymidineuptake
at the acute phase of disease (clinical scores: 1–3). Group 3: asdescribedabove.Forintracellularcytokinestaining,spleen
Mice (𝑛 = 9) were treated i.p. with allogeneic mASCs (106 andDLNcellsuspensions(1×106cells/mL)werestimulated
cellsobtainedfromBalb/cmiceandexpandedinhypoxia)at withPMA(50ng/mL)andionomycin(0.5𝜇g/mL;bothfrom
theonset(clinicalscores:0-1).Group4:mice(𝑛 = 7)were SigmaAldrich)for6hoursinthepresenceof3𝜇Mmonensin
treated i.p. with allogeneic mASCs (106 cells obtained from (eBioscience, San Diego, CA) during the last three hours.
Balb/cmiceandexpandedinhypoxia)attheacutephaseof CellswereprocessedforFACSanalysisasdescribedbelow.
thedisease(clinicalscores:2-3).Group5:mice(𝑛 = 7)were
6
treatedi.p.withhASCs(10 cells)attheacutephaseofdisease 2.9. Isolation and Characterization of DCs from EAE Mice.
(clinicalscores:1-2).ClinicalsymptomsofEAEwerescored To determine the effect of mASCs on DC phenotype and
daily using a 0–8 scale as follows: 0, no detectable signs of function in vivo, EAE mice were treated i.p. with PBS
EAE; 1, affected tail tonus; 2, tail paralysis; 3, mild hind leg (control,𝑛 = 4)orwithallogeneicmASC(𝑛 = 4)afterthe
paresis;4,severehindlegparesis;5,onehindlegparalysis;6, onsetofdisease(clinicalscores:1-2),and7dayslater,DLNs
completehindlegparalysis;7,completehindlegparalysisand wereisolatedanddigestedwith1.6mg/mLcollagenasetype
forelegparesis;and8,death.Fortheacquisitionofcellsand IVand0.1%DNAseI(SigmaAldrich)inRPMI1640medium
∘
tissues, anotherset of mice were used and sacrificed 7 days withoutsupplementsat37 Cfor30minutes.Forintracellular
aftertreatmentwithPBSormASCsasdescribedbelow.Mice TNF-𝛼staining,DLNcellswerewashedtwicewithcomplete
werescoreddailyfordiseasesymptoms.Watergelproducts RPMI1640 and 2 × 106 cells/mouse were plated in 12-well
providingwaterandmoistenedfoodpelletswereplacedon platesinthepresenceof3𝜇Mmonensinfor4h.Cellswere
the cage floor in Petri dishes which were changed daily gently harvested using cell scrapers, washed using FACS
to prevent dehydration. Mice were euthanized if exhibiting buffer, and processed for intracellular staining as described
severehindlegparalysisandforelegparesis(aclinicalscore below. For TNF-𝛼 ELISA, CD11c+ DCs were immunomag-
of7). neticallypurifiedusingCD11c-microbeads(MiltenyiBiotech,
Bergisch Gladbach, Germany) from collagenase type IV-
2.7.HistologicalAnalysisofCellInfiltrationandDemyeliniza- digestedDLNsandplatedat2.5×105cells/mLinthepresence
tion. SpinalcordsfromEAEmicetreatedi.p.withPBS(𝑛 = ofLPS(1𝜇g/mL).Supernatantswerecollectedafter48hours
4)orallogeneicmASC(𝑛=4)attheonsetofdisease(clinical forcytokinedeterminations.
scores: 0-1) were removed 7 days after treatment and pro-
cessedforimmunohistochemistryandKlu¨ver-Barrerastain- 2.10.GenerationofBoneMarrow-DerivedDCs(BM-DCs)and
ing. For light microscopy, cervical and lumbar spinal cord mASC Cocultures. BM-DCs were generated as previously
segmentswerefixedwithbuffered10%formalinfor48hand described[32].Briefly,0.4×106 BMcells/mLfromC57Bl/6
4 StemCellsInternational
micewereculturedincompleteRPMI1640containing20ng/ (BD Pharmingen), IL-10, IL-17, and CXCL10 (eBioscience).
mLGM-CSF(Peprotech,London,UK).After6–8days,non- Platesweredevelopedusingperoxidase-labelledstreptavidin
+ 󸀠
adherent cells were harvested and CD11c immature DCs (Sigma) and 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulpho-
wereimmunomagneticallypurifiedusingCD11c-microbeads nicacid)(ABTS)andreadat405nm.PGE2 levelsinsuper-
(Miltenyi Biotech) according to the manufacturer’s instruc- natantsweremeasuredbyELISA(CaymanChemicals,Ann
tions. Cell preparations consisted of >95% CD11c+ DCs. Arbor,MI)accordingtothemanufacturer’sinstruction.
To assess the effect of mASCs on the maturation of DCs,
different numbers of mASCs were plated in 12-well plates 2.14.GeneExpressionAnalysis. TotalRNAwaspurifiedusing
+
and allowed to adhere for at least 6 hours. CD11c DCs Ultraspec (Biotecx, Huston, TX) from spinal cords isolated
(0.4×106 cells/well)wereseededintowellswithorwithout from PBS-treated (control, 𝑛 = 4) or allogeneic mASC-
mASCsandLPS(1𝜇g/mL)wasaddedfor48hourstoinduce treated (𝑛 = 4) EAE mice (7 days after treatment) or from
DC maturation/activation. In some experiments L-NAME mASCs stimulated with LPS (1𝜇g/mL, Sigma) or TNF-𝛼
(1mM) or indomethacin (20𝜇M) was added to the DC: (10ng/mL,Peprotech)andIFN-𝛾(10ng/mL,BDBiosciences)
mASC cocultures during maturation. DCs were harvested for6,12,and24hours.TotalRNA(1𝜇g/sample)wasreverse
from the cocultures by gently collecting the nonadherent transcribed using M-MuLV RT (Roche Diagnostic, Basel,
DCsfromtheadherentmASCmonolayer.Theacquiredcells Switzerland)andrandomhexamerprimers.Semiquantitative
consistedof>95%CD11c+ DC.Supernatantswerecollected PCR and qPCR were performed using Taq polymerase
and assayed for cytokine levels by ELISA or NO2 levels (Biotools, Madrid, Spain) or Supermix (Bio-Rad, Hercules,
by Griess assay. Cell suspensions were processed for flow CA), respectively. Primer pairs include the following: iNOS
cytometryanalysisofcellsurfaceantigensorusedforMLR FW: 5󸀠-GTTCTCAGCCCAACAATACAAGA-3󸀠; iNOS RV:
asdescribedbelow. 5󸀠-GTGGACGGGTCGATGTCAC-3󸀠; MCP-1 FW: 5󸀠-TTA-
󸀠 󸀠
AAAACCTGGATCGGAACCAA-3 ; MCP-1 RV: 5 -GCA-
2.11. Mixed Leukocyte Reaction. Spleens from BALB/c (H- TTAGCTTCAGATTTACGGGT-3󸀠; TGF-𝛽1 FW: 5󸀠-TGC-
2d) were homogenized and erythrocytes lysed using the GCTTGCAGAGATTAAAA-3󸀠; TGF-𝛽1 RV: 5󸀠-AGCCCT-
󸀠 󸀠
Ammonium-Chloride-Potassium (ACK) lysis buffer. Cells GTATTCCGTCTCCT-3 ;IL-10FW:5 -GGTTGCCAAGCC-
(4 × 106 cells/mL) were plated in Petri dishes and allowed TTATCGGA-3󸀠;IL-10RV:ACCTGCTCCACTGCCTTGCT;
∘ 󸀠 󸀠
to adhere at 37 C for 90 minutes. Nonadherent cells were IDO FW: 5 -GGCTAGAAATCTGCCTGTGC-3 ; IDO RV:
collectedandusedasresponders.C57Bl/6(H-2b)DCsfrom 5󸀠-AGAGCTCGCAGTAGGGAACA-3󸀠; COX-2 FW: 5󸀠-
+ 󸀠 󸀠
theDC:mASCcoculturesorpurifiedCD11c DCsfromthe GGGTTGCTGGGGGAAGAAATGTG-3 , COX-2 RV: 5 -
󸀠 󸀠
DLNsofuntreatedormASC-treatedEAEmicewereaddedin GGTGGCTGTTTTGGTAGGCTGTG-3 ;ArginaseIFW:5 -
flatbottomed96-wellplates(15000DCs/well)togetherwith CAGAAGAATGGAAGAGTCAG-3󸀠; Arginase I RV: 5󸀠-
3 × 105 responder cells. After 4 days, cell proliferation was CAGATATGCAGGGAGTCACC-3󸀠; Foxp3 FW: 5󸀠-TTC-
3 󸀠 󸀠
determinedby H-thymidineuptakeasdescribedabove. ATGCATCAGCTCTCCAC-3; Foxp3 RV: 5 -CTGGAC-
ACCCATTCCAGACT-3󸀠; IFN-𝛾 FW: 5󸀠-ACACTGCAT-
2.12.FlowCytometry. Collagenase/DNAse-treatedDLNcell CTTGGTTTGC-3󸀠;IFN-𝛾RV:5󸀠-TTGCTGATGGCCTGA-
suspensionsormASC-culturedDCswerepreincubatedwith TTGTC-3󸀠; 𝛽-actin FW: 5󸀠-AATCGTGCGTGACATCAA-
anti-Fc𝛾RII/Fc𝛾RIIImAbat2.5𝜇g/mL(2.4G2;BDPharmin- AG-3󸀠;𝛽-actinRV:5󸀠-ATGCCACAGGATTCCATACC-3󸀠.
gen) and 7-aminoactinomycin D (2𝜇g/mL; Sigma Aldrich)
andthenstainedwiththefollowingantibodies:CD11c-APC 2.15. Statistical Analysis. All results are expressed as mean
(N481; eBioscience), CD40-PE (2/23), CD80-PE (16-10A1), (SEM)ofatleast3independentexperimentsunlessotherwise
and/orCD86-PE(GL-1)(BDPharmingen).Forintracellular statedinthefigurelegends.TheMann–Whitney𝑈-testwas
cytokinestainingcellswerefixed,permeabilized,andstained appliedonallinvivoresultsandcell-cultureexperimentsto
for IFN-𝛾-FITC (XMG1.2), IL-17-PE (TC11-18H10), TNF-𝛼- comparenonparametricdataforstatisticalsignificance.A𝑝
PE(MP6-XT22),IL-4-PE(BVD4-1D11),andIL-10-PE(JES5- value<0.05wasconsideredsignificant.
16E3) (all from BD Biosciences, San Diego, CA) using
the BD cytofix/cytoperm kit (BD Biosciences) according to
3.Results
the manufacturer’s instructions. Each sample was stained
with appropriate isotype controls. For the analysis of foxp3 3.1. Immunomodulatory Mechanisms of mASCs In Vitro.
expression cells were stained with CD4-FITC (GK1.5; BD Acquiring high numbers of low passage MSCs with potent
Biosciences) and subsequently processed for foxp3 staining immunosuppressive capacity is crucial for their successful
using the foxp3 staining kit (eBioscience) according to the useasatherapyforinflammatory/autoimmunediseases[33].
manufacturer’s instructions. Stained cells were analyzed on In agreement with previous studies [34, 35], we found that
aFACSCaliburflowcytometer(BDBiosciences). mASCexpandedatlowoxygentension(5%O2)proliferated
atahigherratecomparedtomASCsexpandedatnormoxia
2.13. Cytokine and PGE2 ELISAs. The cytokine content in (Figure 1(a)). Thus, we decided to culture the ASCs at 5%
supernatants from the mASC: splenocyte and DC cultures O2 and use these cells for the subsequent characterization
weredeterminedbysandwichELISAsusingcapture/biotiny- of their immunomodulatory functions in vitro and in vivo.
lated detection antibody pairs for IFN-𝛾, TNF-𝛼, IL-12p40 Whereas mASCs constitutively expressed TGF-𝛽1, COX-2,
StemCellsInternational 5
mber 125 ∗ − LPS TIFNN+F--𝛾𝛼O
nu 100 ∗ Time (h): 0 612246122461224H2
cell 40) 75 ∗ TGF-𝛽1
ative ×1( 50 IL-10
ul 25 IDO
m
Cu 0 COX-2
0 5 10 15 20
iNOS
Time (days)
Arginase I
21%O2 𝛽-Actin
5%O2
(a) (b)
5 5 120
PGE(ng/mL)2 01234 2 S 01234 𝜇(M)NO2 Arginase activity (U/mg protein)Co123456n0000000A − − + ∗*+ Proliferation(% of control) 124680000000 0 10 20 40
OX-1/ iNO SplenmocAytSeCs −+ −+ −+ ++ Number of ASCs (×103)
C mASC
mASC + L-norvaline
(c) (d) (e)
4
∗ L) 4 L) ∗
100 m m 3
ol) 75 ng/ 3 ng/
of contr 2550 𝛾FN-] ( 12 ∗ ]PGE(2 12 ^
n (% 0 ∗ L)[I 0 ∗ [ 102 ∗
Proliferatio 1570050 ∗ ∗ L10] (ng/m 12 ]𝜇(M)NO2 48
25 XC [ ^
0 0 10 20 40 C[Con 0A − + + + + Con 0A − + + + +
Number of ASCs (×103) mASC − − + + + mASC − − + + +
L-NAME − − − + − L-NAME − − − + −
mASC
mASC + INDO INDO − − − − + INDO − − − − +
mASC + L-NAME
(f) (g) (h)
Figure 1: mASCs expanded in hypoxia maintain their immunosuppressive activities. (a) Proliferative response of mASCs expanded in
2
normoxia(21%O2)orinhypoxia(5%O2).mASCswereseededat5000cells/cm inT25flasksandcountedatdifferenttimepointswhen
reaching70–80%ofconfluence.Dataareshownasmean(SD)ofonerepresentativeexperimentoutofthree.∗𝑝 < 0.05versus21%O2.
((b)and(c))ImmunephenotypiccharacterizationofmASCs.mASCswereexpandedunderhypoxicconditionsandthenculturedwith
medium(unstimulated)orstimulatedwithLPS(1𝜇g/mL)orTNF-𝛼(10ng/mL)andIFN-𝛾(10ng/mL)fordifferenttimepoints(24hoursfor
NO2determination).GeneexpressionofdifferentimmunemarkerswasanalyzedusingsemiquantitativePCR.iNOSactivitywasmeasured
inTNF-𝛼/IFN-𝛾-stimulatedmASCsbythedetectionofNO2inculturesupernatantsusingGriessassay.COX-1/2activitywasdetermined
bymeasuringthePGE2 levelsinculturesupernatantsfromunstimulatedmASCs.(d)ArginaseactivityisinducedinmASC:splenocyte
cocultures.SplenocyteswereculturedwithorwithoutmASCsandstimulatedwithConAfor3days.Definedquantitiesofcelllysateswerethen
subjectedtoaninvitroarginaseactivityassay.Dataareshownasmean(SEM)of3independentexperiments.∗𝑝<0.05versussplenocytes
6
+ConA.((e),(f),and(g))mASCssuppressTcellproliferationinvitroviaiNOSandCOX-1/2butnotarginaseactivity.Splenocytes(10
cells/mL) were stimulated with ConA (2.5𝜇g/mL) in the presence of different numbers of mitomycin C-treated mASCs (ratio 20:1 for
IFN-𝛾andCXCL10determination).((e)and(f))TheiNOSinhibitorL-NAME(1mM),theCOX-1/2inhibitorindomethacin(20𝜇M),and
3
thearginaseinhibitorL-norvaline(10mM)wereaddedtocultureswhenindicated.After3days,cellproliferationwasmeasuredby[ H]-
thymidineincorporationand(g)IFN-𝛾andCXCL10contentsinthesupernatantsweredeterminedbyELISA.Dataareshownasmean
(SEM)of3independentexperiments.∗𝑝 < 0.05versusmASCin(f)and∗𝑝 < 0.05versusConAin(g).(h)iNOSandCOX-2activities
6
areincreasedinmASC:splenocytecocultures.ConA-stimulatedsplenocytes(10 cells/mL)wereculturedwithmitomycinC-treatedmASCs
(ratio20:1)inthepresenceofL-NAMEorindomethacinfor3daysandthePGE2andNO2contentsmeasuredintheculturesupernatants.
Resultsareshownasmean(SEM)of3separateexperiments.∗𝑝<0.05versusConA;∧𝑝<0.05versusmASC+ConA.
6 StemCellsInternational
andarginaseImRNAsandPGE2,theexpressionofbothIDO foxp3 mRNAs in mASC-treated mice compared to control
and iNOS mRNA and NO2 production was induced upon EAEmice(Figure3(c)).
stimulation with TNF-𝛼 and IFN-𝛾, but not LPS (Figures
1(b)and1(c),FigureS2).Interestingly,wefoundasignificant 3.4. Allogeneic mASCs Inhibit MOG-Specific Immune Res-
induction of arginase activity in the splenocyte: mASCs ponses but Do Not Induce foxp3+ Tregs. In both EAE and
coculturesincomparisontomASCsorsplenocytescultured MS, autoreactive Th1 and Th17 cells producing IFN-𝛾 and
alone (Figure 1(d)). Although arginase activity has been IL-17, respectively, infiltrate the CNS and promote disease,
shown to inhibit the proliferation of T cells [36], we found whereas treatments that induce a skewing towards an IL-
that the addition of the arginase inhibitor L-norvaline did 4 dominated Th2 response generally suppress EAE [39].
not significantly revert the suppressive activity of mASCs mASC treatment significantly reduced the number of IFN-
in concanavalin A- (ConA-) induced splenocyte prolifera- 𝛾- and IL-17-secreting cells in both DLNs and spleen in
tion (Figure 1(e)). In contrast, inhibition of iNOS activity EAEmice(Figure4(a)andrepresentativegatingstrategyin
with L-NAME or of COX-1/2 activities with indomethacin Figure S3). Moreover, there was a significant reduction in
significantly alleviated the mASC-mediated suppression of theMOG35−55-specificrecallresponse,withrespecttoboth
T cell proliferation (Figure 1(f)). However, none of these proliferation and production of IFN-𝛾 and IL-17 compared
inhibitorsaffectedthemASC-mediatedsuppressionofIFN-𝛾 to control EAE mice. The effect was antigen specific since
ortheinductionofCXCL10inthesecocultures(Figure1(g)). activationoftotalTcellswithanti-CD3Abswhichresultedin
Since inhibition of iNOS may decrease COX-2 expression similarproliferationandcytokineproductionbybothgroups
and PGE2 secretion [37, 38], we measured the PGE2 levels (Figures 4(b) and 4(c)). However, we could not detect an
in mASC: splenocyte cocultures treated with L-NAME. We increaseintheanti-inflammatorycytokinesIL-10andIL-4in
found that L-NAME did not affect the productionof PGE2 mASC-treatedmicecomparedtocontrolmice(Figure4(a)),
by mASCs and indomethacin did not modulate their iNOS suggestingthatthemASCsdidnotinduceaskewingtowards
activity.Insummary,thesedatashowthatlowoxygentension a Th2 response or IL-10 producing Tregs. In addition, we
+ +
increases the expansion of mASCs in vitro and that these could not detect any change in the size of the CD4 foxp3
cellsareeffectiveinsuppressingimmuneresponsesinvitro, TcellpopulationinDLNsorspleensofmASC-treatedEAE
using both constitutive (COX-1/2) and inducible (iNOS) micecomparedtocontrolmice(Figure4(d)).
mechanisms.
3.5. mASCs Inhibit the Maturation/Activation of Dendritic
3.2. Therapeutic Effect of Allogeneic and Xenogeneic ASC Cells In Vitro and In Vivo. Considering the importance of
on Chronic EAE. We next wanted to assess the capacity of DCs in the initiation of the autoimmune response seen in
allogeneicandxenogeneicASCstoinhibitimmuneresponses EAEandMS[40],wedecidedtoevaluatetheeffectofmASC
in vivo. To this end, we induced chronic EAE in C57Bl/6 on the DC activation in vitro and in vivo. To this end, we
mice and administered mASCs to the animals at different cocultured BM-derived immature DCs with different num-
time points after immunization. A single injection of allo- bers of mASCs in the presence of LPS. We found that
geneic mASC at the onset or at the acute phase of disease mASCsignificantlyinhibitedtheLPS-inducedexpressionof
significantlyreducedthediseaseseveritycomparedtocontrol CD40andTNF-𝛼whilenotaffectingtheinductionofCD80
animals receiving PBS (Figure 2(a)). Both early and late and CD86 or the production of IL-10 or IL-12 by BM-DCs
injections of mASCs significantly reduced the cumulative (Figure5(a)andrepresentativegatingstrategyinFigureS4).
diseaseindex(Figure2(b)).XenogeneichASCsinjectedinto ThispartialblockofDCmaturationresultedinasignificant
EAEmiceduringtheacutephaseofthedisease(animalswith decreaseintheirTcellactivatingcapacity(Figure5(b)).Since
a mean score of 1.5) also reduced the mean clinical score both iNOS and PGE2 inhibited the proliferation of spleno-
and significantly decreased the cumulative disease index cytes in vitro and can modulate DC activation [41–44],
comparedtocontrolmice(Figures2(c)and2(d)). we set out to investigate their involvement in the mASC-
mediatedinhibitionofDCmaturation.Wefoundthataddi-
3.3. Allogeneic mASCs Reduce Demyelinization and Inflam- tionofindomethacinbutnotL-NAMEsignificantlyreversed
matoryInfiltrationintheCentralNervousSystemduringEAE themASC-mediatedsuppressionofTNF-𝛼productionand
Progression. WethenwantedtoanalyzeCNStissuesections CD40 expression resulting in a significant increase in their
fromcontrolandmASC-treatedEAEmiceforthepresence immunostimulatorycapacity(Figure5(c)).Theseeffectswere
of inflammatory infiltrates and demyelinating plaques. To observeddespitethepresenceofiNOSactivityinthemASC:
this end, mASCs were injected intraperitoneally into EAE DCcocultures(FigureS2).
miceaftertheonsetoftheclinicalsymptoms(averagescore To study DC activation in vivo, we purified DCs from
1.9 (0.4)) and spinal cords were obtained 7 days later from DLNs of mASC-treated and control EAE mice 7 days after
mASC-treatedmice(averagescore2.2(1.9))andcontrolmice mASCinjectionandanalyzedtheirexpressionofcostimula-
(averagescore3.5(1.1))andprocessedforstaining.Wefound torymarkersandTNF-𝛼production.Consistentwiththein
thatmASC-treatedmiceexhibitedlittlecellinfiltrationinto vitrodata,DCsfrommASC-treatedEAEmiceexpressedsig-
the CNS parenchyma and subsequently few demyelinating nificantlylowerlevelsofCD40,butnotCD86,comparedto
plaques compared to control EAE mice (Figures 3(a) and DCsfromcontrolEAEmice(Figure5(d)).Inaddition,intra-
3(b)).Consistentwiththereducedleukocyteinfiltration,we cellularstainingofTNF-𝛼onwholeLNsuspensionsrevealed
detected lower levels of IFN-𝛾, but also TGF-𝛽1, IL-10, and thatmASC-treatedEAEmicehadfewerCD11c+TNF-𝛼+DCs
StemCellsInternational 7
8
8
6
ore ore
al sc al sc 6
nic 4 nic ∗
n cli n cli 4 mASC ∗
a a
Me Me ∗
2
2
∗
mASC
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Days after immunization Days after immunization
Control
mASC
(a)
160
8
160
Cumulative disease index 1248000 ∗ ∗ Mean clinical score 246 hACSs ∗ Cumulative disease index 1482000 ∗
0 0
0 5 10 15 20 25 30 0
Days after immunization
Control
Control
mASCs at onset Control
mASCs at acute phase hASC hASC
(b) (c) (d)
Figure2:AllogeneicandxenogeneicASCreduceEAEseverity.ChronicprogressiveEAEwasinducedinC57Bl/6micebyimmunization
6
withMOG35−55.AnimalswereinjectedintraperitoneallywithPBS(control)orwithhypoxia-expandedallogeneicmASCs(10 cells/mouse)
eitherattheonsetofEAE(day11afterimmunization)whenthemeanclinicalscorewas0.25inthecontrolgroup(𝑛 = 8mice)and0.22in
themASC-treatedgroup(𝑛=9mice)orattheacutephaseofEAE(day15afterimmunization)whenthemeanclinicalscorewas2.7inthe
controlgroup(𝑛=6mice)and2.8inthemASC-treatedgroup(𝑛=7mice).(a)Clinicalsymptomswerescoreddailyinthegrouptreatedat
onset(leftpanel)andattheacutephaseofEAE(rightpanel).(b)Cumulativediseaseindexisthesumofdailyclinicalscoresobservedbetween
days20and40.Resultsareshownasmean(SD).∗𝑝<0.05versuscontrol.(c)HumanASCsameliorateEAE.MicewithMOG-inducedEAE
wereintraperitoneallyinjectedwithPBS(control,𝑛=7)orwithhASCsexpandedinhypoxia(106cells/mouse,𝑛=6mice)duringtheacute
phaseofthedisease(arrow)whenthemeanclinicalscorewas1.5inbothgroups.Clinicalsymptomswerescoreddaily.(d)Cumulativedisease
indexisthesumofdailyclinicalscoresobservedbetweendays20and40.Resultsareshownasmean(SD).∗𝑝<0.05versuscontrol.
compared to control EAE mice (Figure 5(d) and represen- 4.Discussion
tative dot plots in Figure S5). In order to further analyze
DC function we purified CD11c+ cells from the DLNs of MSCs have emerged as a promising therapy for MS with
controlandmASC-treatedEAEmiceandassessedtheirTNF- some studies suggesting an MSC-mediated modulation of
𝛼 producing capacity upon LPS restimulation in vitro. We theaberrantimmuneresponse[17,21].However,theefficacy
found that DCs from mASC-treated animals produced less hassofarbeenmodestwarrantingthesearchforimproved
TNF-𝛼 upon LPS stimulation in vitro compared to control MSC culture protocols and a greater understanding of the
DCs (Figure 5(d)). Taken together, these data suggest that mechanisms behind the immunomodulatory and trophic
mASCcouldsuppressEAEpartlybypreventingDCfunction effectsofMSCsinvitroandinvivo[16,17,19].Ouranalysis
invivo. oftheimmunomodulatorymechanismsemployedbymASCs
8 StemCellsInternational
Klüver-Barrera staining MBP IHC
Control mASC Control mASC
(a)
40 Cervical SC 50 Lumbar SC U) 300 3 U)
A. A.
Plaque # 1230000 Control mASCs Plaque # 123400000 Control mASCs Relative gene expression ( 1200000 IDO Foxp∗3 TGF∗𝛽 IFN∗𝛾 IL-1∗0 MCP-1 012 Relative gene expression (
Control
mASC
(b) (c)
Figure3:AllogeneicmASCsreducedemyelinizationandinflammatoryinfiltrationinthecentralnervoussystemduringEAEprogression.
MicewithMOG-inducedEAEwereintraperitoneallyinjectedwithPBS(control,𝑛=4)orwithmASCsexpandedinhypoxia(106cells/mouse,
𝑛=4)aftertheonsetoftheclinicalsymptoms,andspinalcordswereobtainedatthepeakofthedisease7dayslater.((a)and(b))Transverse
sectionsofcervicalandlumbarregionsofspinalcordwererandomlyanalyzedforthepresenceofinflammatoryinfiltratesandplaquesof
demyelinizationusingtheKlu¨ver-Barrerastaining(leftpanels)andanti-myelinbasicproteinimmunohistochemistry(rightpanels).Arrows
pointtoareasofdemyelinization.(c)GeneexpressionofdifferentinflammatorymarkerswasdeterminedbyPCRinmRNAsisolatedfrom
thespinalcords.Resultsareshownasmean(SD)of4micepergroup.∗𝑝<0.05versuscontrol.
culturesat5%O2showedthatboththeiriNOSandCOX-1/2 microglia in controlling autoimmune-mediated inflamma-
activitiesareinvolvedintheinhibitionofTcellproliferation tionintheCNSremainstobeelucidated[47,48].
invitro.AlthoughiNOSisusedonlybymurineMSCs,COX- In agreement with previous studies [11, 27], we found
1/2 activity has been shown to be important for the immu- that early injection of mASCs reduced the activation of
nomodulatorycapacityofbothmurineandhumanMSCs[29, MOG35−55-specific T cells and significantly inhibited EAE
45,46].InadditiontoiNOSandCOX-1/2,wealsodetected progressionandseverity.
asignificantinductionofarginaseactivityinthemASC:sple- Regulatory T cells (Tregs) control the severity of EAE
nocyte cultures, which was found predominantly in the throughthepreventionofTcellexpansionintheDLNs[49]
adherent splenocyte fraction and not in the mASC popula- and by inhibiting activated T cells in the CNS [50]. Several
tion(FigureS6).Thisisinagreementwithourrecentfinding studieshavereportedanincreasedfrequencyofTregsinthe
+
thatMSCscaninducearginaseI regulatorymacrophagesin LNsofEAEanimalstreatedwithMSCs[26,51].Interestingly,
vitro[29].Althougharginaseactivitydoesnotappeartobe two studies observed an increase in Treg number [17] or
importantfortheMSC-mediatedinhibitionofTcellprolifer- foxp3 mRNA [21] in PBMCs from MS patients after MSC
ationinvitro,theroleofMSC-educatedmacrophagesand/or administration.Incontrast,wedidnotobserveanincreased
StemCellsInternational 9
DLN Spleen DLN Spleen
Cytokine-producing cells (%) 01234 𝛾IFN∗ IL-4 IL-10 IL-17∗ 𝛾IFN∗ IL-4 IL-10 *IL-17∗ 01234 Cytokine-producing cells (%) 3×10Proliferation (CPM) 123400000 mulated OG3555–∗ nti-CD3 mulated OG3555– nti-CD3 12345060000003×10Proliferation (CPM)
CmoAnStCrosl Unsti M A Unsti M A
Control
mASCs
(a) (b)
DLN Spleen
60
30
L)
𝛾[IFN] (ng/m 2400 ∗ 1200𝛾FN] (ng/mL)
[I
0 0
5
ng/mL) 12 812 g/mL) cells (%) 43
[IL-17] ( 48 ∗ 4 [IL-17] (n ++43Dfoxp 12
∗ C
0 0 0
mulated OG3555– nti-CD3 mulated OG3555– nti-CD3 DLCNontrol Spleen
nsti M A nsti M A mASCs
U U
Control
mASCs
(c) (d)
+
Figure4:AllogeneicmASCsinhibitMOG-specificimmuneresponsesbutdonotinducefoxp3 Tregs.mASCswereinjectedintraperitoneally
inMOG-inducedEAEmiceaftertheonsetoftheclinicalsymptomsandspleensandDLNswereisolatedatthepeakofthedisease.Untreated
EAEmicewereusedascontrols.(a)Thepercentagesofcytokine-producingcellsinspleenandDLNsweredeterminedbyintracellularflow
cytometryasdescribedinMaterialsandMethods.((b)and(c))DLNandspleencellswereculturedwithmedium(unstimulated)orstimulated
withMOG35−55(50𝜇g/mL)oranti-CD3(1𝜇g/mL,usedasunspecificstimulation).Proliferationwasmeasuredafter3daysby[3H]-thymidine
+ +
incorporation(b).Thecontentofcytokineswasdeterminedinculturesupernatantsafter48hours(c).(d)ThepercentagesofCD4 Foxp3
TcellsinspleenandDLNsweredeterminedbyflowcytometry.Resultsareshownasmean(SD)from4micepergroup.∗𝑝 < 0.05versus
control.
+ + +
frequencyofCD4 foxp3 TcellsorIL-10 cellsinspleenand relativetodiseaseprogressionandtothenumbersofMSCs
lymph nodes nor increased foxp3 mRNA levels in the CNS reaching the DLNs [26, 51]. These differences could also
aftermASCinjectionintoEAEmicewhichisinaccordance partiallyexplainwhytheclinicalefficacyofMSCsinMSisnot
withotherstudiesonMSCsandEAE[27,52,53].Thesedis- quiteestablished.Importantly,inanotherMS-trial,Connick
crepanciescouldbeduetodifferencesintheimmunomod- et al. [54] observed a cessation of the protective effects six
ulatorymechanismsemployedbymurineandhumanMSCs, months after intravenous infusion of MSC, suggesting that
underlyingdifferencesinthepathophysiologicalmechanisms a possible induction of Tregs by MSCs was not enough to
between EAE and MS, and the timing of MSC injection restore immune homeostasisin MS patients. Future studies
10 StemCellsInternational
CD40 TNF-𝛼
of control)17050 ∗ ∗ ∗ ∗ ng/mL) 2.0
pression (% 2550 𝛼[TNF-] ( 1.0 ∗ ∗ ∗
x
E 0 0
CD80 IL-10
150 0.8
ol)
r 125
cont 100 mL) 0.6
of g/
pression (% 257505 [IL-10] (n 00..24
x
E 0 0
CD86 IL-12
150 30
ol)
of contr110205 g/mL) 20
pression (% 257505 [IL-12] (n 10
x
E 0 0
0 0.5 1 2 4 0 0.5 1 2
Number of mASCs (×105) Number of mASCs (×105)
(a)
100 #
ol) 80 ∗
r
nt
o 60
c
%
0 ( 40
4
D 20
C
0
100
80
ol)
ntr 60
o
c
% 40
𝛼F ( 20 #
N
T ∗
0
% of control)1680000 % control)1680000 #
Proliferation ( 24000 ∗ Proliferation ( 24000 ∗
Control mASC LPS − + + + +
mASC − − + + +
L-NAME − − − + −
Indo − − − − +
(b) (c)
Figure5:Continued.