ll
muneencephalomyelitis
oRN
ion
lau
EA
ent
EAE
demye
em(C
D4
+
Tc
import
FN-γpr
helper
autoim
elper1
miR-146a,miR-326,miR-124andmiR-92awereidenti?edrecentlyas
JournalofNeuroimmunology266(2014)56–63
ContentslistsavailableatScienceDirect
JournalofNeuro
sevier.c
micecanameliorateseveralautoimmunedisorders,includingexperi-
mentalautoimmuneencephalomyelitis(EAE)(Ivanovetal.,2006).In
addition,IL-17-expressingTcellshavebeenfoundinlesionsofbraintis-
suesfrompatientswithmultiplesclerosis(Tzartosetal.,2008).
MicroRNAs(miRNAs)areaclassofnoncodingRNAsthatmodulate
geneexpressionattheposttranscriptionallevelandareinvolvedinreg-
ulatingseveralaspectsofin?ammation(XiaoandRajewsky,2009;
O''Connelletal.,2010a,b).Whenpoorlyregulated,miRNAsarecritically
involvedinarangeofhumandiseases(Maetal.,2007;Thumetal.,
microRNAswhichwereknowntobeimmunologicallyrelevant.Fur-
thermore,westudiedtheroleofmiR-155inTh1andTh17celldiffer-
entiationandcytokineproductionbyover-expressionandinhibition
ofmiR-155inEAE.
2.Materialsandmethods
2.1.Patientsandcontrols
2008;SlackandWeidhaas,2008)andpotent
andprognosticmarkersortherapeutictargets
andRudensky,2010).
?Correspondingauthorat:TheDepartmentofNeuro
HebeiMedicalUniversity,HepingXiRoad,No.215,Shijia
Tel.:+8631166002122;fax:+8631166002023.
E-mailaddress:docshuangq@126.com(L.Guo).
0165-5728/$–seefrontmatter?2013ElsevierB.V.Allri
http://dx.doi.org/10.1016/j.jneuroim.2013.09.019
7(Th17)cellsarecritical
mation(Bettellietal.,
atinhibitionofIL-17in
etal.,2010;Junkeretal.,2009)anditwasassociatedwithimpairedau-
toimmunedevelopmentinmiR-155de?cientmice.
Inthepresentstudy,weexaminedtheexpressionofeight
mediatorsofchronicandautoimmunein?am
2006).Subsequentstudiesdemonstratedth
1.Introduction
Multiplesclerosis(MS)isachronic
tivediseaseofthecentralnervoussyst
pathogenesisofMSremainsunclear,C
nitywassuggestedasoneofthemost
genesis(SospedraandMartin,2005).I
(Th1)cellswereonetypeofeffector
pathogenesisofMSandexperimental
(EAE).RecentstudysuggestedthatTh
linatingneurodegenera-
NS).Althoughtheexact
ellmediatedautoimmu-
antaspectsofthepatho-
oducingThelpertype1
Tcellsthatmediatethe
muneencephalomyelitis
importantregulatorsforimmunecelldevelopmentandimmunere-
sponses.MiR-155isoneofthemiRNAsthataremosthighlyimplicated
inautoimmunity,anditwasshownmiR-155playsacrucialroleinthe
functionofpathogenicimmunecells,suchasTcells,Bcellsanddendrit-
iccells(DCs)(Calame,2007;O''Connelletal.,2010a,b;Zhouetal.,
2010).Moreover,aberrantexpressionofmiR-155hasbeenobserved
inmanyhumanautoimmuneconditions(Stanczyketal.,2008;Wang
SeveralmiRNAssuchasmiR-155,miR-181a,miR-181b,miR-150,
MicroRNA-155modulatesTh1andTh17ce
multiplesclerosisandexperimentalautoim
JingZhang,YeCheng,WeiCui,MeixiLi,BinLi,LiGuo?
TheDepartmentofNeurologyoftheSecondHospitalofHebeiMedicalUniversity,China
abstractarticleinfo
Articlehistory:
Received6May2013
Receivedinrevisedform24September2013
Accepted30September2013
Keywords:
MicroRNAs
Experimentalautoimmuneencephalomyelitis
Multiplesclerosis
Th1cells
Th17cells
Mammaliannoncodingmicr
foundthatmiR-155express
andmicewithexperimenta
Th1andTh17cellsandmild
155promotedthedevelopm
155conferssusceptibilityto
ofmultiplesclerosis.
journalhomepage:www.el
iallyserveasdiagnostic,
(Luetal.,2005;Littman
logyoftheSecondHospitalof
zhuang,HebeiProvince,China.
ghtsreserved.
As(miRNAs)aresuggestedtobeinvolvedinimmunesystemfunction.We
washighlycorrelatedwithdiseaseseverityinpatientswithmultiplesclerosis
toimmuneencephalomyelitis(EAE).KnockdownofmiR-155resultedinlow
E,anditsoverexpressionledtomoreTh1andTh17cellsandsevereEAE.MiR-
ofin?ammatoryTh17/Th1cellsubsets.These?ndingsdemonstratethatmiR-
byaffectingin?ammatoryTcellresponsesandcanbeanewtargetfortherapy
?2013ElsevierB.V.Allrightsreserved.
differentiationandisassociatedwith
immunology
om/locate/jneuroim
MSpatientswereadmittedintheSecondHospitalofHebeiMed-
icalUniversitybetweenJanuary2010andDecember2012.AllMS
patientsunderwentastandardbatteryofexaminationsandful?lled
theMcDonald''scriteria.TheMScohortconsistedof58%femalesand
42%males.Serawerecollectedfrom31MSpatients,32GBSpatients
(forotherneuroimmunologicaldiseasecontrolstudy)and31healthy
subjects.ThehealthycontrolsubjectswerealsoresidinginHebei
inhibitorwasadministeredwhenaclinicalscoreof1.5wasobserved
(n=5).
2.7.ProliferativeresponsesofTcellandcytokineanalysis
Spleensordraininglymphnodeswereharvestedandpooledfrom
EAEmice,andsingle-cellsuspensionswereprepared.Cellswerecultured
at5×10
7
cells/wellin24-wellU-bottomplateswith10mg/mlMOG35–
55peptideincompleteRPMI1640medium(including10%FCS,100
units/mlstreptomycin,50mMbeta-mercaptoethanol)(n=4).For
ELISAanalyses,supernatantswereharvestedat72hofculture.Thecon-
57J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
regionandmatchedwithcasesintermsofgenderandage(meanage
40.7years;range25–56).Noneofthepatientsreceivedanyimmuno-
modulatorytherapy3weeksbeforethebloodwithdrawal.Moreover,
toavoidpossibleconfoundereffectsduetodiurnalvariationinim-
munefunction,allsampleswerecollectedbetween6and8inthe
morning.ThisstudywasapprovedbylocalEthicalCommittees.All
recruitedsubjectssignedaninformedconsenttoparticipateinthe
study.AllthepatientsandcontrolswereChineseofHanrace.
2.2.Mice
Atotalof82C57BL/6wild-typemicewerepurchasedfromVital
RiverLaboratoryAnimalTechnologyCo.Ltd.Beijing,China.Mice
weremaintainedinaspeci?cpathogen-freecondition.Allmice
were6–8wkoldatthebeginningofexperiments.Allexperiments
wereinaccordancewithGuidelinesfortheCareandUseofLabora-
toryAnimals(Science&TechnologyDepartmentofHebeiProvince,
PRChina).
2.3.InductionandevaluationofEAE
EAEwasinducedinC57BL/6micebyimmunizationwith250μgof
MOGp35–55.AllpeptidesweredissolvedincompleteFreund''sadjuvant
(Sigma,StLouis,MO,USA)containing4mg/mlofheat-killedmycobac-
teriumtuberculosisH37Ra(DifcoLaboratories,Detroit,MI,USA).Atday
0and48hafterimmunization,C57BL/6micewereinjectedwith500ng
ofpertussistoxin(Alexis,SanDiego,CA,USA)inPBS,intraperitoneally
(i.p.).ClinicalassessmentofEAEwasperformedafterdiseaseinduction
bythefollowingcriteria:0,nodisease;1,tailparalysis;2,hindlimb
weaknessorpartialparalysis;3,completehindlimbparalysis;4,fore-
limbandhindlimbparalysis;5,death.Micewererandomlydivided
into?vegroups:15miceinmiR-155controlmimicgroup,19micein
miR-155mimicgroup,20miceinmiR-155controlinhibitorgroup,20
miceinmiR-155inhibitorgroup,and8miceinnotreatmentgroup.
2.4.Histopathology
Forhistopathologicalstudies,spinalcordsweredissectedfrom
femalemice(n=4),?xedin10%formalininPBS,andembeddedina
singleparaf?nblock.The8mmthicksectionswerestainedwithH&E
andluxolfastblue,andstainedsectionswereevaluatedforimmune
cellin?ltrationanddemyelination.
2.5.AnalysisofmiR-155expression
ForanalysisofmiR-155expression,real-timeRT-PCRanalyseswere
carriedoutusingTaqManmiRNAassays(AppliedBiosystems)andrel-
ativeexpressionwascalculatedusingtheCTmethod,andnormalized
touniformlyexpressedU6(AppliedBiosystems)(n=4).
2.6.MiR-155mimicandinhibitortreatment
TheoligonucleotidesofmiR-155mimicandinhibitor(Genepharma,
China)weresynthesizedtooverexpressorknockdownmiR-155expres-
sionsinmice.ThesequenceofmiR-155mimicis:sense(5′–3′)UUAA
UGCUAAUUGUGAUAGGGGUandantisense(5′–3′)CCCUAUCACAAU
UAGCAUUAAUU,whereasmiR-155inhibitorsequenceis:ACCCCUAU
CACAAUUAGCAUUAA,whichisbasedoncomplementaritytosensese-
quenceofthemiR-155maturesequence.2′OMewasusedaschemical
modi?cationoftheinhibitortomakethemodi?edantisenseoligo
playingasteadyroleinthecompetitiveinhibitionofthetargetsense
strand.ForinvivomiR-155treatment,100μlEntranster?—invivo
(EngreenBiosystemCo,Ltd.,Beijing)wasmixedwith50μgmiR-155
mimicorinhibitorortheirrespectivecontrolsaccordingtheinstruction.
Andthecomplexeswereadministeredinvivotothesemiceondays5,7,
9,11,13and15afterimmunization(n=10).ForEAEreversal,miR-155
centrationsofindicatedcytokinesweremeasuredbyquantitativecap-
tureELISA,accordingtotheguidelinesofthemanufacturers(BD
Biosciences)(n=4).Forthedetectionofproliferativeresponses,
splenocytesorLNcellswereseededin96-wellplateat5×10
3
cellsper
wellwith10mg/mlMOG35–55peptideandthenculturedfor72h.
10μlofCCK-8(CellCountingKit-8,DojindoLaboratories,Kumamoto,
Japan)solutionwasaddedtoeachwellofperplate,andincubatedthe
plateat37°Cforthe?nal4h.Theabsorbanceat450nmwasassayed
forproliferation.Experimentswererepeatedfourtimes,andeachseries
wasperformedintriplicate.
2.8.PreparationandevaluationofCNScells
Brainsandspinalcordsofmice,whichwereperfusedwithcoldPBS,
weredissectedandincubatedin2.5mg/mlcollagenaseDfor30minat
37°C.Single-cellsuspensionswereprepared.Cellswerewashedin
RPMI1640medium,andmononuclearcellswereisolatedusingadis-
continuousPercollgradient(Pharmacia,Piscataway,NJ)(n=4).
2.9.Statisticalanalysis
Statisticalanalysiswasperformedusingtheunpairedttest.Pb0.05
isconsideredsigni?cant.Dataarepresentedasmean±SEM.ANOVA
analysiswasusedtocalculatethedifferencesofvarioustreatmentsof
micewithEAE.AlltheanalysesweredoneusingSPSS7.0.
3.Results
3.1.UpregulationofmiR-155inpatientswithMS
TherespectiveclinicalcharacteristicsinpatientswithMS,GBSand
healthyindividualswereshowninTable1.Eightextracellularimmuno-
logicallyrelevantmicroRNAs,includingmiR-155,miR-326,miR-146a,
miR-150,miR-181a,miR-181b,miR-124andmiR-92a,wereinvestigat-
edinserasamplesfromMSandcontrolsubjects.FiveofthosemiRNAs
wereN1foldup-regulationinMScomparedtocontrols,whereasnone
ofthemshoweddown-regulation.MiR-155showedthehighestin-
crease(foldchange=3.65;Pb0.001)(Fig.1A).Fig.1Bshowedthat
miR-155expressionwassigni?cantlyhigherinseraofpatientswith
MSthanthosewithGBSandhealthyindividuals.Detailedanalysis
foundahigherexpressionofmiR-155inMSpatientsduringrelapse
thanthoseduringremission(Fig.1C).
Table1
CharacteristicsofpatientswithMSandcontrols.
MSControlGBS
Total313132
Age44.4±13.240.7±8.441.5±12.2
Sex
Female181614
Male131518
Clinicalstage
Relapsing14Allintheacutephase
Remitting17
isof
Spa
58J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
Fig.1.UpregulationofmiR-155inpatientswithmultiplesclerosis.QuantitativePCRanalys
181a,miR-181b,miR-124,miR-92a),expressioninserafromnormalcontrols(n=31)andM
3.2.RegulationofEAEdevelopmentbymiR-155
ExpressionofmiR-155frommicewithEAEwasincreasedinspleen,
lymphnodeandbrainduringtheacutephasebutdecreasedtonormal
expressionwhendiseaseremitted(Fig.2A).
WetransfectedmiR-155mimic,inhibitorandcontrolsinvivoby
injectionthroughthetailveinandassessedthetransfectef?cacyby
quantitativePCRanalysisinvariousorgans(spleen,lymphnode,brain
andliver)ofthemice(Fig.2B).TransfectionwithmiR-155mimicled
tonearlytwo-foldincreaseinexpressionofmiR-155inspleenand
1.75-foldincreaseinexpressionintheperipherallymphnodes,and
1.43-foldincreaseinthebrain.MicetransfectedwithmiR-155mimic
developedsevereEAE,whereasinhibitortransfectedmicehadsome-
whatmildEAE(Fig.2C).Histologicalanalysisofspinalcordsections
showedthatmicetransfectedwithmiR-155mimicdevelopedpromi-
nentin?ammatoryin?ltrationanddemyelination,whereasinhibitor
transfectedmiceshowedmildCNSpathology(Fig.2E,F).Wefurther
testedtheef?cacyofmiR-155inhibitoronEAEaftertheonsetofclinical
symptoms.WefoundthattreatmentwithmiR-155inhibitorenhanced
clinicalrecoveryfromEAE(Fig.2D).
3.3.PromotionofTh-1cellsandTh-17cellsdifferentiationby
miR-155inEAE
WeexaminedthepresenceofIL-17(Th17)orinterferon-γ(IFN-γ)
(Th1)-producingCD4
+
TcellsduringEAEinlymphnodes,spleenand
theCNSinthefourgroupsoftransfectedmice(miR-155mimic,inhibi-
tor,controlmimicandcontrolinhibitor).Intracellularcytokinestaining
showedthatthefrequenciesofbothTh-1andTh-17cellsinthe
splenocytesandlymphnodescellswerehigherinmiR-155mimic
transfectedmicebutonlythefrequencyofTh-17cellsinmiR-155
fromnormalcontrols(n=31),patientswithGBS(n=32)andMS(n=31);C.miR-155expre
17).Pb0.05.
eightimmunologicallyrelevantmicroRNAs(miR-155,miR-326,miR-146a,miR-150,miR-
tientsduringrelapse(n=14);B.QuantitativePCRanalysisofmiR-155expressioninsera
inhibitortransfectedmicewaslowerthanthecontrols(Fig.3A,B,D
andE).MiR-155inhibitortransfectedmicehadlowerfrequencyof
Th17cellsinCNSmononuclearcellscomparedtotheirrespectivecon-
trol.ThefrequencyofTh-17cellsinCNSishigherinmiR-155mimic
transfectedmice.ButfrequencyofTh-1cellswasnotchangedinthe
CNSamongthefourgroups(Fig.3C,F).
CD4
+
TcellsinmiR-155mimictransfectedmiceunderwentcelldi-
visionsinvitrowithMOG35–55,whereasCD4
+
TcellsinmiR-155in-
hibitortransfectedmicehadareducedproliferation(Fig.4A,B).In
parallel,CCK8assaysviasplenocytesproducedsimilardifferences
(Fig.4C).SplenocytesandLNcellsofmiR-155inhibitortransfected
miceshoweddecreasedproductionofbothofIL-17AandIFN-γcom-
paredtotheircontrols,whereastheproductionofthesecytokinesin-
creasedinmiR-155mimictransfectedmice(Fig.4D,E).Genes
controllingexpressionofIL-17andIFN-γweresigni?cantlyupregulated
inmiR-155mimictransfectedmice,butdownregulatedinmiR-155in-
hibitortransfectedmice(Fig.4F,G).
3.4.MiR-155promotesTh-1cellsandTh-17cellsdifferentiationduringthe
inductionphaseofEAE
Next,weinvestigatedwhetheradefectinin?ammatoryTcelldevel-
opmentcouldalsobedetectedduringtheinductionphaseofEAE.Mice
wereharvestedonday13afterimmunizationwithMOG35–55.In?am-
matoryTcelldevelopmentintheimmunologyorgansandtheCNSin
thefourgroupswereassessed.Intracellularcytokinestainingshowed
thatboththeproportionsofTh-1andTh-17cellsinthesplenocytes
werehigherinmiR-155mimictransfectedmiceandlowerinmiR-155
inhibitortransfectedmicethantheirrespectivecontrols(Fig.5A).
InLNscells,Th17andTh1cellswerepresentatlowerproportion
amongCD4
+
TfrommiR-155inhibitortransfectedmice,whereasthe
ssioninserafromMSpatientsduringrelapse(n=14)andpatientsduringremission(n=
59J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
frequencyofTh1cellsamongCD4
+
TwasequivalentbetweenmiR-155
mimictransfectedmiceandcontrols(Fig.5B).TheproportionofTh-17
cellsincreasedobviouslyamongtotalCD4
+
TcellsintheCNSofmiR-
Fig.2.RegulationofEAEdevelopmentbymiR-155.A.QuantitativePCRanalysisofmiR-155exp
n=4micepergroup);B.QuantitativePCRanalysisofmiR-155expressioninspleen,lymphno
pergroup);C.ClinicalscoresforEAEinmicetransfectedwithmiR-155mimic,inhibitor,contro
withmiR-155inhibitoraftertheonsetofEAE(n=5micepergroup);E.Histologyofparaf?nsec
day25afterimmunization.SpinalcordsectionsstainedwithH&Eandluxolfastbluetoaccessi
ationinthesections,presentedasin?ltratespermm
2
(left)anddemyelinationarearelativeto
155mimictransfectedmicecomparedtocontrolmiceanddecreased
inmiR-155inhibitortransfectedmice.ButtheproportionofTh-1cell
wasnotchangedintheCNSamongthefourgroups(Fig.5C).
ressioninspleen,lymphnodeandbrainduringEAEdevelopment(non-treatmentgroup,
de,brainandliveraftertransfectedwithmiR-155mimicandinhibitorinvivo(n=4mice
lmimicandcontrolinhibitor(n=10micepergroup);D.Clinicalscoresformicetreated
tionsofspinalcordsisolatedfromoligonucleotide-infectedmice(n=5micepergroup)on
n?ammationanddemyelination;F.Quanti?cationofspinalcordin?ltratesanddemyelin-
totalanalyzedarea(right).n=5,Pb0.05,Pb0.01.
60J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
TherecallresponsetoMOG35–55wasalsotestedwithsplenocytes
onday13EAEmiceviaCCK8essays.Splenocytesexhibiteddiminished
proliferationinMiR-155inhibitortransfectedmice,whereasMiR-155
mimictransfectedmiceexhibitedincreasedproliferationinsplenocytes
(Fig.5D).
IL-17AandIFN-γproductionswereassayedbyELISAinculturesu-
pernatantswithMOG35–55stimulation72h.SplenocytesofmiR-155
inhibitortransfectedmiceshoweddecreasedproductionofbothof
IL-17AandIFN-γcomparedtotheircontrols,whereastheproduction
ofthesecytokinesincreasedinmiR-155mimictransfectedmice.Similar
de?cienciesinTh1andTh17cellswerealsoobservedintheLNs(Fig.5E,
F).Th17andTh1expressionsweredetectedbyquantitativePCR(qPCR)
inthefourgroups.Consistentwiththat,thegenescontrollingtheex-
pressionofIL-17andIFN-γweresigni?cantlyupregulatedinmiR-155
mimictransfectedmice,butdownregulatedinmiR-155inhibitor
transfectedmice(Fig.5G,H).
Fig.3.InvivogenerationofTh-17cellsandTh-1cellsinEAEmiceenhancedbymiR-155.A–C.In
mononuclearcells(C)(n=4micepergroup);D–F.ThefrequenciesofTh-17cellsandTh-1ce
respectively(n=4micepergroup).
4.Discussion
ThisstudyhasshownthatmiR-155expressioninserawasassociat-
edwithMSpatientduringrelapse,andknockdownoroverexpressionof
miR-155canalleviateoraggravatediseaseseverityinEAEmode,sepa-
rately.Furthermore,westudiedtheroleofmiR-155inTh1andTh17cell
differentiationandcytokineproductionbyover-expressionandinhibi-
tionofmiR-155inEAE.Tosumup,ourresultssuggestthatmiR-155isa
Tcell-associatedmicroRNAthatplaysaroleinthepathogenesisofmul-
tiplesclerosis.
MicroRNAisagroupofsmallnoncodingRNAswhicharethoughtto
regulategeneexpressionsposttranscription.DysregulationofmiRNAs
hasbeenassociatedwithseveralautoimmunediseases(Tangetal.,
2009).ElevatedexpressionofmiR-155hasbeenobservedinplasma
andbrainlesionsfrommultiplesclerosis(MS)patients(Junkeretal.,
2009;Siegeletal.,2012).
tracellularstainingofIL-17andIFN-γinspleencells(A),LNcells(B)andCNSin?ltrating
llsinspleencells(D),LNcells(E)andCNSin?ltratingmononuclearcells(F)areanalyzed
61J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
Althoughothertissuesandcelltypesmightbemorere?ectiveof
MSpathogenicchanges,wechosetoinvestigatemiRNAexpression
patternsinsera,whicharereadilyaccessibleandcouldpotentially
representanoptimalclinicalbiomarker.Cell-freemiRNAscanbede-
tectedinseveralhumanbody?uidswhicharecorrelatedwithdis-
easeactivityandprognosis,particularlyincancer,cardiovascular
diseasesandbraininjury(Mitchelletal.,2008;Boerietal.,2011;
Redelletal.,2009).Interestedly,circulatingmiRNAsareexceptional-
lystableinbiological?uids,indicatingthatmiRNAsarereleased
fromcellsinmembrane-derivedvesicles(exosomes)whichcanpro-
tectthemfrombloodRNaseactivity(Mitchelletal.,2008).This
stronglysuggeststhatcirculatingmiRNAcanbeusedasapotential
clinicalbiomarker.
Toourknowledge,fewstudieshaveexaminedtheextracellular
miRNAlevelsinplasmasamplesfromacohortofMSandcontrol
subjects(Siegeletal.,2012).6outof900miRNAstestedwerefound
tobesigni?cantlyupregulated(miR-614,miR-572,miR-648,miR-
1826,miR-422aandmiR-22)whereasoneplasmamiRNA(miR-1979)
wassigni?cantlydownregulatedinMSpatients.Interestingly,both
miR-422aandmiR-22havepreviouslybeenimplicatedinMS(Junker
etal.,2009;DeSantisetal.,2010;Lindbergetal.,2010).
SomestudieshaveshownthatmiR-155expressionisrequiredfor
normalinnateandacquiredimmunity(Tilietal.,2007).Andprevious
workalsohasdemonstratedthatmiR-155canimpactTh1celland
Th2celllineageskewinginvitro(Rodriguezetal.,2007;Thaietal.,
2007).InhibitionofmiR-326viaamiRNA“sponge”wasprovedto
Fig.4.miR-155affectsin?ammatoryTcelldevelopmentinvivoduringEAE.Apresentsthespl
CD4
+
proliferatingcellsfromthefouroligonucleotide-transfectedgroupswasassayedby?ow
frequencyofCD4
+
proliferatingcellsispresentedinB.TheharvestedsplenocytesinAwasalso
37°Cforthe?nal4h.Cellproliferationwasevaluatedbystimulationindex(n=4),andtheav
splenocytes(D)andLNcells(E)fromoligonucleotide-transfectedEAEmice(n=4);FandGsho
determinedbyrealtimePCR(n=4).
suppressTh17celldifferentiationandrelieveEAEsymptomsthrough
aTcell-intrinsicmechanism(Duetal.,2009).
PresentstudiesshowedtherolesofmiR-155inTcellpolarization
andEAEbyusingmir-155KOanimals.AlleviationofEAEandlessin-
?ammationintheCNShaveshowninMiR-155de?cientmice,which
wasassociatedwithadecreaseinTh1andTh17responses(O''Connell
etal.,2010a,b;Murugaiyanetal.,2011).Currently,oneofthemost
promisingmethodsofmiRNAmanipulationisdeliveryofmodi?edoli-
gonucleotidesmimickingorinhibitingspeci?cmiRNA.Thisstudyhas
improvedinvivodeliveryofthemiR-155mimicandmiR-155inhibitor
byencapsulationintoananoparticle-baseddeliverysystem.Theresult
isconsistentwiththeprevious?ndingsandprovidesexactevidence
thatmiR-155playsanimportantroleintheregulationofMS.Mice
treatedwithmiR-155inhibitorexhibitadelayedcourseandreducedse-
verityofdiseaseandlessin?ammationintheCNS.
IFN-γ-producingTh1andIL-17-producingTh17cellsarekeyproin-
?ammatorymediatorsofcellularimmunitythatunderliecrucialevents
duringdevelopmentofEAE.TheTh1lineagecytokinecanhelpTh17
cellsinvadethebrainandspinal,thustriggerEAE(Reboldietal.,
2009).ThehighpercentageofTh17cellshasanimpactonthein?am-
mationinthebrainandtheseverityofdisease(Stromnesetal.,2008).
ThisstudyhasshownthattheattenuationofEAEinmiR-155inhibitor
miceisassociatedwithadecreaseinIL-17andIFN-γexpressioninthe
peripherallymphoidorgansandCNS.TheincreasedTh1andTh17re-
sponsesinmiR-155overexpressionmicesuggestthatmiR-155en-
hancesthedifferentiationofTh17andTh1cells,whichsustained
enocytesharvestedfrommice25daysafterEAEinductionlabeledwithCFSE.CFSElossby
cytometryafterrestimulationwithMOG35–55(10mg/ml)for72h(n=4);Theaverage
restimulatedwithMOG35–55(10mg/ml)for72h,andaddedCCK-8solutionincubatedat
eragefrequencyispresentedinC;DandEpresenttheproductionofIL-17AandIFN-γby
wthegenesrelativeexpressionofIL-17AandIFN-γofsplenocytes(F)andLNcells(G)was
62J.Zhangetal./JournalofNeuroimmunology266(2014)56–63
in?ammationresponseandaggravateclinicalsignsofEAEmode.Based
uponthestudy,miR-155maybeaneffectivetherapeutictargetinthe
treatmentofMS.
AppendixA.Supplementarydata
Supplementarydatatothisarticlecanbefoundonlineathttp://dx.
doi.org/10.1016/j.jneuroim.2013.09.019.
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