expression
Received25April2015
Availableonline25August2016
Keywords:
CD25
CD25siRNAtreatment
t
(IFN-g,IL-1b,andTNF-a).Inthecurrentstudy,cornealtransplantationwasperformedonWistarratsas
etal.,2009),whereasTh2andTregcytokinesare
t
T-relatedcyto-
pathwayisa
regsthroughJAK/
Tregssecretean
TGF-bandIL-
cells)into
ofTregsis
0werede?nedas
DCs,andsecretesubstantialpro-in?ammatorycytokines,suchas
IL-1bandTNF-a,triggeringTh1responseanditssecretionofIFN-g,
whichregulatesthein?ammatoryresponse(Horwitzetal.,2008).
Therefore,thecytokinesmentionedaboveareimportantpartici-
pantsinimmunetolerance.CD25playsapivotalroleintheregu-
lationandexpressionofothercytokines.However,therelationship
amongcytokineshasseldombeenstudied.
Correspondingauthor.DepartmentofOphthalmology,TheFirstAf?liated
HospitalofChongqingMedicalUniversity,1YouYiRoad,YuZhongDistrict,
Chongqing400016,China.
E-mailaddress:minzhaomz@outlook.com(M.Zhao).
1
ContentslistsavailableatScienceDirect
ExperimentalEye
journalhomepage:www.elsevier.com/locate/ye
ExperimentalEyeResearch151(2016)134e141
QinQinandDanLuocontributedequallytothispaper.
largenumberofcytokinesthatparticipateintherejectionprocess.
Th1-typeandTh17cytokinesacceleratecornealrejection(Chen
Tregcytokines(Lietal.,2011).However,whenthesuppressive
environmentisbroken,tolerogenicDCsconvertintoimmunogenic
thepatients''qualityoflife.Cyclosporine,FK506(tacrolimus),
rapamycin,andotherimmunosuppressiveagentshavebeen
extensivelyusedtopreventandcurecornealrejection(Yuanetal.,
2008).However,theseagentsonlyalleviatebutnotcompletely
inhibittheoccurrenceofcornealrejection.Inacomplexprocess,
CD4
t
Tcellscontributetocornealallograftrejection(Jiaetal.,
2012).Whenactivated,CD4
t
TcellsdifferentiateintoTh1,Th2,
Th17,andTreg(CD4
t
CD25
t
Foxp3
t
regulatoryT)cellsandsecretea
signaling,whichregulatesthesecretionofCD4
kines.Inaddition,anotherCD25-mediatedsignaling
prerequisiteforsurvivalanddifferentiationofT
STATsignals(Wuestetal.,2008).Whenactivated,
abundanceofsuppressivesolublecytokinesincluding
10,thusinducingimmatureDCs(immaturedendritic
tolerogenicDCs.Therefore,thesuppressivefunction
TGF-bandIL-10dependent.Thus,TGF-bandIL-1
Cornealallograftrejectionistheprimarycauseofcornealgraft
failure(CosterandWilliams,2005),whichseriouslydeteriorates
pleiotropic.Thea-chainorCD25ofIL-2Risconsideredakey
mediatorofCD4
t
TcelldifferentiationbydownstreammTOR
Th1cytokines
Tregcytokines
1.Introduction
http://dx.doi.org/10.1016/j.exer.2016.08.010
0014-4835/?2016ElsevierLtd.Allrightsreserved.
donorsandSprague-Dawleyratsasrecipients.ThesurvivalcurvesindicatedthatCD25siRNAtreatment
signi?cantlyprolongedgraftsurvivaltime(meansurvivaltime[MST],14.8±0.7days)ascomparedwith
controls(MST,7.6±0.7days;n?12,p<0.01).HEstainingshowedthatCD25siRNAalleviatedin-
?ammatorycellin?ltration.Atdays3,7,14,and21,themRNAandproteinexpressionofCD25inthe
CD25siRNAgroupswerelessthanthoseofthecontrolgroup,althoughthemostsigni?cantdecreaseof
CD25proteinwasatday3.TheexpressionofIL-10andTGF-bintheCD25siRNAgroupincreased,while
IFN-g,IL-1b,andTNF-aexpressiondecreased,aswellasnosigni?cantchangesinFoxp3expressionwere
observedatday14post-operation.Inconclusion,CD25siRNAgenetherapyplayedaprotectiverolein
cornealgraftrejectionviaup-regulationofTregcytokineexpressionanddown-regulationofTh1cyto-
kineexpression.
?2016ElsevierLtd.Allrightsreserved.
immunosuppressive(DeolandTuch,2000).
Cytokinesareroutinelyregulatedbyothercytokinesandare
4June2016
Acceptedinrevisedform23August2016
Receivedinrevisedform
inductionofcornealgraftrejectionbyregulatingCD4Tcellfunction.Furthermore,CD25-mediated
signalingiscloselyassociatedwiththeexpressionofTregcytokines(IL-10,TGF-b)andTh1cytokines
Researcharticle
CD25siRNAinducesTreg/Th1cytokine
transplantationmodels
QinQin
1
,DanLuo
1
,YunjieShi,QingqingZhao,Yuan
DepartmentofOphthalmology,TheFirstAf?liatedHospitalofChongqingMedicalUniversity
Ophthalmology,ChongqingEyeInstitute,Chongqing,China
articleinfo
Articlehistory:
abstract
Cornealgraftrejectionisthe
inratcorneal
Chen,JingWu,MinZhao
,ChongqingEyeBank,ChongqingKeyLaboratoryof
majorreasonfortransplantfailure.CD25playsanimportantroleinthe
Research
xer
eResear
GenetherapyinvolvesthetransferofDNAorRNAfragments
intospeci?ccellsusingvectors,andhasbeenusedsuccessfullyin
thetreatmentofophthalmicdiseases(Mohanetal.,2012).The
corneaiswell-suitedtogenetherapybecauseitisaccessible,and
thecornealepitheliumisamenabletopersistentgeneexpression
becauseofitslowdifferentiationrate(Ritteretal.,2007).Visionis
easilyregulatedbygenetherapy(Sharmaetal.,2011).RNAinter-
ference(RNAi)isanewtechnologytosilencetargetgeneexpres-
sionbyinducingRNAdegradation.SmallinterferingRNA(siRNA)
hasbeenusedextensivelytotreatdiseases.siRNAisatypeRNAi
whichisdouble-stranded,20e25nucleotidesinlength(Zhaoetal.,
2013).Therefore,weadoptedEntranster?-CD25siRNAtomodify
CD25expressioninratcornealgrafts,totestwhetherdown-
regulationofCD25expressioncouldinhibitcornealallograft
rejectionandtostudythedynamicsofTreg/Th1cytokines.Inthis
study,weshowedthatCD25siRNAgenetherapywaseffectivein
preventingcornealgraftrejection,mediatedbytheup-regulation
ofTregcytokinesanddown-regulationofTh1cytokines.
2.Methods
2.1.Animals
FemaleSprague-Dawleyrats(200e220g)andWistarrats
(200e220g)wereusedasrecipientsanddonors,respectively.Rats
werepurchasedfromtheLaboratoryAnimalCenterofChongqing
MedicalUniversityandBeijingMedicalUniversity.Ratswere
housedinstandardconditions(light/dark:12/12h,temperature:
25
C14
C,andrelativehumidity:40e60%)andwerefedwithastan-
dardpelletdietandwateradlib.Allexperimentswereincompli-
ancewiththeAssociationforResearchinVisionand
Ophthalmology(ARVO)statementfortheUseofAnimalsin
OphthalmicandVisionResearchandapprovedbytheEthics
CommitteeoftheFirstAf?liatedHospitalofChongqingMedical
University.
2.2.Cornealtransplantation
Orthotopiccornealtransplantationwasperformedaspreviously
described(WilliamsandCoster,1985).Brie?y,thedonorand
recipientratswereanesthetizedwithanintraperitonealinjection
of3%pentobarbitalsodium(1ml/kg,Sigma,UK).Therecipient
pupilwascompletelydilatedusingcompoundtropicamideeye
drops(ShenyangXinqiPharmaceuticalCo.,Ltd,China).Wistar
donorcorneaswereexcisedusinga3.5mmtrephine,preservedin
cornealstoragemedia(ChongqingEyebank,China),andgrafted
intothe3.0mmrecipientbedswith8interrupted10-0nylon
sutures.
2.3.Genetransferprotocol
CD25siRNAwasdeliveredinvivofollowingthemanufacturer''s
instructionsforEntranster?transfectionreagent(Engreen,Beijing,
China).Thetransfectioncomplexwaspreparedasfollows.Reagent
3waspreparedbymixing25mlCD25siRNAsolutionwith25ml
Entranster?transfectionreagentatanconcentrationof0.5mg/ml.
Reagent2waspreparedbymixing25mlscrambledsiRNAsolution
with25mlEntranster?transfectionreagentatthesameconcen-
tration.Reagent1wascomposedofnormalsaline(50ml).Afterthe
corneasweregrafted,therecipientcornealepitheliumwas
removedasdescribedpreviously(Sharmaetal.,2011).Allallograft
recipientswererandomlydividedinto3groups.Reagents1,2,and
3wereappliedfor5minontothecorneasofgroup1(control),
group2(scrambledsiRNA)andgroup3(CD25-siRNA),respectively.
Q.Qinetal./ExperimentalEy
Afterremovingexcessreagent,erythromycinophthalmicointment
(ChongqingKeruiPharmaceuticalCo.,Ltd,China)wasinstantly
appliedtorecipients''eyes.
2.4.Clinicalassessment
Orthotopicgraftswereexamineddailybyslitlampmicroscopy
forupto21days.Threeindiceswereusedtoevaluatethegrafts
accordingtoLarkin''sscoringcriteria(Larkinetal.,1997).Scoresfor
cornealopacity(0e4),edema(0e2),andneovascularization(0e4)
weregradedasshowninTable1.Rejectedgraftswereidenti?ed
witheitherasumofthethreeindicatorsC215oranedemadegreeup
to3.Atotalof28animalsdevelopedcomplications,consistingof
peripheralanteriorsynechiae,cataract,anteriorchamberloss,
persistenthyphema,orinfectionwereexcluded.Newmodelswere
supplemented.
2.5.Histologicalinspection
Afteranesthesia,recipientcorneasfromthreeratspergroup
wereharvestedandembeddedinparaf?natday14post-grafting.
Usingamicrotome,5mm-thicknesssectionswerecutandstained
withhematoxylinandeosin.Thesectionswereexaminedwitha
lightmicroscopic.
2.6.Immunohistochemicalstaining
Threeratspergroupweresacri?cedatday14post-surgery.
5mm-thicknesssectionswerecutfromparaf?n-embeddedcor-
neasandendogenousperoxidasewasblockedwith3%H
2
O
2
.The
slideswereincubatedovernightat4
C14
Cwithmonoclonalantibody
forFoxp3(ab22510,1:50,Abcam,Cambridge,UK),IL-10(ab192271,
1:200,Abcam),IL-1b(ab9722,1:100,Abcam),TNF-a(ab6671,1:100,
Abcam),IFN-g(ab9657,1:50,Abcam),TGF-b(ab66043,1:50,
Abcam)andCD25(sc-666,1:100,SantaCruz,CA,USA).After
washingwithphosphate-bufferedsaline(PBS,GibcoBRL,USA),
sectionsweretreatedwithappropriateHRP-conjugatedsecondary
antibodies.ThesampleswerevisualizedwithDABsolutionand
counter-stainedwithhematoxylin.Negativecontrolswereob-
tainedbyreplacingtheprimaryantibodywithPBS.
2.7.Westernblot
Threeeyespergroupwereexcisedatdays3,7,14,and21,
respectively.ProteinwasextractedusingRIPAlysisbuffer(Beyo-
timeInc.,China)followingthemanufacturer''sinstructions,then
separatedon12%SDS-polyacrylamideelectrophoresisgelsand
transferredtopolyvinylidene?uoridemembranes.Afterblocking
with5%skimmilk,themembraneswereincubatedwithCD25
antibody(sc-666,1:200,SantaCruz,CA,USA)asaprimaryantibody
andprocessedwithhorseradishperoxidase(HRP)-conjugated
secondaryantibody(SantaCruz).Immuno-reactivitywasdetected
usingECLsolutionandb-actinwasusedasaloadingcontrol.All
experimentsweredoneinduplicate.
2.8.Real-timePCR
Threecorneaspergroupwereexcisedatdays3,7,14,and21,
respectively.TissueRNAwaspreparedwithTrizolreagent(Invi-
trogen,USA)accordingtothemanufacturer''sinstructionsand
cDNAwaspreparedfrom1mgtotalRNAusingreversetranscription.
TargetRNAwasampli?edinthe10mLreactionmixtureusingan
ABI7500real-timePCRsystem(AppliedBiosystems,Germany)and
thecomparativethresholdcyclequanti?cationmethodwasusedto
normalizeGAPDHlevels.Experimentswereperformedinduplicate
ch151(2016)134e141135
witheachsample.CD25PrimerSequenceswere:CD25Forward:5
0
-
Table1
Gradingofclinical?ndingsusedindiagnosisofgraftrejection.
Opacity0:completetransparency
1:minimallossoftransparency
2:moderatelossoftransparency,butirisvesselsvisibleonretroillumination
3:irisvesselsnotvisible,butpupiloutlinevisible
4:pupiloutlinenotvisible
Edema0:noedema
1:moderateedema
2:markededemawithobviousgraftthickening
Neovascularization0:noneovascularizationofgraft
1:vesselgrowthto25%ofgraftradiusinanyquadrant
2:vesselgrowthto50%ofgraftradius
Q.Qinetal./ExperimentalEyeResearch151(2016)134e141136
GAGGAAGAGCAGAAGAAC-3
0
,Reverse:5
0
-GTCTCGGGACTTCATAAC-
3’.
2.9.Flowcytometry
Peripheralbloodfromthreeratspergroupwascollectedfrom
thecontrolandCD25-siRNAgroups,andmononuclearcellswere
separatedusingFicoll-Paquegradientcentrifugationfollowingthe
manufacturer''sinstructions(TianJinHaoYangBiologicalManu-
factureCo.,Ltd,China).CD4
t
Tpopulationsweredeterminedby
?uoresceinisothiocyanate-conjugatedCD4(eBioscience,SanDiego,
CA)andanalyzedusingaFACScan?owcytometer(BDBiosciences).
2.10.Statisticalanalysis
Alldatawereexpressedasthemean±standarddeviation.Graft
survivaltimewasdeterminedbyKaplan-Meieranalysisandlog-
ranktest.Multiplecomparisonsweredetectedbyone-way
ANOVAandpost-hoctesting.DifferencesinCD4
t
Tcellpercent
wereanalyzedbyatwo-tailedMann-WhitneyUtest.Dataevalu-
ationwasperformedusingSPSS11.5software(SPSS,Inc.,Chicago,
Illinois,USA),withaP<0.05consideredstatisticallysigni?cant.
3.Results
3.1.CD25siRNAreducedgraftneovascularizationandmaintained
transparency
Inthecontrolgroupatday3,smallnewvesselssproutedinthe
graftlimbustowardsthesuture,withtransparentcorneas.The
vesselsgraduallyincreasedandtransparencygraduallydecreased.
Atday10,numerousnewvesselsgrewintothecenterofthegrafts
andthecorneasbecamecloudy.Bycontrast,intheCD25siRNA
groupatday10,onlyafewsmallvesselsinthegraftlimbuswere
observed,andthecorneaswereinmildedema.Therewasnosig-
ni?cantdifferenceinthemorphologybetweenthecontroland
scrambledsiRNAgroups(Fig.1).
Fig.1.Representativemicrographsofcornealgraftmorphologyatday10post-operation.At
cloudyincontrolandscrambledsiRNAgroups,whileonlyafewsmallvesselsandslightopacity
3.2.CD25siRNAprolongedgraftsurvival
CD25siRNAtreatmentremarkablyprolongedthesurvivalofrat
allografts(MST,14.8±0.7days)comparedwiththecontrolgroup
(MST,7.6±0.7days,P<0.05)andscrambledsiRNAgroup(MST,
7.8±0.6days,P<0.05).CD25siRNAtreatmentsigni?cantly
reducedgraftopacity,edemaandneovascularizationcompared
withothergroupspost-surgery(Table2,Fig.2).
3.3.CD25siRNAamelioratedin?ammatoryin?ltrationand
neovascularization
Duringtheacuterejectionphase,alargeamountofin?amma-
torycellsandnewvesselswereseeningraftsfromthecontrol
group,mostlyincornealstromawithsevereedema.Thenumberof
in?ammatorycellsgraduallydecreasedbutthenumberofvessels
graduallyincreased.IntheCD25-siRNAgroup,graftsexhibited
fewerin?ammatorycellsandlessedema.Therewasnosigni?cant
differenceinHEmorphologybetweenthecontrolandscrambled
siRNAgroups(Fig.3).
3.4.CD25siRNAdown-regulatesCD25proteinandmRNA
expression
Immuno-histochemicalstainingshowedthatCD25expression
intheCD25siRNAtreatmentgroupwaslowerthaninothergroups
(P<0.05)at14dpost-surgery.A‘ttt’levelofbrownstaining
mainlyincornealepitheliumandcornealstromawasobservedin
thecontrolgroup,whereas,a‘t’levelofbrownstainingwas
observedintheCD25-siRNAgroup,mainlyinthecornealstroma.
Therewasnosigni?cantdifferenceinthelevelofbrownstaining
betweencontrolandscrambledsiRNAgroups.Duringtheacute
graftstage,theexpressionofCD25mRNAgraduallyincreased,
3:vesselgrowthto75%ofgraftradius
4:vesselgrowthtocenterofgraft
peakingat14dpost-surgeryinallgroupsandthendecreased.
However,ateachtime-point,thelevelofCD25mRNAwaslowerin
theCD25-siRNAgroupcomparedwithothergroups(P<0.05).No
signi?cantdifferencewasobservedbetweenthetwocontrol
day10,numerousnewvesselsgrewintothecenterofthegraftsandthecorneaswere
wereobservedintheCD25-siRNAgroup.
survival
eResear
Table2
Ratcornealgraftmeansurvivaltime.
GroupGraft
Controlgroup(group1)8,8,7,7,7,7,8,9,8,7,8,7
scrambledsiRNAgroup(group2)8,7,8,8,8,8,7,7,8,9,8,8
CD25-siRNAgroup(group3)15,16,14,16,15,15,14,15,14,15,14,15
Q.Qinetal./ExperimentalEy
groups.ThechangeinthelevelofCD25proteinexpressionwas
similartothatofCD25mRNAexpression(Fig.4).
At14daftergraftsurgerywhenallgraftswererejected,
immuno-histochemicalstainingrevealedextensiveamountsof
TNF-a,IFN-g,andIL-1binthecornealepitheliumandstromainthe
controlgroups,withbrownstaininglevelsof‘ttt’.LessTNF-a,
IFN-g,andIL-1bwereseeninthecornealstromafromtheCD25-
siRNAgroupwithabrownstaininglevelof‘t’.IL-10andTGF-b
cellswerepresentinthecornealepitheliumoftheCD25-siRNA
groupwithabrownstaininglevelof‘ttt’,withrelativelyfewer
cellsobservedinthecontrolgroupswithabrownstaininglevelof
‘t’.Interestingly,noremarkabledifferenceinFoxp3expressionwas
observedamongthegroups,andbrownstainingwasobservedin
thecornealepitheliumandstroma.Therewasnosigni?cantdif-
ferenceinthecytokineexpressionbetweencontrolandscrambled
siRNAgroups(Fig.5).
ToquantifyCD4
t
Tcellpopulationchangeswithrejection,we
Fig.2.Effectofgenetherapyonallograftssurvival.(a)CD25siRNAtreatmentremarkablyprolong
withthecontrolgroup(Group1,MST?7.6±0.7days,n?12,P<0.05)andscrambledsiRNA
edema,neovascularization,andrejectionindexinGroup3werelowerthaninGroups1and
Fig.3.Histopathologyofcornealgraftsstainedwithhematoxylin-eosinatday14(originalmagni
graftsfromthecontrolandscrambledsiRNAgroup.Fewerin?ammatorycellsandlessedema
[days]Mean±SD
7.6±0.7
7.8±0.6
14.8±0.7
ch151(2016)134e141137
collectedperipheralbloodmononuclearcellsfromcontroland
CD25-siRNAgroupsat3,7,14,and21daftergraftsurgery.The
percentageofCD4
t
Tcellsgraduallyincreasedinallratsfollowed
bytherejectionphaseuntil21daftergraftsurgery.CD4
t
Tcell
populationswerelowerintheCD25-siRNAgroupcomparedwith
thecontrolgroup(P<0.05)atalltime-points.At14daftergrafting
whenallthegraftswererejected,thepercentageofCD4
t
Tcellsin
thecontrolgroupwas65%,butonly48%intheCD25-siRNAgroup
(Fig.6).
4.Discussion
Cornealtransplantationistheonlytreatmentforcorneal
blindness,whichisthesecondleadingcauseofblindness,nextto
cataract(Tanetal.,2012).About400,000patientssufferfrom
cornealblindnessinChina.However,only5000cornealtrans-
plantationsareperformedeachyearduetoalackofdonorcorneas
edthesurvivalofratallografts(group3,MST?14.8±0.7days,n?12)compared
group(Group2,MST?7.8±0.6days,n?12,P<0.05).(b)Themeanscoresofopacity,
2ateachtime-period(P<0.05).
?cation,200C2).Alargeamountofin?ammatorycellsandnewvesselswereseenin
wereobservedintheCD25-siRNAgroup(arrowshowsnewvessels).
Q.Qinetal./ExperimentalEyeResearch151(2016)134e141138
(LiandXie,2011).Thesuccessrateofcornealtransplantationisnot
ashighasonewouldexpectmainlybecauseofgraftrejection
(CosterandWilliams,2005),whichcompoundstheproblemofthe
limitednumberofdonorcorneas.
Inthecurrentstudy,weusedRNAitechnologytosilenceCD25
geneexpressiontostudytheinteractionsbetweenTregandTh1
cytokines.OurresultsshowedthatCD25siRNAtreatmentresulted
insubstantiallyreducingexpressionofCD25for21days,especially
Fig.4.CD25siRNAdown-regulatedCD25proteinandmRNAexpression.(a)Immuno-histochemical
400C2).TheCD25expressionintheCD25-siRNAgroupwaslowerthaninothergroups(P<
groupweredetectedbyWesternblotanalysisatdays3,7,14,and21post-surgery.(c)CD25/
levelsofCD25ingraftsweremeasuredbyRT-PCRatdays3,7,14,and21post-surgery.Signi
(controlgroup),Group2(scrambledsiRNAgroup)andGroup3(CD25-siRNAgroup).
atday3post-surgery.Inourpreviousstudy(Qinetal.,2015),we
foundasecondgenetransferatsevendayspost-operationdidnot
signi?cantlyprolonggraftsurvivalcomparedwithonlyonetreat-
ment.ThisindicatesthatIL-2/CD25signalingmaybeinvolved
primarilyattheearlystageofgraftrejection.Moreover,IL-2/CD25/
mTORmediatedsignalswereessentialforCD4
t
Tcell
differentiation.
ThepercentageofCD4
t
Twassigni?cantlylowerintheCD25-
stainingofCD25wasobservedat14dpost-surgery(originalmagni?cation,
0.05,arrowsindicatedpositivelystainedcells).(b)TheproteinslevelsofCD25ofeach
b-actindensityratiosbydensitometricanalysisofWesternblots.(d)mRNAexpression
?cantlydifferent,P<0.05.Errorbarsarethestandarddeviationofthemean.Group1
eResearQ.Qinetal./ExperimentalEy
siRNAgroupcomparedwiththeothertwogroupsatalltime-points
andCD25wasexpressedmainlyinthecornealepitheliumand
stroma.DespitethereductioninCD25expression,graftrejection
wasinducedbythebandgchainsofIL-2R(Nashanetal.,1995),as
wellastheremainingCD25andothermTOR-dependentcommon
signals.Nonetheless,CD25siRNAtreatmentsigni?cantlyprolonged
graftsurvival,maintainedbettergrafttransparencyandstructure,
mainlyduetotheinhibitionofthein?ammatoryresponseacti-
vatedbyCD4
t
Tcells.
Inthecurrentstudy,allgraftswererejected14dpost-graft
Fig.5.Expressionlevelsofimmunoregulationrelatedcytokinesinthegraftsatday14post-operation.
immunohistochemicalstaining(originalmagni?cation,400C2.Arrowsindicatedpositivelystained
siRNAgroupcomparedwiththeothertwogroups.IL-10andTGF-bexpressionwerehigherin
remarkabledifferenceinFoxp3expressionwasobservedamongthesethreegroups.
ch151(2016)134e141139
surgery.HigherlevelsofIL-10andTGF-bwereobservedafter
CD25siRNAtreatment,butthelevelofFoxp3remainedunchanged
inanyofthegroupsatanytimeaftersurgery.AsthelevelofFoxp3
isassociatedwiththefunctionofTregs,ourresultssuggestedthat
thedown-regulationofCD25expressionhadnoeffectonTregs
activity.TregssuppressedCD4
t
Tcellfunctionbycompetingfor
bindingtoIL-2producedbyothercells,thuspreventingIL-2
bindingtoCD25ontheCD4
t
Tcells.However,CD25onlyactsas
abindingsite,andthebandgchainofIL-2Rareresponsiblefor
signaltransduction.TheexpressionofFoxp3dependsontheIL-2Rb
ThelevelsofFoxp3,IL-10,TGF-b,IL-1b,TNF-aandIFN-gweremeasuredby
cells).LessTNF-a,IFN-g,andIL-1bwereseeninthecornealstromaintheCD25-
thecornealepitheliumoftheCD25-siRNAgroupthanthatoftheothertwogroups.No
eResear
signaling(Burchilletal.,2007).Additionally,thegchainofIL-2Risa
commoncytokine-receptor,whichalsoparticipatesinother
cytokine-mediatedsignalsforthegenerationofTregs(Wangetal.,
2008),suchasTGF-bandIL-10(Zhengetal.,2002).Additionally,
mucosalCD103
t
DCsmayconvertvitaminAintoretinoicasacidas
asubstituteforIL-2andpromptthegenerationofTregsaccom-
paniedbyTGF-b(Coombesetal.,2007).AlthoughCD25expression
wasabsent,ourresultsshowedthatinvivo,itseffectwas
compensatedforbyothercytokines.Weusedonly50mgsiRNAfor
genesilencinganditisunknownwhetherthechangesinTregs
differentiationandfunctionmaybein?uencedbyotherCD25
concentrations.
OurstudyshowedthatCD25siRNAtreatmentcausedthedown-
regulationofpro-in?ammatorycytokinesTNF-a,IFN-g,andIL-1b
14dpost-surgery,apointintimewhenallofthegraftswere
rejected.In?ammationaftercornealtransplantationisacontrib-
utingfactorfortheapoptosisofcornealendothelia(Claerhoutetal.,
Fig.6.Quanti?cationofCD4
t
Tcellinperipheralbloodineachgrouponpostoperative
days3,7,14,and21.CD4
t
Tcellpopulationanalyzedby?owcytometrywaslowerin
theCD25-siRNAgroup(Group3)comparedwiththecontrol(Group1,P<0.05)atall
time-points.
Q.Qinetal./ExperimentalEy140
2008)inducedbyallofthecytokinesmentionedabove(Claerhout
etal.,2004;Dinarello,2009).InCECs,IL-1bstimulationfor10min
signi?cantlyincreasedtheactivityofPI3-kinaseinvitro(Leeetal.,
2004),followingactivationbymTORthatinducedTcelldifferen-
tiation.TNF-apromotedmacrophagefunction,upregulatedmajor
histocompatibilitycomplexclassIIantigenexpressionandstimu-
latedcornealcellapoptosis(Hegdeetal.,2005),thusinducing
rejection(Nishiyamaetal.,2005).IL-1bandTNF-atriggerTh1
responseandinducethesecretionoflargeamountsofIFN-g(Li
etal.,2011).IFN-gisconventionallyapro-in?ammatorycytokine
thatcontributestocornealrejectionanddestruction(Zhengetal.,
2010),up-regulatingtheexpressionofMHCclassIandII,and
intercellularadhesionmolecule1(ICAM-1)(Penninoetal.,2010).In
addition,IFN-ginhibitstheTGF-bsignalingpathwaybyinduction
ofSmad7expressioninhepaticstellatecells(Wengetal.,2007).In
thecurrentstudy,theproductionofTNF-a,IFN-g,andIL-1bwas
down-regulatedat14dpost-operation,bytheinhibitionofthe
transcriptionfactorNF-kBbyTGF-bandIL-10(Kimetal.,2005)and
thedepletionineffectorTcellpopulations(Rosaliaetal.,2013),
thusprotectingcornealgrafts.WhenTregsareunabletoexpress
Foxp3,theymayinduceIFN-gmediatedimmuneresponse
(Radhakrishnanetal.,2008).Nevertheless,IFN-g/TNF-amodelsof
synergismhavebeenshowntoprotectpancreaticb-cellsbyNF-kB
activation(Changetal.,2003)suggestingthatthefunctionofNF-kB
wasamphitropic.Wecouldnotcon?rmifIFN-gorNF-kBwere
bene?cialordetrimentaltocornealgraftsinourcurrentmodels.
Inconclusion,CD25siRNAtreatmentsigni?cantlyprolongedthe
survivaltimeofcornealgraftsviainhibitionoftheproliferationof
CD4
t
Tcells,up-regulationofTregcytokinesanddown-regulation
ofTh1cytokines.However,graftrejectionwasstillinducedby
commonsignals,suchasthosemediatedbymTORandNF-kB.
Therefore,furtherstudiesareneededtoelucidatethemechanisms
ofinteractionamongcytokinesandthedevelopmentofstrategies
topreventcornealrejection.
Acknowledgments
ThisstudywassupportedbyNaturalScienceFoundationof
China(No.81170822andNo.81201771).
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