Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model

21 Pages·2015·6.3 MB·English

RESEARCHARTICLE
Paradoxical Effects of All-Trans-Retinoic Acid
on Lupus-Like Disease in the MRL/lpr Mouse
Model
XiaofengLiao1,JingjingRen1,Cheng-HsinWei2,A.CatharineRoss2,ThomasE.Cecere1,
BernardS.Jortner1,S.AnsarAhmed1,XinM.Luo1*
1 DepartmentofBiomedicalSciencesandPathobiology,CollegeofVeterinaryMedicine,VirginiaTech,
Blacksburg,VA,24061,UnitedStatesofAmerica,2 DepartmentofNutritionalSciences,PennsylvaniaState
University,UniversityPark,PA,16802,UnitedStatesofAmerica
* [email protected]
Abstract
Rolesofall-trans-retinoicacid(tRA),ametaboliteofvitaminA(VA),inbothtolerogenicand
immunogenicresponsesaredocumented.However,howtRAaffectsthedevelopmentof
systemicautoimmunityispoorlyunderstood.HerewedemonstratethattRAhaveparadoxi-
caleffectsonthedevelopmentofautoimmunelupusintheMRL/lprmousemodel.Wead-
ministered,orally,tRAorVAmixedwith10%oftRA(referredtoasVARA)tofemalemice
OPENACCESS startingfrom6weeksofage.Atthisage,themicedonotexhibitovertclinicalsignsoflupus.
Citation:LiaoX,RenJ,WeiC-H,RossAC,Cecere However,theimmunogenicenvironmentprecedingdiseaseonsethasbeenestablishedas
TE,JortnerBS,etal.(2015)ParadoxicalEffectsof evidencedbyanincreaseoftotalIgM/IgGintheplasmaandexpansionoflymphocytesand
All-Trans-RetinoicAcidonLupus-LikeDiseaseinthe
dendriticcellsinsecondarylymphoidorgans.After8weeksoftRA,butnotVARAtreatment,
MRL/lprMouseModel.PLoSONE10(3):e0118176.
significantlyhigherpathologicalscoresintheskin,brainandlungwereobserved.These
doi:10.1371/journal.pone.0118176
wereaccompaniedbyamarkedincreaseinB-cellresponsesthatincludedautoantibody
AcademicEditor:ColetteKanellopoulos-Langevin,
productionandenhancedexpressionofplasmacell-promotingcytokines.Paradoxically,
XavierBichatMedicalSchool,INSERM-CNRS-
UniversitéParisDiderot,FRANCE thenumberoflymphocytesinthemesentericlymphnodedecreasedwithtRAthatledtosig-
nificantlyreducedlymphadenopathy.Inaddition,tRAdifferentiallyaffectedrenalpathology,
Received:July12,2014
increasingleukocyteinfiltrationofrenaltubulointerstitiumwhilerestoringthesizeofglomer-
Accepted:January5,2015
uliinthekidneycortex.Incontrast,minimalinductionofinflammationwithtRAintheab-
Published:March16,2015
senceofanimmunogenicenvironmentinthecontrolmicewasobserved.Altogether,our
Copyright:©2015Liaoetal.Thisisanopenaccess resultssuggestthatunderapredisposedimmunogenicenvironmentinautoimmunelupus,
articledistributedunderthetermsoftheCreative
tRAmaydecreaseinflammationinsomeorganswhilegeneratingmoreseveredisease
CommonsAttributionLicense,whichpermits
inothers.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Introduction
Funding:Theauthorsreceivednospecificfunding
forthiswork. VitaminAplaysanimportantroleinthedevelopmentofabalancedimmunesystem[1,2].
All-trans-retinoicacid(tRA),apredominantvitaminAmetabolite,exertsmostofthefunc-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. tionsattributedtovitaminA[3].RecentstudiesofintestinalmucosahaveshownthattRA
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 1/21
RetinoicAcidParadoxicallyAffectsLupus
secretedbygut-specificCD103+dendriticcellscanmodulatetheThelper(Th)17-regulatoryT
cell(Treg)balance[4–6].tRAhasalsobeenshowntoinducegut-tropic,IgA-producingBcells
[7].Systemically,tRAisknowntoregulateTh1-Th2balance[8,9]andincreaseantigen-specif-
icantibodyresponsebypromotingtheactivationandthedifferentiationofBcellsintoplasma
cells[10–12].Morerecently,tRAhasbeenshowntobeessentialforthedifferentiationofcon-
ventionaldendriticcells[13].TheseevidencesimplythattRAmayaffectautoimmunitybut
whetherandhowtRA,orvitaminAingeneral,maydosoisnotclearlyunderstood.
Systemiclupuserythematosus(SLE)isanautoimmunediseasewithpersistentinflamma-
tionthatdamagesmultipleorgansincludingkidney,skin,lung,heart,jointsandbrain[14].A
majorityofpatientsarewomenofchildbearingage[14,15].SLEisinitiatedbyabreachof
immunotolerancetoself,whichpromotesthegenerationofhighaffinityautoantibodiespri-
marilyagainstnuclearcomponentsandphospholipids[16,17].Theautoantibodiesrecognize
andbindselfantigens,formingimmunecomplexes(IC)thatdepositintheperipheraltissues.
ThecomplementsystemissubsequentlyactivatedbyICinsituandinducesinflammation,
whichamplifiesitselfbyrecruitinginflammatoryleukocytes[18,19].Commonly-useddrugs
forthetreatmentofSLEincludenonsteroidalanti-inflammatorydrugs,antimalarialmedicine,
glucocorticoids,andimmunosuppressivedrugs[14].Recently,severalantibodyproductsspe-
cificallyperturbingautoimmunereactionshavebeendevelopedtoreplacethetraditional,more
toxicchemicalagents[14,20,21].However,theymaycompromisethenormalimmunere-
sponsetoinfections[22,23].Therefore,thereisaneedfornew,morenaturalinterventions
withminimalsideeffects.
AbeneficialeffectoftRA,aloneorincombinationwithlow-doseimmunosuppressive
drugs,onlupusnephritishasbeenreportedinbothmousemodelsandSLEpatients[24–28].
However,SLEisasystemicautoimmunediseaseinvolvingmanyotherorgansbesidesthekid-
ney.EvidenceislackingonhowtRAaffectsotherSLE-manifestedorgans,suchasthebrain
andlung.WehereindemonstratethecomplexeffectsoftRAondifferentperipheraltissues
usingtheclassicallupus-proneMRL/lprmousemodel.
MaterialsandMethods
Ethicsstatement
ThisstudywascarriedoutinstrictaccordancewiththerecommendationsintheGuideforthe
CareandUseofLaboratoryAnimalsoftheNationalInstitutesofHealth.Theprotocolwasap-
provedbytheInstitutionalAnimalCareandUseCommittee(IACUC)ofVirginiaTechCol-
legeofVeterinaryMedicine(AnimalWelfareAssuranceNumber:A3208-01).Allanimal
experimentswereconductedunderIACUCprotocol#12-062.Foranesthesiaandeuthanasia,
isofluraneandCO wereused,respectively,accordingtotheIACUCprotocol.
2
MiceandvitaminAadministration
MRL/Mp(MRL)andMRL/Mp-Faslpr(MRL/lpr)micewerepurchasedfromTheJacksonLabo-
ratory(BarHarbor,ME),andbredandmaintainedinaspecificpathogen-freefacilityfollowing
therequirementsofInstitutionalAnimalCareandUseCommittee(IACUC)atVirginiaPoly-
technicInstituteandStateUniversity.All-trans-retinoicacid(tRA)andall-trans-retinylpalmi-
tate(RP)werepurchasedfromSigma(St.Louis,MO),andpreparedandusedinthedarkto
avoidexposuretolight.Bothretinoidsweredissolvedincanolaoil(vehicle)andadministered
orally(directlyintothemouth)tofemalemicefrom6till14weeksofage.FortRAtreatment,
6mgtRA/kgbodyweight(BW)wasusedperday.Thisdosewasreducedfromthereported
doseof10mgtRA/kgBW[27]thatledtoskinlesionsinMRL/lprmiceinourpilotstudy,
whichcouldaffectouranalysisoftheskin.FordailyVARAtreatment,11.2mgRP/kgBW
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 2/21
RetinoicAcidParadoxicallyAffectsLupus
(equivalentto6mgretinol/kgBW)wasmixedwith0.6mgtRA/kgBW(10%oftheamountof
retinol)beforebeinggiventothemice.Micewereweighedtwiceweeklyandtheretinoiddoses
wereadjustedaccordingly.
Leukocyteisolationandflowcytometry
Forbonemarrowcells,bonesfrombothhindlimbsofeachmousewerecrackedgentlyina
mortarcontainingphosphatebufferedsaline(PBS)byusingapestle.Bonemarrowwasre-
leasedbygentlestirringaftertheadditionofC10medium(RPMI1640,10%fetalbovine
serum,1mMsodiumpyruvate,1%100MEMnon-essentialaminoacids,10mMHEPES,55μM
2-mercaptoethanol,2mML-glutamine,100U/mlpenicillin-streptomycin,allfromLifeTech-
nologies,GrandIsland,NY).Thesuspensionwasclearedbypassingthrougha70-μmsterilecell
strainerandcarefullylayeredonthetopofFicoll-PaquePlus(GEHealthcare,Pittsburg,PA).
Aftercentrifugationat1,363×gfor30minatroomtemperature,mononuclearcellsinthebuffy
coatlayerwerecollected.Spleenandalllymphnodesinthemesentericregion(MLN)werecol-
lectedandmashedin70-μmcellstrainerswithC10.Forsplenocytes,redbloodcellswerelysed
withRBClysisbuffer(eBioscience,SanDiego,CA).Forsurfacemarkerstaining,cellswere
blockedbyanti-mouseCD16/32(eBioscience),stainedwithfluorochrome-conjugatedantibod-
ies,andanalyzedwithBDFACSAriaIIflowcytometer(BDBiosciences,SanJose,CA).Forintra-
cellularstaining,Foxp3Fixation/Permeabilizationkit(eBioscience)wasused.Anti-mouse
antibodiesusedinthisstudyinclude:B220-FITC,CD3-FITC,I-E/I-A-FITC,CD25-AlexaFluor
488,CD11c-PE,CD19-PerCP-Cy5.5,CD4-PerCP-Cy5.5,ratIgG2a-APC,CD40-APC,and
Foxp3-PE-Cy7(eBioscience);CD27-PE,Siglec-H-PerCP-Cy5.5,CD138-APC,CD44-APC-Cy7,
andCD62L-BV510(Biolegend,SanDiego,CA);mouseIgG2a-PE,I-Ek-PE,CD11c-PE-Cy7,
Ly6C-PE-Cy7,CD11b-APC-Cy7,CD8a-V450,I-A/I-E-V500,andB220-V500(BDBiosciences);
CD3e-APC(MiltenyiBiotec,Auburn,CA).FlowcytometrydatawereanalyzedwithFlowJo.
Enzyme-linkedimmunosorbentassay(ELISA)
Bloodwascollectedintoanticoagulant-coatedCapijecttubes(TerumoMedical,Somerset,NJ)
andcentrifugedat15,000×gfor30sec.Plasmawascollectedandstoredat-80°C.Fordetection
ofanti-double-strandedDNA(dsDNA)IgG,theplatewascoatedwith0.1mg/mlofcalfDNA
(Sigma)in1saline-sodiumcitrate(SSC)bufferat4°Covernight,followedbywashingwithPBS
containing0.05%Tween-20(PBS-T).WellswerethenblockedwithPBScontaining1%bovine
serumalbumin(BSA)for1hatroomtemperatureandwashed.Sampleswereaddedandincu-
batedfor1hatroomtemperature.Afteradditionalwashing,HRP-conjugatedgoatanti-mouse
IgG-Fcsecondaryantibody(BethylLaboratories,Montgomery,TX)wasaddedandincubated
0 0
for1hatroomtemperature,followingbymorewasheswithPBS-T.3,3,5,5-Tetramethylben-
zidine(TMB)substrate(Biolegend)wasthenadded.Afterthereactionwasstopped,theplate
wasreadat450nmwithSpectraMaxplatereader(MolecularDevices,Sunnyvale,CA).Total
IgGandIgMconcentrationsweredeterminedwithmouseIgGandIgMELISAkitsaccording
tothemanufacturer’sinstructions(BethylLaboratories).
Histology
Allfixedtissueswereparaffin-embedded,sectioned,andstainedforHematoxylinandEosin
(H&E)orPeriodicAcid-Schiff(PAS)attheHistopathologyLaboratoryatVirginia-Maryland
RegionalCollegeofVeterinaryMedicine.Afterimmersion-fixationin10%neutralbuffered
formalin,brainsweresectionedinthetransverseplaneatlevelsofthefollowingstructures:ol-
factorybulb,headofcaudatenucleus,rostrallevelofhippocampus,caudallevelofhippocam-
pus,midlevelofcerebellumwithunderlyingmedullaoblongata,caudallevelofcerebellum
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 3/21
RetinoicAcidParadoxicallyAffectsLupus
withunderlyingmedullaoblongata.Inaddition,longitudinalsectionsofthetrigeminalgangli-
onandadjacentnervewerealsoobtained.BrainslideswerereadwithNikonECLIPSECi-Lmi-
croscopeandpicturesweretakenbyusingNIS-ElementsD3.264-bitsoftwareunder20×
objectivelense(NikonPlan20×/0.40,OFN22WD1.2)atroomtemperature.Inflammatoryle-
sionsweregradedas0(nolesions),1,2or3(increasingseverityoflesions).Allbrainslides
werescoredbyaboardcertifiedveterinaryneuropathologist(Jortner)inablindedfashion.
Kidneyswerefixedinformalinimmediatelyafterisolation,whilelungtissueswereinflated
withformalinthroughthetracheabeforesubmergedinformalin.Lungandkidneyslideswere
readwithOlympusBX43microscopeandpicturesweretakenbyusingOlympuscellSenssoft-
ware.Lunglesionswerescoredsemiquantitatively(0–4)basedontheextentofperibronchial,
perivascular,orinterstitiallymphocyticinfiltrationaspreviouslydescribed[29].Glomerularle-
sionsweregradedonascaleof0–3forincreasedcellularity,increasedmesangialmatrix,necro-
sis,percentageofscleroticglomeruli,andpresenceofcrescents[27].Similarly,
tubulointerstitiallesionsweregradedonascaleof0–3forinterstitialmononuclearinfiltration,
tubulardamage,interstitialfibrosis,andvasculitis.Slideswerescoredbyaboardcertifiedveter-
inarypathologist(Cecere)inablindedfashion.
Immunohistochemistry
KidneyswereembeddedinTissue-TekO.C.T.Compound(SakuraFinetek,Torrance,CA)and
rapidlyfrozeninafreezingbathofdryiceand2-methylbutane.FrozenOCTsampleswere
cryosectionedandunstainedslideswerestoredat-80°C.Frozenslideswerewarmedtoroom
temperatureandletdryfor30min,followedbyfixationin-20°Ccoldacetoneatroomtemper-
aturefor10min.AfterwashinginPBS,slideswereblockedwithPBScontaining1%BSAand
anti-mouseCD16/32for20minatroomtemperature.Slideswerethenincubatedwithfluoro-
chrome-conjugatedantibodymixturefor1hatroomtemperatureinadarkhumidbox.Slides
weremountedwithProlongGoldcontainingDAPI(LifeTechnologies).Thefollowinganti-
mouseantibodieswereusedinimmunohistochemicalanalysis:complementC3-PE(Cedar-
lane,Burlington,NC);IgG-FITC(Sigma);CD11c-PE,CD3e-FITC(eBioscience);andCD138-
APC(Biolegend).Slidesstainedwithanti-complementC3andanti-IgGwerereadandpictured
withEVOSFLmicroscope(AdvancedMicroscopyGroup,GrandIsland,NY)anda20×objec-
tive.SlidesstainedwithantibodiesagainstCD11c,CD3eandCD138werereadandpictured
withBX51uprightOlympusmicroscope(Olympus,CenterValley,PA),a20×objectiveand
StereoInvestigatorsoftware(MBFBioscience,Williston,VT).
Reversetranscription-quantitativepolymerasechainreaction(RT-
qPCR)
SpleenandMLNwerehomogenizedwithBulletBlenderhomogenizer(NextAdvance,Averill
Park,NY)andtotalRNAwasextractedwithRNeasyPlusMiniKit(Qiagen,Valencia,CA)ac-
cordingtothemanufacturers’instructions.GenomicDNAwasremovedbydigestionwith
RNase-freeDNaseI(Qiagen).ReversetranscriptionwasperformedbyusingiScriptcDNA
SynthesisKit(Bio-Rad,Hercules,CA).QuantitativePCRwasperformedwithiTaqUniversal
SYBRGreenSupermix(Bio-Rad)andABI7500FastReal-TimePCRSystem(AppliedBiosys-
tems,GrandIsland,NY).RelativequantitieswerecalculatedusingL32asthehousekeeping
gene.PrimersequencesformouseIL-6,IL-21,IFN,andL32canbefoundinS1Primer
Sequences.
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 4/21
RetinoicAcidParadoxicallyAffectsLupus
Othermeasurements
ProteinuriawasmeasuredweeklywithChemstrip2GP(Roche,Indianapolis,IN).Ascaleof
0–4wasusedthatcorrespondedtonegative,trace(5–20mg/dL),30mg/dL,100mg/dL,and
500mg/dLtotalprotein,respectively.Dermatitisonthebackoftheneckand/orfaceofthe
micewasobservedandrecordedinablindedfashion.Totalretinolfromliversampleswas
quantifiedbyUltraPerformanceLiquidChromatography(UPLC)afterextractionandsaponi-
fication.Briefly,portionsofeachsample(around0.05g)weresaponifiedin5%potassiumhy-
droxide,1%pyrogalloland98%ethanol,at55°C.Afterextractionintohexanesandphase
separationwithwater,analiquotoftheupperphaselipidextractwasmixedwithaknown
amountofinternalstandard,trimethylmethoxyphenyl-retinol(providedbyM.Klaus,Hoff-
mann-LaRoche,Basel,Switzerland).Samplesweredriedundernitrogenandreconstitutedin
methanolforUPLCanalysisusingaC-18reversed-phasecolumnandmobilephaseof92.5%
methanoland7.5%waterataflowrateof0.6ml/minwithmonitoringat325nm.Theliver
totalretinolconcentrationswerecalculatedbasedonareasofthepeaksfortrimethylmethoxy-
phenyl-retinol(knownamount)andtotalretinol.
Statisticalanalysis
Forthecomparisonoftwogroups,unpairedstudent’st-testwasused.Forthecomparisonof
morethantwogroups,one-wayANOVAandTukey’spost-testwereused.Resultswerecon-
sideredstatisticallysignificantwhenP<0.05.Insomeexperiments,Spearmancorrelationtest
andGrubbs’testforidentificationofoutlierswereused.Allanalyseswereperformedwith
Prismsoftware.
Results
IdentifyinganappropriateageoffemaleMRL/lprmiceforintervention
Dependingontheimmunologicalstate,tRAcanpromoteeitherimmunogenicortolerogenic
immuneresponses[2].Itisimmunosuppressiveundersteadystate[30–32].However,under
aninflammatoryenvironment,evidencehasshownthattRAcanbeimmunogenicanddeterio-
ratepre-existinginflammation[4,33,34].AlthoughabeneficialeffectoftRAonlupusnephri-
tishasbeenreported[24–27],whetheritwouldbeofbenefitordetriment,systemically,toSLE
patientswithearlystagesoflupuswhereinflammationhasinitiatedbutclinicalsignsaremini-
malisunclear.Tofindanappropriateexperimentalmodeltomimicthesepatients,wefirstas-
sessedtheimmunologicalenvironmentinyoung,femaleMRL/lprmicethatwerereaching
sexualmaturity(i.e.,around6weeksold[35]).Wefoundthat,unlike9-and17-week-oldmice,
6-week-oldMRL/lprmicehadacomparablelevelofanti-dsDNAIgGintheplasmaasage-
matchedMRLcontrols(Fig.1A).Nokidneypathologywasobserved,suggestingminimalclini-
calsignsoflupusatthisage[27,36,37].However,lymphoproliferationhadalreadyinitiatedas
evidencedbyhigherlevelsoftotalIgGandIgMintheplasmaof6-week-oldMRL/lprmice
thanthecontrols(Fig.1B)andaccumulationsofBcellsanddouble-negative(DN)Tinthe
spleenandMLNoflupus-pronemice(Fig.1C).Thesetwocelltypescancontributetolupus
pathogenesisbyproducingautoantibodiesandtheproinflammatorycytokineIL-17,respec-
tively[38–42].Dendriticcells,recentlyshowntobeastrongmediatorinlupusdevelopment
[43,44],werealsoinvestigated.Wefoundthatsignificantlymoreplasmacytoiddendriticcells
(pDCs)werepresentinthebonemarrowandMLNof6-week-oldMRL/lprmicethanage-
matchedMRLcontrols(Fig.1D).Inaddition,althoughthenumberofpDCsinthespleendid
notdiffer,thepercentageofsplenicpDCsthatwereMHC-II+CD40+orMHC-IIhighwashigher
inlupus-pronemice(Fig.1E),suggestingtheiractivation[45,46].Moreover,inbothspleen
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 5/21
RetinoicAcidParadoxicallyAffectsLupus
Fig1.Immunogenicenvironmentin6-week-oldMRL/lprmice.(A)Anti-dsDNAIgGintheplasmaof6-week-,9-week-and17-week-oldMRLandMRL/lpr
miceasdetectedbyELISA.Datashownasrelativelevels(RL)werenormalizedtotheaverageabsorbancevalueofMRLmiceatthesameage,whichwas
definedas1.(B)TotalIgGandIgMconcentrationsintheplasmaof6-week-oldmiceasdetectedbyELISA.(C-I)ThepercentagesandabsolutenumbersofT,B,
anddendriticcellsinthespleen,mesentericlymphnode(MLN),andbonemarrow(BM)of6-week-oldmiceasdeterminedbyflowcytometry.(C,upperplots)
ThepercentagesofBcells(CD19+)andCD4-CD8-Tcells(DNTcells,pre-gatedonCD3+)intheMLN.(C,lowerplots)Theabsolutecellnumbersand
percentagesofDNTcellsinthespleen.(D)pDCs(definedasCD11c+CD11b-B220+Siglec-H+)intheBMandMLN.(E)Theabsolutenumbersandpercentages
ofactivatedpDCs(MHC-II+CD40+orMHC-IIhighpDCs)inthespleen.RepresentativeflowcytometryplotsofMRLandMRL/lprareshown.(FandG)The
absolutecellnumbersandpercentagesofCD11b-cDCs(CD11c+CD11b-B220-Siglec-H-MHC-II+)andCD11b+cDCs(CD11c+CD11b+B220-MHC-II+)inthe
spleenandMLN.(H)PercentagesofLy6C+cellsinCD11b+cDCsinthespleenandMLN.(I)ThepercentageofCD40+cellsinCD11b+cDCsinthespleenand
thepercentageofMHC-IIhighcellsinLy6C+CD11b+cDCsintheMLN.RepresentativeflowcytometryplotsofMRLandMRL/lprareshown.ns:notsignificant,*
P<0.05,**P<0.01,***P<0.001,student’st-test.Dataareshownasmean+standarderrorofthemean(SEM),n=3miceineachgroup.
doi:10.1371/journal.pone.0118176.g001
andMLN,accumulationofCD11b-(Fig.1F)andCD11b+(Fig.1G)conventionaldendritic
cells(cDCs)wasobservedfor6-week-oldfemaleMRL/lprmice.MostaccumulatedCD11b+
cDCswereLy6C+(Fig.1H),suggestingthattheymayhavederivedfrommonocytes[47,48].
LikepDCs,CD11b+cDCsinMRL/lprmiceappeartobeactivatedbasedonupregulatedCD40
andMHC-IIexpression(Fig.1I).Takentogether,theseresultssuggestthatanimmunogenic
environmenthasbeenestablishedin6-week-oldfemaleMRL/lprmicealbeitthelackofovert
clinicalsignsoflupus.Therefore,wedecidedtoinvestigatetheeffectsoftRAonthesemicethat
mimicpatientswithearly-stagelupus.
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 6/21
RetinoicAcidParadoxicallyAffectsLupus
Inflammationoftheskin,brainandlungwithtRA
ToevaluatetheeffectoftRAonlupuspathogenesis,wetreated6-week-old,femaleMRL/lpr
mice,orallyanddaily,withvehicle,tRAorVARAtill14weeksofage.ComparedtotRAalone,
VARAcontainedbothtRAandretinylpalmitate,thelatterbeingaprimaryingredientinvita-
minAsupplementsandthusmoreclinicallyrelevantthantRA.MRLmicetreatedwithvehicle
wereusedasthecontrol.RetinolanalysisshowedthatvitaminAaccumulatedintheliverof
VARA-treatedmice(S1AFig.).BecausetRAisametaboliteofretinol[49–51],tRA-treated
micedidnothaveretinolaccumulationinliver.Resultsofiverfunctiontests(S1BFig.)and
bodyweight(S1CFig.)werenotdifferentamongMRL/lprgroups,suggestingthattheadminis-
tereddosesoftRAandVARAwerenottoxictothemice.
tRAofcomparabledosesandwithsimilartreatmenttimeframewaspreviouslyreportedto
improverenalpathologyinthesamemousemodel[27].However,wenotedthattRAincreased
serumconcentrationsoftotalantibodiesandautoantibodiesinthereportedstudy,andwon-
derediftheantibodieswouldaffectthemiceatthesystemiclevel,asSLEisasystemicautoim-
munedisease.Strikingly,wefoundthattRAworsenedlupus-likediseaseintissuesnot
investigatedinthepreviousstudy,includingskin,brainandlung.Thepercentageofmicewith
dermatitis(S1DFig.),andtheleukocyteinfiltrationscoresofthebrain(Fig.2Aand2C)and
lung(Fig.2Band2D)increasedsignificantlywithtRAtreatmentcomparedtoMRL/lprmice
treatedwithvehicle.Forthebrain,lesionsweremostprofoundinthetelachoroideaofthe3rd
ventricleandadjacentleptomeninges,andinchoroidplexusofthe4thventricleincludingcho-
roidinthelateralrecessesandadjacentleptomeninges.Forthelung,althoughnormalperi-
bronchiallymphoidaggregateswerepresentintheMRLcontrolmice,allthreeMRL/lpr
groupsexhibitedincreasedseverityofperibronchiallymphoidinfiltrationcharacterizedby
mixedsmallandlargelymphocytesandfewMottcells.Perivascularandinterstitialinfiltrates
oflymphoidcellswerealsoobservedinthelungsofthesemice.Incontrast,theeffectofVARA
onthebrainandlungofMRL/lprmicewascomparabletothevehiclecontrol(Fig.2Cand2D).
Fig2.tRA-inducedpathologyinthebrainandlung.Startingfrom6weeksofage,MRLandMRL/lprmiceweregiven,orallyanddaily,vehicle(canolaoil),
tRA(6mg/kgBW),orVARA(6mgretinoland0.6mgtRAperkgBW)till14weeksoldwhentissueswerecollected.n=6miceineachgroup.(A)H&Estains
ofthebrain.Representativemicrographsareshown.Barequals100m.(B)H&Estainsofthelung.Representativemicrographsareshown.Barequals50
μm.Arrowsindicateareasofinfiltration.(C-D)Leukocyteinfiltrationscoresofthebrain(C)andlung(D)accordingtoH&Estains.*P<0.05,**P<0.01,
***P<0.001,one-wayANOVA.Dataareshownasmean+SEM.
doi:10.1371/journal.pone.0118176.g002
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 7/21
RetinoicAcidParadoxicallyAffectsLupus
TheseresultssuggestthattRAcanincreaseinflammationinlupus-affectedtissuesotherthan
thekidney.
DifferentialeffectsoftRAonglomeruliandtubulointerstitiumofthe
kidney
NephritisisoneofthemostcommonsymptomsinSLEthatcancausedeath[52].Although
theeffectoftRAonglomerularpathologyhadbeenreportedinlupus-pronemice[27],itsef-
fectontubulointerstitiumofthekidneywasunclear.Wefoundaslight,albeitstatisticallynon-
significant,increaseofleukocytesinfiltratingthetubulointerstitialregionofthekidneyintRA-
treatedMRL/lprmicecomparedtolupus-pronemicetreatedwithvehicle(Fig.3Aand3B).
Theseinterstitiallesionscontainedmultiplecoalescingperivascularandperitubularinfiltrates
ofmononuclearcellsandlownumbersofMottcells.Cellularinfiltrationwasmostseverein
themedullabutextendedintothecortex.Inaddition,immunohistochemicalstainingshowed
theaccumulationofalargenumberofTcellsanddendriticcells,andtoalesserextent,ofplas-
macells,intheinterstitium(Fig.3C).Incontrast,althoughnotstatisticallysignificant,lessse-
vereglomerulonephritiswasobservedwithtRAtreatmentcomparedtovehicle-treatedMRL/
lprmice(Fig.3D),whichwasaccompaniedbyaslightdecreaseinproteinuria(Fig.3E).Inad-
dition,thesizeofglomerulithatwasenlargedinMRL/lprmice(ascomparedtoMRL)de-
creasedwithtRAtreatment(Fig.3Fand3G),consistentwithpriorreports[25,27].IgGand
complementC3levelsintheglomerularregionwerecomparableamongthethreeMRL/lpr
mousegroups(Fig.3H).Takentogether,theseresultssuggestthattheeffectoftRAonthekid-
neymayberegion-specific.Itincreasesleukocyteinfiltrationinthetubulointerstitiumwhilere-
storingthesizeofglomeruliintherenalcortex.TreatmentwithVARAdidnotaffect
kidneypathology.
ReducedlymphadenopathywithtRA
MRL/lprmicehadsignificantlylargerlymphnodesinthemesentericregionthanMRLmice
(Fig.4Aand[53]).WefoundthattreatmentwithtRAsignificantlydecreasedthesizeofMLN
inMRL/lprmice(Fig.4A),consistentwithapriorreport[27].Aslightdecreaseinspleensize
wasalsoobservedbutnotstatisticallysignificant(S2AFig.).Closeranalysisrevealedthatthe
changeofMLNsizewasduetodecreasednumbersofTcellsandBcells,whichtogetherrepre-
sentednearlyallmononuclearcellsintheMLN(Fig.4B).VARAexertedsimilar,butlessobvi-
ous,effects.Interestingly,theproportionsofdifferentT-cellsubsets,includingCD4+,CD8+,
DNTcells(S2BFig.)andnaïve,centralmemory,effectormemoryTcells(S2CFig.),didnot
changewithtRAorVARAtreatment.BecausetRAiscriticalforthegenerationofgut-tropic
Tregsthatcansuppressintestinalinflammation[31],weexaminedwhetherachangedpropor-
tionofTregsinthesecondarylymphoidorgansaccompaniedtRA-mediatedreductionof
lymphadenopathy.ItwasfoundthattRAdidnotchangethepercentageofTregsinthespleen
orMLN(S2DFig.).Thiscouldbeduetothepresenceoftransforminggrowthfactor(TGF)β,a
cytokineknowntobeatanincreasedlevelinMRL/lprmice[54],thatwasrecentlyshownto
suppresstRA-mediatedexpansionofTregsfromperipheralbloodCD4+Tcellsisolatedfrom
SLEpatients[55].Inaddition,thepercentageofactivatedBcellsintheMLNwasnotaffected
bytRA,either(seebelowinFig.5B).TheseresultssuggestthattRAmighthavedecreasedthe
numberoflymphocytesintheMLNwithoutchangingtheircompositionoractivation.One
possibleexplanationforthisphenomenonmaybeincreasedtraffickingoflymphocytesfrom
MLNtononlymphoidorgans,suchasthebrainorlung.Dendriticcells,whichrepresented
<1%oftotalcellsintheMLNof14-week-oldMRL/lprmice,werenotaffectedbyeithertRA
orVARAtreatment(S2EFig.).
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 8/21
RetinoicAcidParadoxicallyAffectsLupus
Fig3.tRA-mediatedmodulationofkidneypathology.(A-C)Leukocyteinfiltrationofthetubulointerstitialregion.(A)Cellinfiltration(CI)scoresaccording
toH&Estains.(B)RepresentativemicrographsofH&Estainsofthetubulointerstitialregion.Barequals100μm.Areasofinfiltrationareindicatedbyarrows.
(C)ImmunohistochemicalstainsofTcells(CD3-blue),dendriticcells(CD11c-red),andplasmacells(CD138-green).Representativeimagesareshown.Bar
equals100μm.(D-H)Glomerularanalysis(GA).(D)AverageGAscoresofhypercellularity,mesangialmatrixexpansion,necrosis,percentageofsclerotic
glomeruli,andglomerularcrescents.(E)Analysisofproteinuria.TheleveloftotalproteinintheurinewasmeasuredweeklywithChemstrip2GP.Thedataof
6-to8-week-oldtimepointswerecombinedastheearlystage,andthoseof12-to14-week-oldtimepointswerecombinedasthelatestage.(F)
RepresentativeH&Estainsshowingkidneyglomeruli.Barequals60μm.(G)PASstainsshowingkidneyglomeruli.Barequals40μm.(H)
ImmunohistochemicalstainsofIgG(green)andcomplementC3(red)depositioninthekidneycortex.Barequals200μm.ns:notsignificant,*P<0.05,
**P<0.01,***P<0.001,one-wayANOVA.Dataareshownasmean+SEM(n=6miceineachgroup).
doi:10.1371/journal.pone.0118176.g003
IncreasedcirculatingautoantibodieswithtRAandVARA
WenextinvestigatedthehumoralimmuneresponsethatisthecauseoftypeIIIhypersensitivi-
tyinSLEdisease[56].Bothanti-dsDNAandtotalIgGincreasedsignificantlyinthetRA-and
VARA-treatedgroupscomparedtothevehiclecontrolinMRL/lprmice(Fig.5A).Interesting-
ly,theratioofanti-dsDNAIgGtototalIgGdidnotchange,suggestingthattRAandVARAaf-
fectedantibodyresponseingeneralanddidnotspecificallytargetautoantibodiesfor
expansion.Inaddition,whileneithertRAnorVARAaffectedthenumbersofantibody-secret-
ingBcellsintheMLNandbonemarrow,thenumberofplasmacellsinthespleenofVARA-
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 9/21
RetinoicAcidParadoxicallyAffectsLupus
Fig4.tRA-mediateddecreaseoflymphocyteaccumulationintheMLN.(A)MLNweight,MLN/body
weightratio,andtotalnumberofcellsinMLN.(B)AbsolutenumbersofTandBcellsintheMLN.*P<0.05,
**P<0.01,***P<0.001,one-wayANOVA.Dataareshownasmean+SEM(n=6miceineachgroup).
doi:10.1371/journal.pone.0118176.g004
treatedmicewassignificantlygreaterthanthatofvehicle-treatedMRL/lprmice(Fig.5B).
Moreover,tRAandVARArespectivelyincreasethemRNAlevelsofIL-21andIL-6inthe
spleen(Fig.5C).However,anothercytokineknowntopromoteplasmacellformation,IFN,
waslowerinMRL/lprthanMRLmiceanddidnotchangewithretinoidtreatments.Thiswas
notentirelysurprisingbecauseIFNαproductionhadbeenshowntodiminishinoldmicewith
latestageoflupusdisease[57].IntheMLN,tRAsignificantlyincreasedIL-6andIL-21mRNA
perunitweightbutnotonthetissuelevel(S3AFig.)duetosignificantlysmallerMLNwith
tRAtreatment(Fig.4A).TheseresultsindicatethattRAandVARAincreasedcirculatingauto-
antibodiesinMRL/lprmice,andtheymightdosothroughenhancingIL-6/IL-21production
and/orinducingplasmacelldifferentiationinlymphoidtissues.
Todeterminewhethertheincreaseincirculatingautoantibodiescorrelatedwithincreased
pathologicalscoresforthebrain,lungandkidney,weperformedSpearmancorrelationanalysis
(Fig.6).Intheanalysis,weexcludedoneoutlieridentifiedbyGrubbs’test(S3BFig.).Itwas
foundthatamongthe3organs,thebrainpathologyscorehadthestrongestcorrelationwith
autoantibodyaccumulationintheblood(R2=0.53,P<0.001).Thelunghadaweakercorrela-
tion(R2=0.42,P<0.01),whereasnocorrelationwasobservedforthekidney.Thelackofcor-
relationbetweenkidneypathologyandcirculatingautoantibodiesisconsistentwithrecent
recognitionthatpathogenicIgGinthekidneyarelikelyproducedinsituratherthanfromthe
circulation[58–60].Theseresultsandanalysesindicatethatcirculatinganti-dsDNAantibodies
aredetrimentaltothebrainandlunginlupus-pronemice.Itisunclear,however,whyVARA
increasedautoantibodylevelsinthecirculationwithoutaffectingbrainorlungpathology
(Fig.2).ItispossiblethattheretinolcomponentoftheVARAformulationexertedcertainpro-
tectiveeffectsintheseorgans.Thiswillbeinvestigatedinthefuture.
PLOSONE|DOI:10.1371/journal.pone.0118176 March16,2015 10/21