Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice

12 Pages·2014·3.37 MB·English

by  F Ewald

OPEN Citation:CellDeathandDisease(2014)5,e1168;doi:10.1038/cddis.2014.138
&2014MacmillanPublishersLimited Allrightsreserved 2041-4889/14
www.nature.com/cddis
Constitutive expression of murine c-FLIP causes
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autoimmunity in aged mice
FEwald1,2,MAnnemann1,2,MCPils3,CPlaza-Sirvent1,2,FNeff4,CErck5,DReinhold1andISchmitz*,1,2
Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this
pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-
mediatedapoptosis.Theonlyshortmurinec-FLIPsplicevariantisc-FLIP (c-FLIP ).Toinvestigatethefunctionalroleof
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c-FLIP in the immune system, we used the vavFLIP mouse model constitutively expressing murine c-FLIP in all
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hematopoieticcompartments.LymphocytesfromthesemiceareprotectedagainstCD95-mediatedapoptosisandactivation-
induced cell death. Young vavFLIP mice display normal lymphocyte compartments, but the lymphocyte populations alter
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withage.WeidentifiedreducedlevelsofTcellsandslightlyhigherlevelsofBcellsin1-year-oldvavFLIP micecomparedwith
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wild-type(WT)littermates.Moreover,bothBandTcellsfromagedvavFLIP animalsshowactivatedphenotypes.Serafrom
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1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these
autoantibodiesweredetectedinvavFLIP sera.Furthermore,tissuedamageinkidneysandlungsfromagedvavFLIP animals
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was observed, indicating that vavFLIP mice develop a systemic lupus erythematosus-like phenotype with age. Taken
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together, these data suggest that c-FLIP is an important modulator of apoptosis and enforced expression leads to
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autoimmunity.
CellDeathandDisease(2014)5,e1168;doi:10.1038/cddis.2014.138;publishedonline10April2014
SubjectCategory:Immunity
It is crucial that excessive lymphocytes are deleted by receptoroligomerisationandformationofthedeath-inducing
apoptosis after efficient antigen clearance to maintain cell signallingcomplex(DISC)throughrecruitmentoftheproteins
homeostasis.1 Moreover, insufficient apoptosis of auto- Fas-associated death domain-containing protein (FADD),
reactive immune cells may result in autoimmune diseases.1 procaspase-8, -10 and cellular FLICE-inhibitory protein
Theextrinsicapoptosispathwayistriggeredbyligandbinding (c-FLIP).10 Autoproteolytic processing of procaspase-8 at
ofdeathreceptors,suchasCD95(Fas/APO-1),andregulates theDISCgeneratesactivecaspase-8,11,12whichcleavesand
theremovalofunwantedlymphocytes.2EliminationofTcells activates caspase-3 and -7. The effector caspases process
isachievedbyactivation-inducedcelldeath(AICD),whichis further substrates, eventually leading to apoptosis.13,14 The
triggereduponrestimulationofTcellswithoutco-stimulation.2 c-FLIPsinhibitapoptosisbycompetingwithprocaspase-8for
Notably,theCD95deathreceptorpathwaywasshowntobe binding sites at the DISC and additionally interfere with
important for AICD.3–6 Furthermore, dysregulation of the caspase-8 processing.15–17 So far, three c-FLIP isoforms
CD95 signalling pathway was reported to result in severely expressed on the protein level have been reported:
alteredlymphoproliferation(Lpr)andautoimmunityinbothlpr c-FLIP (c-FLIP ), c-FLIP (c-FLIP ) and c-FLIP
Long L Short S Raji
and generalised lymphoproliferative disorder (gld) mice, (c-FLIP ).15,17,18Thelongisoformresemblescaspase-8,but
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which have mutated CD95 receptor or ligand, respectively, lackscatalyticfunctionbecauseofsubstitutionofamino-acid
aswellasinpatientssufferingfromthehomologoushuman residues critical for enzymatic activity.18,19 The two short
diseaseautoimmunelymphoproliferativesyndrome.7,8CD95 isoforms are distinguished by a functional nucleotide poly-
isthebest-characteriseddeathreceptoranditisactivatedby morphism and mainly consist of the death effector domains
bindingofitscognateligandCD95L.9Ligand-bindingresultsin with unique C-terminal tails.20 The structure of the murine
1InstituteofMolecularandClinicalImmunology,Otto-von-Guericke-UniversityMagdeburg,LeipzigerStr.44,Magdeburg,Germany;2ResearchGroupofSystems-
OrientedImmunologyandInflammationResearch,DepartmentofImmuneControl,HelmholtzCentreforInfectionResearch,Inhoffenstr.7,Braunschweig,Germany;
3MousePathology,AnimalExperimentalUnit,HelmholtzCentreforInfectionResearch,Inhoffenstr.7,Braunschweig,Germany;4InstituteofPathology,Helmholtz
CentreMunich,IngolstaedterLandstr.1,Neuherberg,Germanyand5CellularProteomeResearch,DepartmentofStructureandFunctionofProteins,HelmholtzCentre
forInfectionResearch,Inhoffenstr.7,Braunschweig,Germany
*Correspondingauthor:ISchmitz,InstituteofMolecularandClinicalImmunology,Otto-von-Guericke-UniversityMagdeburg,LeipzigerStr.44,Magdeburg,Germanyor
ResearchGroupofSystems-OrientedImmunologyandInflammationResearch,DepartmentofImmuneControl,HelmholtzCentreforInfectionResearch,Inhoffenstr.7,
D-38124,Braunschweig,Germany.Tel:[phone];Fax:[phone];E-mail:[email protected]
Keywords:c-FLIP;apoptosis;CD95;autoimmunity
Abbreviations: AICD, activation-induced cell death; ANA, anti-nuclear antibody; BALT, bronchial-associated lymphoid tissue; Cflar, caspase-8 and FADD-like
apoptosisregulator;c-FLIP,cellularFLICE-inhibitoryprotein,longisoform;c-FLIP ,cellularFLICE-inhibitoryprotein,shortisoform;c-FLIP ,cellularFLICE-inhibitory
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protein,Rajiisoform;CNS,centralnervoussystem;DC,dendriticcell;DISC,death-inducingsignallingcomplex;DN,doublenegative;EAE,experimentalautoimmune
encephalomyelitis;FADD,Fas-associateddeathdomain-containingprotein;Gld,generalisedlymphoproliferativedisorder;IFN-g,interferon-g;Lpr,lymphoproliferation;
MOG,myelinoligodendrocyteglycoprotein;MS,multiplesclerosis;SLE,systemiclupuserythematosus;WT,wildtype
Received05.9.13;revised27.2.14;accepted28.2.14;EditedbyMLeverkus
c-FLIP inautoimmunity
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Cflar (caspase-8 and FADD-like apoptosis regulator) gene B220þ abTcells.7,30c-FLIP inhibitsapoptosisbycompet-
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differs from the human gene locus, in that only c-FLIP and ing with caspase-8 for binding sites at the DISC.17,21 The
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c-FLIP can be expressed.21 Although the short c-FLIP mouse model vavFLIP , in which murine c-FLIP is under
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isoformshavebeendescribedasanti-apoptotic,c-FLIP was control of the vav-promoter to ensure expression in all
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reported to act both pro- and anti-apoptotic depending on hematopoietic compartments,28 was used to study the
expression level and strength of receptor stimulation.22–24 functional role of murine c-FLIP in the immune system. As
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Transgenic overexpression of c-FLIP in mice resulted in thymocytes as well as B and T cells from vavFLIP mice
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autoimmunity, although only in the Balb/c background.25 In were protected against CD95-induced apoptosis,28 we
contrast,miceoverexpressinghumanc-FLIP didnotdevelop investigated if the impaired lymphocyte apoptosis in
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autoimmune disease.26,27 Not much is known about the vavFLIP mice would lead to elevated numbers of lympho-
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physiologicalfunctionofc-FLIP .Toinvestigatethefunctional cytes and altered lymphocyte populations with age. No Lpr
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role of c-FLIP in the immune system, we used a mouse wasidentifiedin12–14monthsoldvavFLIP animals.Infact,
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model, called vavFLIP , with constitutive expression of the number of cells in the peripheral lymph nodes (pLNs)
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murine c-FLIP in all hematopoietic compartments. We from vavFLIP mice was reduced compared with WT
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previouslyreportedthatthesetransgenicmicehaveabetter littermates (Figure 1a). Furthermore, pLN cells of WT and
clearance of the Gram-positive bacteria Listeria monocyto- vavFLIP mice were analysed for DN B220þ T cells
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genescomparedwithinfectedwild-type(WT)mice.28Herewe compared with 3 months old MRL/lpr mice as controls. The
showthatagedmiceconstitutivelyexpressingmurinec-FLIP characteristicDNB220þ populationwasidentifiedinMRL/lpr
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have altered lymphocyte populations with higher levels of mice,butwasnotobservedineitherWTorvavFLIP animals
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activated B and T cells compared with WT littermates. (Figure1b).ThelackofDNB220þ TcellsinvavFLIP mice
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Moreover, vavFLIP animals spontaneously develop auto- issimilartothephenotypeofc-FLIP transgenicmice31and
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immunity with age, showing features of systemic lupus mice with transgenic expression of human c-FLIP .27 Thus,
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erythematosus(SLE). vavFLIP micedonotdevelopanlpr/gld-like phenotype.
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Results AlteredlymphocytepopulationsinagedvavFLIPRmice.
YoungvavFLIP micedisplayednormallymphocytepopula-
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vavFLIP micedonotrecapitulatethelpr/gldphenotype. tions.28 We analysed aged WT and vavFLIP mice to
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Insufficientcelldeathcanleadtoautoimmunityasaresultof investigate if the constitutive expression of c-FLIP alters
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altered immune cell populations. Lpr and gld mice, with lymphocyte populations over time. Dendritic cells (DCs),
mutations in the CD95 receptor and ligand, respectively, macrophages and granulocytes have been reported to be
develop Lpr and autoimmunity.7,29 Moreover, these mice regulated through death receptors and c-FLIP proteins.32,33
accumulate unusual CD4– CD8– double negative (DN) We therefore analysed the frequencies of CD11cþ DCs,
Figure1 One-year-oldvavFLIP micedisplaynormalcellularityinlymphoidorgansanddonotaccumulateDNB220þ cells.(a)AbsolutecellnumbersinpLNsand
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spleensofWT(n¼13)andvavFLIP (n¼15)miceat12–14monthsofage.Symbolsrepresentindividualmice,horizontallinesdisplaythemean±S.E.M.pooledfromfive
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independentexperiments.Statisticalanalysiswasperformedwithtwo-tailednonparametricMann–WhitneyU-test.(b)AnalysisofDNB220þcellsinpLNsfrom1-year-oldWT
andvavFLIP animals.RepresentativedotplotsareshownforMRL/lpr(n¼2),WT(n¼6)andvavFLIP (n¼7)mice.ThreemonthsoldMRL/lprmicedevelopthe
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characteristicDNB220þ cells(upperpanel).ThissubsetcouldnotbeidentifiedineitherWT(midpanel)orvavFLIP mice(lowerpanel)
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CD49bþ naturalkiller(NK)cells,F4/80þ macrophagesand expression regulates homeostasis of peripheral CD8þ
Gr1þ granulocytes in 1-year-old mice. The populations of T cells. Taken together, the B- and T-cell populations are
CD11cþ, CD49bþ, F4/80þ and Gr1þ cells from WT and altered in aged vavFLIP mice, whereas other haemato-
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vavFLIP were comparable (Supplementary Table 1). Flow poietic cells are unaffected.
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cytometryanalysesoflymphocytesub-populationsinspleen
and pLNs from 12 to 14 months old mice revealed reduced Activated phenotypes of T and B cells from vavFLIP
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frequencies of vavFLIP CD3þ T cells compared with WT mice. The activation status of T cells in young vavFLIP
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CD3þ T cells (Figures 2a and b), consistent with reduced mice was comparable to WT littermates.28 As AICD is
absolute CD3þ T-cell number in pLNs from vavFLIP impaired inTcellsfromvavFLIP mice,28weinvestigated if
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animals (Figure 2c). Furthermore, an increased percentage theactivationstatuschangedwithage.Weobservedhigher
ofvavFLIP CD19þ BcellsinpLNscomparedwithWTwas percentagesofvavFLIP CD4þ cellsexpressingtheactiva-
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observed (Figures 2a and d). Frequencies and absolute tion marker CD25 in comparison with WT CD4þ cells
numbersofCD19þ Bcellswerenotsignificantlyalteredin (Figures3aandb),withabsolutecellnumbersbeingsimilar
the spleen (Figure 2e). Neither did we detect alterations between 12 and 14 months old WT and vavFLIP mice
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in germinal centre formation in aged transgenic mice (Figure 3c). As CD25 is not only a marker for activated
(data not shown). The CD4þ/CD8þ cell profiles within T cells, but is also highly expressed on regulatory T (T )
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the CD3þ T-cell compartment were comparable between cells,34 we analysed the expression of the master transcrip-
WT and vavFLIP mice (Supplementary Figure 1). tionfactorofT cells,Foxp3.35Indeed,mostoftheCD25þ
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Nevertheless, lower frequencies of splenic CD8þ T cells CD4þ Tcellsco-expressedFoxp3indicatingthat1-year-old
wereobservedaswellasreducedabsolutecellnumbersof vavFLIP micehave higher levels of T cellsin both pLNs
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CD8þ cells in pLNs (Supplementary Figure 1b). As we (Figure3d)andspleen(datanotshown)comparedwithWT
previously reported that thymocyte development in littermates. Interestingly, we also observed an increase in
vavFLIP mice is normal,28 we suggest that c-FLIP CD25þ TcellsthatwerenegativeforFoxp3suggestingthat
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Figure2 Alteredlymphocytepopulationsin12–14monthsoldvavFLIP mice.(a)RepresentativedotplotsofCD3-andCD19-stainedWTandvavFLIP pLNcells.
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FrequencyandabsolutecellnumberofCD3þ cells(bandc)andCD19þ cells(dande)infreshlyisolatedpLNsandspleensfromWT(n¼13)andvavFLIP (n¼15)
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littermates.Individualmicearerepresentedasseparatesymbols.Horizontallinesshowthemeanpooledfromfiveindependentexperiments;errorbarsdisplaytheS.E.M.
Statisticalanalyseswereperformedwithtwo-tailednonparametricMann–WhitneyU-tests
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Figure3 IncreasedlevelsofCD25þTcellsandT cellsin1-year-oldvavFLIP animals.(a)RepresentativedotplotsofWT(upperpanel)andvavFLIP (lowerpanel)
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CD3þcellsinpLNs.Frequency(b)andabsolutecellnumber(c)ofCD4þCD25þcellsinWT(n¼13)andvavFLIP (n¼15)pLNandspleens.Symbolsrepresentindividual
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mice.Horizontallinesdisplaythemean±S.E.M.Statisticalanalyseswereperformedwithtwo-tailednonparametricMann–WhitneyU-tests.(d)DotplotsshowingCD25þand
Foxp3þ TcellswithintheCD4þ compartmentrepresentativeforthreeWT(left)andfourvavFLIP (right)mice
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conventional T cells may indeed be more activated naive (CD62Lþ) cells were identified compared with WT
(Figure 3d). Therefore, the memory/naive T-cell profile (Figures 4c and d). The absolute cell numbers of CD62Lþ
was analysed in 12–14 months old WT and vavFLIP cells were reduced for both CD4þ and CD8þ T-cell
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animals. Strikingly, elevated frequencies of CD44þ anti- populations (Supplementary Figures 2b and c). These data
gen-experienced cells within the CD3þ compartment were indicatethatTcellsfromvavFLIP micearehighlyactivated
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identified in both pLNs and spleen from vavFLIP mice incomparisonwithWTTcells.
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(Figures 4a and b). We also observed reduced numbers SimilartoTcells,BcellsfromvavFLIP miceareprotected
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of CD3þCD62Lþ naive T cells in vavFLIP animals against apoptosis.28 The activation status of B cells was
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(Supplementary Figure 2a). Notably, both within the CD4þ examined by flow cytometry. CD40, CD80 and CD95 had
helper T-cell population and the CD8þ cytotoxic T-cell similarexpressionlevelsinvavFLIP andWTanimals(data
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population from vavFLIP pLNs and spleens, higher levels notshown),whereasanalysisoftheactivationmarkersCD69,
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of antigen-experienced (CD44þ) cells and lower levels of CD86andmajorhistocompatibilitycomplexclassIImolecule
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Figure4 TcellsfromvavFLIP miceat12–14monthsofagearehighlyactivated.(a)RepresentativedotplotsofpLNcells(leftpanel)andsplenocytes(rightpanel)from
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WT(upperpanel)andvavFLIP animals(lowerpanel).FrequenciesofCD44þ,CD44þCD62LþandCD62LþcellswithinCD3þcells(b),CD4þcells(c)andCD8þcells
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(d)frompLNandspleen.(b-d)SymbolsrepresentindividualWT(b:n¼13,candd:n¼6)andvavFLIP (b:n¼15,candd:n¼6)mice.Horizontallinesrepresent(b)the
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meanpooledfromfiveindependentexperimentsor(candd)themeanofoneexperimentrepresentativefortwoindependentexperiments;errorbarsdisplayS.E.M.Statistical
analyseswereperformedwithtwo-tailednonparametricMann–WhitneyU-tests
I-A/I-Erevealedanincreasedexpressionofthesemarkersin Disease progression in MOG-EAE is comparable
vavFLIP BcellscomparedwithWTBcells(Figure5).This between WT and vavFLIP mice. As constitutive expres-
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indicatesthatBcellsfrom12to14monthsoldvavFLIP mice sionofc-FLIP alterslymphocytepopulationsandactivation
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are more activated in comparison with B cells from WT status with age, we investigated if this has functional
animalsat1yearofage.TheactivatedphenotypesofBandT relevance in T-cell-mediated autoimmune disease.
cellsidentifiedin1-year-oldvavFLIP miceareconsistentwith Experimental autoimmune encephalomyelitis (EAE) is a
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a previous study, which reported that B and T cells from widely accepted animal model for studying the human
c-FLIP transgenicmiceonaBalb/cbackgroundwerehighly demyelinating inflammatory disorder multiple sclerosis
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activated.25 (MS).36 T 1 and T 17 effector T cells have been shown to
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Figure5 vavFLIP Bcellsfrom12to14monthsoldmiceshowanactivatedphenotype.(a–c)pLNcellsandsplenocytesfromWTandvavFLIP mice(n¼6)were
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isolatedandanalysedbyflowcytometry.TheactivationstatusofCD19þBcellswasassayedbythemarkersCD69(a),CD86(b)andI-A/I-E(c).Representativehistograms
withfluorescentminusone(FMO)ascontrol(leftpanel)anddiagramsdisplayingthemeanfluorescentintensity(MFI;rightpanel)±S.E.M.areshown.Statisticalanalyses
wereperformedwithtwo-tailednonparametricMann–WhitneyU-tests
contributetothepathologyinEAE37andc-FLIP transgenic frequencies of IFN-gþ and IL-17Aþ cells from vavFLIP
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micewerereportedtoberesistanttomyelinoligodendrocyte pLNs in comparison with WT 11 days after immunisation
glycoprotein (MOG)-EAE because of enhanced T 2, and (Figure 6e). This effect was levelled out at higher
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thus diminished T 1, effector responses.38 To assess if MOG peptide concentrations on day 14 (Figure 6e). The
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c-FLIP influences the differentiation of T 0, T 1, T 2 or frequenciesofIL-4-producingTcellsinpLNsfromvavFLIP
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T 17cells,weculturednaiveWTandvavFLIP Tcellsunder and WT mice were comparable (Figure 6e). Similar
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T 1, T 2 or T 17 polarising conditions in vitro. The results were obtained when T cells were restimulated in a
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frequencies of interferon-g (IFN-g)-producing T 1 cells were polyclonal manner, that is, with anti-CD3 and anti-CD28
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comparablebetweenWTandvavFLIP differentiatedTcells (SupplementaryFigure3).Insummary,ourdataindicatea
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(Figures 6a and b). Interestingly, frequencies in IL-17A- minor defect in vavFLIP effector T-cell responses that
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producing T 17 cells and IL-4 mRNA induction in T 2 cells results in a slight delay in EAE disease progression in
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werelowerinvavFLIP Tcellsculturedundertherespective vavFLIP mice.
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polarisingconditions (Figures6a,bandc).
WTandvavFLIP micewereimmunisedwiththeMOG Elevated ANA-titres and tissue damage of kidneys and
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peptide in complete Freund’s adjuvant in conjunction with lungs in aged vavFLIP mice. B-cell-mediated auto-
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pertussis toxin to investigate if constitutive murine c-FLIP immunity is characterised by autoantibodies targeting the
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expressioninTcells,macrophagesandDCsinfluencesEAE body’s own tissues. As vavFLIP mice had slightly higher
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disease.However,bothWTandvavFLIP animalsdeveloped B-cell levels with activated phenotype, we analysed sera
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diseasewithneurologicaldefectsresultinginparalysisofthe from 1-year-old WT and vavFLIP mice for anti-nuclear
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tail and muscle weakness with comparable severity of antibodies (ANAs) using an indirect immunofluorescence
disease. Only a slightly slower disease progression was assay with HEp-2 cells as source of nuclear antigens.
observed in vavFLIP mice (Figure 6d). To investigate Negativefluorescentpatternwasmostoftenobservedwhen
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whether T-cell effector responses were affected by the sera from WT mice were incubated on HEp-2 cells
transgenic expression of c-FLIP in vivo, we analysed (Figure 7a), whereas homogenous fluorescent pattern was
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cytokine production in MOG-peptide restimulated T cells detectedforthemajorityofserafromvavFLIP miceevenat
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from MOG-immunised mice. We observed slightly reduced high serum dilution (Figure 7b). Hence, significantly more
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Figure6 ComparablediseaseprogressioninMOG -peptide-inducedEAEinWTandvavFLIP mice.(a–c)NaiveTcellsfromWTandvavFLIP micewereisolated
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andculturedunderT 0,T 1,T 2andT 17polarisingconditions,followedbyflowcytometryanalysisofIFN-gandIL-17A.(a)RepresentativedotplotsofT 1andT 17
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differentiatedcellsareshown.(b)Dataarerepresentedasthemean±S.E.M.fromtwoindependentexperiments(n¼5forWTandvavFLIP cells).Statisticalanalyseswere
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performedwithtwo-tailednonparametricMann–WhitneyU-tests.(c)Quantitativereal-timePCRanalysisofIL-4mRNAexpressioninTH2differentiatedWTandvavFLIP
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Tcells.Dataarerepresentedasthemean±S.E.M.fromtwoindependentexperiments(n¼4forWTandn¼5forvavFLIP ).(d)EAEwasinducedbyinjectingWTand
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vavFLIP mice(n¼11)withtheMOG -peptideandtwofurtherinjectionsofpertussistoxin.Subsequently,theclinicalscorewasmonitoredfor40days.Dataarepooled
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fromtwoindependentexperimentsandrepresentedasthemean±S.E.M.(e)WTandvavFLIP animalswereinjectedwiththeMOG -peptideincompleteFreund’s
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adjuvant.pLNcellsisolated11(WTn¼4,vavFLIP n¼4)and14(WTn¼5,vavFLIP n¼3)daysafterinjectionwererestimulatedwiththeindicatedconcentrationsof
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MOGpeptidefor24h,followedbyflowcytometryanalysisofIFN-g,IL-17AandIL-4-producingTcells
autoantibodies were identified in sera from vavFLIP mice micewerestainedwithperiodicacid–schiff(PAS)followedby
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comparedwithWTmice(Figure7c). histologicalscoring.Themajorityofglomeruliinkidneysfrom
Multiple organs can be affected in systemic autoimmune WTmicewerenormal(Figure7d).Incontrast,thickeningof
diseases. We therefore examined several organs from the Bowman’s capsule and protein deposition in glomeruli
1-year-old WT and vavFLIP animals by histology. No were observed in kidneys from vavFLIP mice, indicating
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differences between WT and vavFLIP mice could be nephropathy (Figure 7e). Glomerular deposition of
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observed in brains, spleens and pancreas stained with PAS-positivematerialiscommoninagingmice.39However,
haematoxylin and eosin (H&E) and in spleens stained for B significantly more tissue damage was detected in vavFLIP
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cellsandmacrophages(datanotshown).Kidneysfromaged kidneys with 63.6% of the kidney sections displaying a
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Taken together, the elevated levels of ANAs and tissue
damageinkidneysandlungsaresymptomssimilartohuman
SLE.41,42 Hence, the kidney damage may be related to the
ANAsandrepresentamildformofSLEnephropathy.
Discussion
CD95-mediated apoptosis has a crucial role in controlling
immune responses and preventing autoimmune diseases.1
c-FLIPproteinsprotectcellsagainstCD95-inducedapoptosis
attheleveloftheDISC.2Althoughtheisoformsc-FLIP and
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c-FLIP have been thoroughly characterised, not much is
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known about the functional role of c-FLIP in the immune
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system.Inthisstudy,wereportthatconstitutiveexpressionof
murinec-FLIP resultsinalteredlymphocytepopulationsand
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lupus-likesymptomswithage.
LprandgldmicewithmutationsintheCD95receptorand
ligand,respectively,developlymphoproliferativediseaseand
autoimmunity with accumulation of unusual DN B220þ
T cells.7,29,30,43 c-FLIP proteins inhibit the same apoptosis
pathwaybypreventingcaspase-8cleavageattheDISC.15,16
Indeed,T-cell-specificc-FLIP transgenicmicewerereported
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to develop autoimmunity when bred on Balb/c, but not
C57BL/6, background.25 However, neither altered Lpr nor
Figure7 AutoantibodyproductionandnephropathyinagedvavFLIP mice.
R accumulation of DN B220þ T cells was identified in up to
(a–c)QuantificationofANAsinserafrom1-year-oldWT(n¼11)andvavFLIP
R 1-year-old vavFLIP mice, consistent with transgenic mice
(n¼14)littermates.Representativeexamplesof(a)negativefluorescentpattern R
(WT)and(b)homogenousANApattern(vavFLIPR).TheANA-IgGtitreisshownin with T-cell-specific expression of murine c-FLIPL or human
(c).Symbolsrepresentindividualmice.Horizontallinesrepresentthemean;error c-FLIP in the C57BL/6 background.26,27,31 Hence, neither
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bars display S.E.M. Statistical analysis was performed with two-tailed nonpara- constitutive expression nor overexpression of c-FLIP
metric Mann–Whitney U-test. (d–f) Kidneys from 1-year-old WT (n¼18) and proteins,atleastintheC57BL/6background,issufficientfor
vavFLIP (n¼22)animalswereanalysedbyhistology.(dande)Representative
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histologicalsectionsofparaffin-embeddedkidneysstainedwithPASareshownfor
TheautoimmuneinflammationinEAEismainlycausedby
(d)WTglomeruluswithmildthickeningoftheBowman’scapsuleand(e)vavFLIP
glomerulus with moderate thickening of the Bowman’s capsule and moderateR autoreactiveTH1andTH17effectorcells.37Lprmiceshoweda
glomerularproteindeposition.Scalebarrepresents25mm.(f)Histologicalscoreof prolongedandenhancedinflammationinthecentralnervous
PAS-stainedkidneys.Individualmicearedisplayedasseparatesymbols.Horizontal system (CNS) upon EAE induction.44 Hence, CD95/CD95L
linesrepresentthemean;errorbarsshowS.E.M.Animalsweredividedupinto interactions are of importance to delete disease-initiating,
groups of normal alterations (histological score o3) and moderate-to-severe autoreactiveTcellsintheCNSinthecourseofEAE.Bothlong
alterations (histological score 3 or higher), followed by statistical analysis with
and short c-FLIP isoforms have been reported to be
Fisher’sexacttest
upregulatedinactivatedTcellsfromthecerebralspinalfluid
inpatientswithactiveMS.45,46Interestingly,transgenicmice
histological score of three or higher, implying mild-to- overexpressinghumanc-FLIP wereprotectedagainstEAE
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moderate alterations(Figure 7f). In comparison, only 27.8% because of enhanced T 2 effector responses and conse-
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ofkidneysfromWTmicemanifestedsimilardamage(Figure7f). quently reduced T 1 effector responses.38 Transgenic
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Thus, vavFLIP mice have a greater degree of nephropathy expression of murine c-FLIP did not alter the cytokine
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comparedwithWTanimalsat12–14monthsofage. production of T cells in a similar way, which may be one
Thehistologicalanalysesoflungsfromagedmicerevealed reason why the enforced expression of c-FLIP only had a
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thatthreeofsixvavFLIP lungsstainedwithH&Edisplayed minoreffectontheprogressionofEAE.Yuetal.47 reported
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markedfocalinterstitialpneumoniacomparedwithoneoffive high expression levels of c-FLIP in T 17 cells, which
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WT lungs. Increase in bronchial-associated lymphoid tissue protected these cells against AICD. This was proposed to
(BALT)hasbeendescribedinagingmice.40Nevertheless,the be a mechanism for the high pathogenicity of T 17 cells in
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amount of BALT was massively increased in four of six autoimmunediseases.Expressionlevelsofc-FLIP inT 17
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vavFLIP animals (67%) in comparison with two of five WT cells was not investigated in this study, nevertheless, the
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mice (40%). Although the composition of BALT was increased expression of c-FLIP in this cell population may
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not obviously altered, we detected more IgG-containing override the effect of the constitutive c-FLIP expression in
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immunecomplexes(Figure8).Infiltratingmacrophageswere vavFLIP miceinthecourseofEAE.However,theimpaired
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observed in regions of interstitial pneumonia by immuno- abilityofnaivevavFLIP TcellstodifferentiateintotheT 17
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histochemical staining for B cells (B220), T cells (CD3) and subset in vitro and the minor reduction of effector T-cell
macrophages(MAC2)invavFLIP lungs(Figure8).Ofnote, responses observed in MOG-immunised vavFLIP mice
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interstitial pneumonia has occasionally been described couldpossiblyexplaintheslightlyslowerdiseaseprogression
forSLE.41 invavFLIP micecomparedwithWTlittermates.
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Figure8 HistologicalalterationsinlungsofagedvavFLIP mice.Lungswerepreparedfrom1-year-oldWT(n¼5)andvavFLIP (n¼6)miceandparaffinembedded.
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SectionswerestainedwithH&E,theB-cellmarkerB220,theT-cellmarkerCD3andthemacrophagemarkerMAC2,respectively.DepositionofIgG-containingimmune
complexeswasdetectedwithgoat-anti-mouseantibodies.Representativetissuesectionsareshown.ArrowsindicateincreasedBALTandinterstitialpneumonia,respectively.
Scalebarsrepresents200mmforH&Esectionand50mmforallothersections
B- and T-cell numbers and distribution were normal in proportion of vavFLIP animals displayed focal interstitial
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young vavFLIP mice28 and no differences in DCs, macro- pneumoniaandanincreasedamountofBALTcomparedwith
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phages,granulocytesorNKcellscouldbeidentifiedevenin WT littermates. Moreover, elevated levels of anti-nuclear
miceat1yearofage.Interestingly,agedvavFLIP micehad autoantibodies were identified in vavFLIP sera, indicating
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alteredlymphocytepopulationswithlowerlevelsofTcellsand thatvavFLIP micedevelopalupus-likephenotypewithage.
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higherfrequenciesofBcellscomparedwithWTlittermates. Hence, our findings imply that the altered lymphocyte
Moreover, reduced levels of CD8þ cells were observed, populations with activated phenotypes in aged vavFLIP
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whichisconsistentwithareportofWuetal.48wherereduced mice are of functional relevance. Most probably the auto-
peripheral CD8þ cell numbers were described in mice with immunediseaseobservedinvavFLIP animalsiscausedbya
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transgenicexpressionoftheviralFLIPMC159undercontrol combination of autoreactive B and T cells, with persisting
oftheCD2enhancercassette.Furthermore,alterationsinthe CD4þ helper T cells, which may prime B cells to produce
ratioof antigenexperiencedto naiveTcellswereidentified, autoantibodies,andincreasedlevelsofBcells.Insupportof
mostprobablybecauseofdefectiveAICD.Humanc-FLIP is this notion, lupus-like disease was described in c-FLIP
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upregulated in the activation and expansion phase of an transgenicmice onthe Balb/cbackground andthe develop-
immuneresponse,butisthereafterdownregulatedtoenable ment of autoimmunity in this study was shown to require
AICD and elimination of effector T cells.49 We previously CD4þ T cells, which were proposed to result from impaired
describedasimilarkineticformurinec-FLIP ,whichexplains thymic selection.25 Notably, elevated gene expression of
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thedefectinAICDuponconstitutivec-FLIP expression.28In CFLARinCD33þ myeloidcells,CD4þ TcellsandCD19þ B
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this study, increased frequencies of activated T helper cells cellswasreportedinSLEpatients.50Furthermore,Xuetal.51
andantigen-experiencedTcellsaswellaslowerfrequencies observed that human lupus T cells did not downregulate
andnumbersofnaiveTcellsinvavFLIP micecomparedwith c-FLIP after initial activation, which suggests that c-FLIP
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WT mice were observed. This is consistent with a previous proteinsareofimportanceinthedevelopmentofSLEdisease.
reportwhereanincreasedmemoryT-cellpoolwasidentified The higher level of T cells in aged vavFLIP mice is
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inc-FLIP transgenicmicecomparedwithWTanimalsafter presumably a compensatory mechanism because of the
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immunisation with the superantigen staphylococcal entero- observed autoimmunity. Imbalance of T cells has been
reg
toxin B.27 Similarly to the activated T-cell phenotype, describedinvariousautoimmunediseases.52Reducedlevels
vavFLIP B cells expressed activation markers to a higher or conflicting data have been reported in patients with, for
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extent than WT B cells. Strikingly, histological analyses of example, juvenile idiopathic arthritis, autoimmune liver
kidneysandlungsfrom12to14monthsoldmicerevealeda diseaseandmyastheniagravis.52Consistentwithourfindings,
higherdegreeofnephropathyinvavFLIP miceandalarger increased levels of T cells were identified in patients with
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primarySjo¨gren’ssyndrome53andinexperimentalarthritisin PBSat41C.Thereafter,cellswerewashedandFcgIII/IIreceptorswereblocked
mice.54TheexpandedT cellpopulationobservedin1-year- byincubationwithanti-CD16/CD32(2.4G2,BDBiosciences).Subsequently,cells
reg
oldvavFLIP animalsmostprobablydampenstheseverityof werewashedandstainedwithantibodiesinPBScontaining2%BSAfor20minat
R 41C.The followingantibodieswereused:CD3-HorizonV450(17A2),CD3-FITC
theSLE-likephenotype.
(145-2C11), CD4-HorizonV500 (RM4-5), CD8-FITC (53-6.7), CD19-FITC (1D3),
We previously reported that vavFLIP mice are better
R CD25-PE Cy7 (PC61.5) (all from BD Biosciences); CD4-Pacific Blue (RM4-5),
protectedagainstListeriamonocytogenesinfectioncompared CD8-APC (53-6.7), CD86-APC (GL-1), I-A/I-E-Alexa Fluor 700 (M5/114.15.2)
with WT littermates.28 Hence, c-FLIPR expression is bene- (allfromBioLegend);CD11c-APCeFluor780(N418),CD19-PerCPCy5.5(1D3),
ficial in an acute infection, whereas we in this study CD44-PE (IM7), CD45R (B220)-APC (RA3-6B2), CD49b-APC (Dx5), CD62L-
demonstratethatchronicexpressionresultsinautoimmunity. PerCPCy5.5(MEL-14),CD69-PECy7(H1.2F3),F4/80-PE(BM8)andGr1-Pacific
Blue (RB6-8C5) (all from eBiosciences, San Diego, CA, USA). Samples were
To conclude, aged vavFLIP mice have altered lymphocyte
R analysedbyLSRIIorLSRFortessaflowcytometers(BDBiosciences).TheFlowJo
populations with increased levels of antigen-experienced
software(TreeStar,Ashland,OR,USA)wasusedtoanalysethedata.
T cells and higher activation status of both B and T cells.
Intracellularproteinswerestainedaftersurfacemarkerstaining.Forstainingof
Moreover, these animals develop lupus-like symptoms with Foxp3-AlexaFluor488,cellswerefixedandpermeabilisedforintracellularcytokine
age.Our results showthat c-FLIP has an important role in stainingbyusingtheFoxp3StainingBufferSet(MiltenyiBiotec,Auburn,CA,USA)
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thecontrolofautoimmunity. according to the manufacturer’s protocol. For intracellular cytokine staining of
IFN-g-FITC(XMG1.2,BioLegend),IL-4-PE(11B11,eBioscience)andIL-17A-APC
(eBio17B7,eBioscience),cellswerestimulatedfor4hwithPMAandionomycin
MaterialsandMethods (SigmaAldrich,Munich,Germany),withadditionofBrefeldinA(SigmaAldrich)for
Mice. vavFLIPRmicewerepreviouslydescribed.28WTlittermateswereusedas thelast2h,beforethestainingwasperformedasforFoxp3.
controlanimals.Allanimalswerekeptinaspecificpathogen-freeenvironmentinthe
animalfacilityofHelmholtzCentreforInfectionResearch,Braunschweig.55C57BL/6 Real-timedetectionPCR. cDNAwasusedasatemplateforreal-timePCR
micewerepurchasedfromHarlanLaboratories(Indianapolis,IN,USA)andCharles usingSYBRGreen(Roche,Mannheim,Germany).UbiquitinC(UBC)wasusedas
River(Wilmington,MA,USA).MRL/lprmicewereakindgiftfromDrDetlefNeumann, referencegenefornormalisation.Measurementswereperformedinduplicatesin
HannoverMedicalSchool,Hannover,Germany.Allbreedingandexperimentswere the LightCycler 96 system (Roche) using the following primers: UBC fwd 50-A
performedinaccordancewiththeguidelinesofnationalandlocalauthorities. AGAGAATCCACAAGGAATTGAATG-30; UBC rev 50-CAACAGGACCTGCTGA
ACACTG-30; IL4 fwd 50-CATCGGCATTTTGAACGAG-30; IL4 rev 50-CGAGC
Cellpreparation. Lymphoidorgans(pLNs,mesentericlymphnodes,spleen TCACTCTCTGTGGTG-30.
andthymus)wereisolatedfrommicekilledviaCO.Organswerehomogenised
2
througha70mmnylonmeshandwashedwithPBS.Erythrocyteswereremoved Experimentalautoimmuneencephalomyelitis. EAEwasinducedin
by 2-min incubation in ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM 10–12weeksoldWTandvavFLIPRmicebyinjecting200mgMOG35-55-peptide
EDTA,pHadjustedto7.3withNaOH)atroomtemperaturefollowedbyafurther (MEVGWYRSPFSRVVHLYRNGK) together with 4 mg/ml mycobacteria in
washing step. Primary murine cells were cultured in RPMI 1640 (Gibco – Life completeFreund’sadjuvantsubcutaneouslyatfoursites.Inall,200ngpertussis
technologies, Grand Island, NY, USA) supplemented with 10% FCS (PAA, toxinwasinjectedintraperitoneallytoopentheblood–brain-barrier.Thepertussis
Pasching, Austria), 50mg/ml of penicillin and streptomycin (Gibco – Life toxininjectionwasrepeated2dayslater.Miceweremonitoreddailyforclinical
technologies), 1% non-essential amino acids (Gibco – Life technologies), 2mM signs of EAE from days 3 to 40 with 0 – no symptoms,0.5 – partial limp tail,
L-glutamine(Gibco–Lifetechnologies)and1mMsodiumpyruvate(Gibco–Life 1–limptail,2–delayedrotationfromdorsalposition,2.5–hindlegweakness,
technologies). 3–completehindlegparalysis,3.5–startingforelegweakness,4–paralysisof
oneforeleg,5–moribund,death.Micewithascorehigherthan3werekilled.
InvitrogenerationofThelpercellsubsets. Fordifferentiation ofT Foranalysisofcytokine-producingTcells,micewereimmunisedwithMOG35-55-
helper subsets, CD4þCD62LhighCD25(cid:2) naive T cells were sorted by using a peptideasabove,butwithoutpertussistoxintopreventthemigrationofprimedTcells
FACS Aria II (BD Biosciences, Heidelberg, Germany) or MoFlo (Beckman and intothebrain.pLNswereisolatedfrommicekilled11and14daysafterinjection.Inall,
Coulter, Indianapolis, IN, USA). Cells were seeded directly after sorting with 5(cid:3)105 cells were seeded per well in 96-well plates and either left untreated or
2(cid:3)105cellsperwellin96-wellplatesandactivatedwithplate-boundanti-CD3 restimulated with plate-bound anti-CD3 (2mg/ml; 145-2C11, BioLegend) and
anti-CD28(2mg/ml;37.51,BioLegend)orupto50mg/mlMOG -peptidefor24h
and anti-CD28 in the presence of priming cytokines and inhibitory antibodies 35-55
accordingto the respectiveT helpersubset: T 0: 2mg/ml anti-CD3(145-2C11, withadditionof10mg/mlBrefeldinA(SigmaAldrich)forthelast2h.IFN-g-,IL-4-and
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BioLegend,SanDiego,CA,USA),2mg/mlanti-CD28(37.51,BioLegend),10mg/ IL-17A-producingTcellswereanalysedbyflowcytometry.
mlanti-IL-4(11B11,self-purified)and10mg/mlanti-IFN-g(XMG1.2,self-purified);
TH1: 2mg/ml anti-CD3 (145-2C11, BioLegend), 2mg/ml anti-CD28 (37.51, Analysis of ANAs. ANAs in sera from 1-year-old WT and vavFLIPR mice
BioLegend), 10mg/ml anti-IL-4 (11B11, self-purified) and 10ng/ml IL-12 (R&D wereanalysedsemiquantitativelybyincubatingHEp-2cellsseededonmicroscope
Systems, Minneapolis, MN, USA); T 17: 3mg/ml anti-CD3 (145-2C11, BioLe- slides (Generic Assays,Dahlewitz, Germany) with sera diluted 1:80–1:1280 for
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gend), 5mg/ml anti-CD28 (37.51, BioLegend), 10mg/ml anti-IL-2 (JES6-1A12, 30minatroomtemperature.Slideswerewashedtwotimes5mininPBSfollowed
BioLegend), 10mg/ml anti-IFN-g (XMG1.2, self-purified), 2ng/ml pTGF-b (R&D by incubation with FITC-conjugated donkey anti-mouse IgG (Dianova, Hamburg,
Systems), 30ng/ml IL-6 (R&D Systems), 10ng/ml IL-1-b (R&D Systems) and Germany)for30mininthedark.Thereafter,slideswerewashedtwotimes5minin
20ng/mlTNF-a(Preprotech,RockyHill,NJ,USA).T 2cellsweredifferentiatedas PBS,transferredtocoverslipsandsealed.Slideswereanalysedbyfluorescence
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follows: CD4þCD62LhighCD25(cid:2) naive T cells were sorted as above. In all, microscopywhereahomogenouspatternwasconsideredaspositiveforANAs.
2(cid:3)106cellswereseededaftersortingin24-wellplatesandactivatedwithplate-
bound 5mg/ml anti-CD3 (145-2C11, BioLegend) and 2mg/ml anti-CD28 (37.51, Histology. Kidneys,lungs,livers,spleens,pancreasandbrainswereisolated
BioLegend).After3days,2(cid:3)105cellsweretransferredto96-wellplateinthe from 1-year-old WT and vavFLIP mice. Organs were fixed in 4% neutrally
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presenceofthefollowingprimingcytokinesandinhibitoryantibodiesandcultured bufferedformaldehydeandembeddedinparaffinforhistologicalanalysis.Inall,
foradditional3days:10ng/mlmurineIL-2(402-ML,R&DSystems),10ng/mlIL-4 3mmsectionsfromorganswerestainedwithH&E.Thedegreeoffocalinterstitial
(11B11,self-purified),5mg/mlanti-IFN-g(XMG1.2,self-purified)and5mg/mlanti- pneumoniaandBALTwasdeterminedinH&E-stainedlungsections.Lungswere
IL12p40 (C17.8, R&D Systems). Subsequently, the differentiated cells were immunohistochemicallystainedwithB220(RA3-6B2;BDBiosciences),CD3(SP7;
stainedforCD4andeitherIFN-g,IL-4orIL-17A(seestainingprocedureinthe NeoMarkers,Fremont,CA,USA)andMAC2(M3/38;CEDARLANE,Burlington,
nextparagraph)followedbyflowcytometryanalysisafter4daysofdifferentiation. ON,Canada)todeterminelymphocyteandmacrophageinfiltration.IgGcomplexesin
lung sections were stained with goat-anti-mouse (TM-060-BN, Thermo Fisher
Flow cytometry. Cells were stained with Live/Dead near IR or blue Scientific,Waltham,MA,USA).KidneysectionswerestainedwithPASstaining
fluorescent reactive dyes (Life Technologies) by incubation for 30min in and the sections were analysed for thickening of the Bowman’s capsule and
CellDeathandDisease