IkBNS Regulates Murine Th17 Differentiation during Gut Inflammation and Infection

12 Pages·2015·2.39 MB·English

k
I B Regulates Murine Th17
NS
Differentiation during Gut Inflammation and
Infection
Michaela Annemann, Zuobai Wang, Carlos Plaza-Sirvent,
This information is current as
Rainer Glauben, Marc Schuster, Frida Ewald Sander,
of January 5, 2019.
Panagiota Mamareli, Anja A. Kühl, Britta Siegmund,
Matthias Lochner and Ingo Schmitz
J Immunol published online 18 February 2015
http://www.jimmunol.org/content/early/2015/02/18/jimmun
ol.1401964 DD
oo
ww
nn
lolo
aa
dd
ee
dd
Supplementary http://www.jimmunol.org/content/suppl/2015/02/18/jimmunol.140196 fro fro
Material 4.DCSupplemental mm
h h
ttpttp
://w://w
Why The JI? Submit online. ww
ww
• Rapid Reviews! 30 days* from submission to initial decision .jim.jim
mm
uu
nn
• No Triage! Every submission reviewed by practicing scientists oo
l.orgl.org
• Fast Publication! 4 weeks from acceptance to publication b/ b/
yy
g g
*average ueue
ss
t ot o
nn
Subscription Information about subscribing to The Journal of Immunology is online at: J J
aa
nn
http://jimmunol.org/subscription uu
aa
ryry
5 5
Permissions Submit copyright permission requests at: , 2, 2
http://www.aai.org/About/Publications/JI/copyright.html 0101
99
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Published February 18, 2015, doi:10.4049/jimmunol.1401964
The JournalofImmunology
k
I B Regulates Murine Th17 Differentiation during Gut
NS
Inflammation and Infection
Michaela Annemann,*,† Zuobai Wang,‡ Carlos Plaza-Sirvent,*,† Rainer Glauben,x
Marc Schuster,*,† Frida Ewald Sander,*,† Panagiota Mamareli,‡ Anja A. Ku€hl,x
Britta Siegmund,x Matthias Lochner,‡ and Ingo Schmitz*,†
IL-17–producingTh17cellsmediateimmuneresponsesagainstavarietyoffungalandbacterialinfections.SignalingviaNF-kB
hasbeenlinkedtothedevelopmentandmaintenanceofTh17cells.WeanalyzedtheroleoftheunusualinhibitorofNF-kB,IkB ,
NS
intheproliferationandeffectorcytokineproductionofmurineTh17cells.OurstudydemonstratesthatnuclearIkB iscrucial
NS
for murine Th17 cell generation. IkB is highly expressed in Th17 cells; in the absence of IkB , the frequencies of IL-17A–
NS NS
producingcellsaredrasticallyreduced.ThiswasmeasuredinvitrounderTh17-polarizingconditionsandconfirmedintwocolitis
models.Mechanistically,murineIkB 2/2Th17cellswerelessproliferativeandexpressedmarkedlyreducedlevelsofIL-2,IL-10,
NS
MIP-1a,andGM-CSF.CitrobacterrodentiumwasusedasaTh17-inducinginfectionmodel,inwhichIkB 2/2micedisplayedan D
NS o
w
increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in n
pathogen clearance, as well as in inflammation-associated pathology. We identified IkBNS to be crucial for the generation and loa
d
function of murine Th17 cells upon inflammationand infection. Our findings may have implications for the therapyof autoim- e
d
muneconditions,suchasinflammatoryboweldisease,andforthetreatmentofgut-tropicinfections. TheJournalofImmunology, fro
2015, 194:000–000. m
h
ttp
T heidentificationoftheproinflammatoryTh17cellsubset transcriptionfactorRORgt(6)andprimarilyproducethecytokine ://w
markedasignificantbreakthroughinourunderstandingof IL-17A, which is a potent inducer of other proinflammatory w
w
theThcellcompartment(1,2),expandingtheestablished cytokines, such as IL-6, IL-1b, and TNF-a, in a variety of IL- .jim
Th1/Th2paradigm(3).Previously,onlytheregulationofcellular 17R–expressing cell types, including endothelial cells, epithelial
m
immune responses against viruses by Th1 cells and humoral cells, and macrophages (7, 8). IL-17A is also involved in the re- u
n
responsesbyTh2cellsinresponsetoextracellularpathogenswere cruitment and activation of neutrophils to sites of infection (7). ol.o
known(3).Today,weknowthatTh17cellsareessentialforhost Moreover, Th17 cells secrete GM-CSF, which controls gran- rg
protection against a variety of extracellular bacteria and fungi ulocyteandmacrophagematuration.ExpressionofGM-CSFwas b/
y
locatedpredominantlyinthegastrointestinaltract,skin,andlungs. reported to be responsible for the pathogenicity of Th17 cells g
u
Consequently,mostTh17cellsarefoundinthesemucosaltissues during experimental autoimmune encephalomyelitis (EAE) (9, e
s
upon infection; however, some reside in the lamina propria (LP) 10),themurinediseasemodelformultiplesclerosis.Ofnote,IL- t o
n
under steady-state conditions (4, 5). Th17 cells depend on the 17–producing T cells can be generated invitro by treating naive J
a
CD4+cellswithTGF-bandIL-6(11,12)becausebothcytokines nu
a
induce theexpression of RORgt(6). ry
*IGmSeymrmsuteanmneys;C-Oo†rInintersnotitlte,udtHeIemlfmomrhuoMnlotozloleCgcyeunlaatnrredafInondrflaICmnlfimenciatctiaioolnnIRmReemsseeuaanrrocclhho,gG3y8,ro1Ou2p4tt,oBD-vreoapunan-rGstmcuheewrnietcikogef, forNlFy-mkpBhoicsytaendeivnedluocpimbleenttraanndscfruipntciotinonf(a1c3to).rInanTd ciesllsc,ruaccitai-l 5, 201
University, 39120 Magdeburg, Germany; ‡Institute of Infection Immunology, vationofNF-kBsignalingisgovernedbytheCARMA1,MALT1, 9
TWINCORE,CentreforExperimentalandClinicalInfectionResearch,ajoint and BCL10 complex that activates the inhibitor of kB kinase
venture between the Medical School Hannover and the Helmholtz Centre for
Infection Research, 30625 Hannover, Germany; and xMedizinische Klinik I, (IKK) complex (14). The IKK complex phosphorylates IkBa,
Charite´–Universita¨tsmedizinBerlin,12203Berlin,Germany which binds to NF-kB transcription factors and causes cytoplas-
ReceivedforpublicationAugust4,2014.AcceptedforpublicationJanuary3,2015. mic retention of NF-kB by blocking the nuclear-localization sig-
nal (15). After phosphorylation by IKKb, IkBa becomes poly-
ThisworkwassupportedbytheCenterofDynamicSystemsMagdeburg,theFritz
Thyssen Stiftung, and the Deutsche Forschungsgemeinschaft (SFB 854, project ubiquitinatedand,finally,undergoesproteasomaldegradation(13).
A23N [to I.S.], as well as SI 749/5-3 and 749/6-1 [to B.S.]). M.A., C.P.-S., and Consequently,NF-kBisreleased,entersthenucleus,andregulates
F.E.S.weresupportedbythePresident’sInitiativeandNetworkingFundoftheHelm-
holtzAssociationofGermanResearchCentersundercontractnumberVH-GS-202. gene expressionviabinding tokBsites.NF-kBtranscriptionfac-
tors are dimers composed of two members of the Rel-protein
AddresscorrespondenceandreprintrequeststoDr.IngoSchmitz,Systems-Oriented
Immunology and Inflammation Research Group, Department of Immune Control, family, comprising p50, p52, p65/RelA, c-Rel, and RelB (13). In
HelmholtzCentreforInfectionResearch,Inhoffenstrasse7,D-38124Braunschweig, the nucleus, these transcription factors are regulated via atypical
Germany.E-mailaddress:[email protected]
IkBproteins,whichincludeIkB (encodedbythegeneNfkbid),
Theonlineversionofthisarticlecontainssupplementalmaterial. NS
IkBz,BCL-3,andIkBh(16).
Abbreviationsusedinthisarticle:ChIP,chromatinimmunoprecipitation;DSS,dex-
We reported recently that the atypical NF-kB inhibitor IkB
tran sodium sulfate; EAE, experimentalautoimmune encephalomyelitis; fwd,for- NS
ward;HZI,HelmholtzCentreforInfectionResearch;IKK,inhibitorofkBkinase; regulates regulatory T cell (Treg) generation (17). Although
LP, lamina propria; LPL, LP lymphocyte; MFI, mean fluorescence intensity; rev, IkB 2/2 mice display a severereduction in Tregnumbers, they
reverse;Treg,regulatoryTcell;UBC,ubiquitinC. NS
donotdevelopsignsofspontaneousautoimmunity(17).Thus,we
Copyright(cid:1)2015byTheAmericanAssociationofImmunologists,Inc.0022-1767/15/$25.00 speculated that IkBNS might be important for the activation,
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401964
2 IkB REGULATESTh17DIFFERENTIATION
NS
proliferation, or cytokine production of proinflammatory effector IsolationofLPmononuclearcells
Tcells.Indeed,weobservedstrongexpressionofIkB inCD4+
NS ToisolatecellsfromLP,colonswereplacedinto15mlice-chilled0.5mM
cells and strongly impaired Th17 responses in vitro and in vivo. EDTAfor30min(onice),followedbysufficientrinsingwith13PBSto
Our results demonstrate that IkB is required for Th17 differ- removeresidualepithelium.Thenthetissuewascutintofinepiecesand
NS
entiation andfunction. digested in DMEM (Life Technologies) supplemented with 1 mg/ml
Collagenase D (Roche) and 0.1 mg/ml DNase I (Roche) three times for
30minat37˚C.Aftereachroundofincubation,cellsweresuspendedby
Materials and Methods passingthemthrougha100-mmmesh.Aftercentrifugation,thecellpellet
Mousestrains wasresuspendedina40%isotonicPercollsolution(GEHealthcare)and
underlain with an 80% isotonic Percoll solution. After centrifugation
B6.129/SV-NFKBID(tm1Clay)mice,laterreferredtoasIkB 2/2mice,were (900 3 g, room temperature for 20 min), LP lymphocytes (LPLs) were
describedpreviously(18).IkB 2/2micewerecrossedtoFNoSxp3DTR-eGFP yieldedfromtheinterfacecellringofthe40–80%Percollgradient.Cells
mice, as previously describedN,Slater referred as IkB 2/2Foxp3DTR-eGFP werewashedwithPBScontaining2%FCSandusedforfurtherstudies.
reportermice(17).IkB 2/2,IkB 2/2Foxp3DTR-eGFNP,SandB6.PL-Thy1a/
CyJmice(Thy1.1mice)NwSerebredNuSnderspecificpathogen–freeconditions Invitro generationofThcellsubsets
iBn6.t1h2e9San7i-mRaagl1ftamc1iMliotymo(fRAthGe1H2e/2lm)hmoilctzeC(1e9n)treweforer IbnrfeedctaiotnthReesaenairmchal. CD4+CD62LhighCD252 naive T cells were sorted via flow cytometry
(FACSAria II; BD Biosciences or MoFlo; Beckman and Coulter) and
facility of the Charite´ – Universita¨tsmedizin Berlin. All animal experi-
resuspended in primary T cell medium (IMDM containing 10% FCS,
mentsandbreedingwereperformedinaccordancewiththeguidelinesof
50 mg/ml penicillin/streptomycin, 25 mM HEPES, 0.05 mM 2-ME, 1%
nationalandlocalauthorities. nonessentialaminoacids,1mMsodiumpyruvate).Two3105cellswere
Chronicdextransodiumsulfate–inducedcolitis seededperwell(96-wellplate)andwereactivatedwithplate-boundanti-
CD3 and anti-CD28 (both from BioLegend) in the presence of priming
Toinducecolitis,micewerefedinfourcycles,eachcomprising7d,with cytokinesandinhibitoryAbs,accordingtotherespectiveThsubset:Th0- D
drinkingwatercontaining3%(w/v)dextransodiumsulfate(DSS)asaninitial 2 mg/ml anti-CD3 (145-2C11; BioLegend), 2 mg/ml anti-CD28 (37.51; o
w
doseand2%(w/v)DSSforallfurthertreatments,followedby14doffeeding BioLegend),10mg/mlanti–IL-4(11B11;madein-house),and10mg/ml n
withDSS-freewater.Inflammationofthecolonwasassessedattheendofthe anti–IFN-g (XMG1.2; made in-house); Th1- 2 mg/ml anti-CD3 (145- lo
a
last cycle. Body weight was measured during the progression of colitis. 2C11; BioLegend), 2 mg/ml anti-CD28 (37.51; BioLegend), 10 mg/ml de
Cvioonussilsyte(n1c7y).ofAtthethestoeonldanodf trheectaelxpbelereimdienngt,wthereelsecnogrtehdoafstdheesccroibloendpwraes- atenmti–s)I;La-4nd(1T1hB1171-;3mmagd/emlina-nhtoiu-CseD),3a(n1d451-20C1n1g;/mBlioILLe-g1e2nd(R),&5DmgS/yms-l d fro
measuredfromthececumtotheanus.Toanalyzehistologicalchangesinthe anti-CD28 (37.51; BioLegend), 10 mg/ml anti–IL-2 (JES6-1A12; Bio- m
cpaonaldroasnffit,anic,noealdnodnwcistuhatmHinp&tloeEs2.-Fwmiexmreedsecficoxtleioodnnssi.naTm4hp%elecsopwlaorenarfesoesrccmotiaroelnddseihwnyedarebe,lidenemdpeabdreadmfdfiaenndinzeeirnd, LcILien-ge1ebnTdG()R,F&1-b0D(mRSg&y/msDtelmaSnyst)si,t–eaImFnNds)-,2g300(XnngMg/m/Gml1l.T2I;NL-mF6-aa(dRe(&PineD-phrSooyuTsseetce)hm,)2.s)C,nge1/l0mlsnlwgp/eomrre-l http://w
asdescribedearlier(17).ThehistologicalscoreofDSScolitisisthesumof analyzedonday4. w
w
theindividualscoresforinflammatorycellinfiltrationsandtissuedamage. Invitro expansionofCD4+CD252Tcells .jim
Adoptive-transfercolitis After sorting of CD4+CD252 T cells, 4 3 106 cells were expanded in mu
Foradoptive-transfercolitis,spleenandlymphnodeswereusedtoisolate RPMI 1640 supplemented with 10% FCS, 1 mM sodium pyruvate, 13 no
wCTrDeerg4es+sCforDrot2emd52wviiaTldfl-cotyewpllecsyoftrroomImkeBtrNwySi2(ldF/2-AtFyCpoSexApor3irDaTIIRkI;-BeBGNFDSP2Br/2eipoosmcrtiieecrnecmeoisrc)eCo.rDTb4hy+eGucsFeinPllg+s p2n-loaMnteeE-s.bseoFnuotnriadelxapanamtnii-snCiooDna,3cciaednlslds,w520emrengg/sm/tmimllsuoplaleutnebidcleiilnlainant/sis-itCxre-Dwp2teo8llmipynlcattihene,wapintrheds1e5nm0cgem/mMofl bl.org/
y
MACSseparationkits(CD4TCellIsolationKit,CD25-PEKit;Miltenyi 10ng/mlmurineIL-2.Onday3,cellsweretransferredintoa10-cmdish g
Biotec),asdescribedinthesupplier’smanual.Atotalof200ml13PBS inatotalvolumeof15mlfreshRPMI1640withsupplements.Onday6, u
e
cCoDn4ta+iGniFnPg+5Tr3eg1s,0w5eCrDe4in+jCecDte2d52i.pT.icnetollsR,AalGo1n2e/o2rmtoigcee.thBeordwyiwthe5igh3tw10as5 5fo3r 210m6itnoa1t0830010rp6mcelaltsrwoeormesteeemdpederoatnurae.12C-ewllesllwplearteesatnimducleantterdifuwgiethd st on
monitoredduringcolitisprogression.Consistencyofthestoolandrectal coatedanti-CD3(10mg/ml)plusanti-CD28(5mg/ml)for15or30minor J
a
bleedingwerescoredasdescribedearlier(17).Thelengthofthecolonwas for1,2,or4h;stimulatedwithPMA(10ng/ml)plusionomycin(1mM) nu
measuredfromthececumtotheanus,andcolonsampleswerescoredin for 2 h; or left untreated. Subsequently, cells were analyzed by Western ary
ablindedmanner,asdescribedpreviously(17). blotting. 5
Citrobacterrodentium infection ProliferationanalysisbyCFSEstaining , 20
1
9
MicewereinfectedwithC.rodentiumstrainICC180expressingthelux- For proliferation analysis, sorted naive T cells were stained with CFSE
operon(courtesyofS.Wiles,Auckland,NewZealand).C.rodentiumwere (eBioscience).Briefly,Tcellswerewashedtwotimeswith13PBSand
culturedinLennoxbrothmedium(Roth)at37˚Covernight.Thenextday, resuspendedin13PBStoaconcentrationof23106/ml.CFSEwasadded
thebacteriawereadjustedto131011CFUin1ml13PBS.Atotalof toafinalconcentrationof5mM.Cellswereincubatedfor10min(CFSE)
131010C.rodentiumwasadministeredtomiceorally.Bodyweightwas at 37˚C in the dark and cultured under Th0-, Th1-, or Th17-polarizing
monitoredduringthe progressionofinfection. Bacteria-derived biolumi- conditions. On day 4, cells were stained for surface markers and intra-
nescenceduringinfectionwasmeasuredinvivobybioluminescenceim- cellularproteinsandanalyzedbyflowcytometry.
aging using IVIS Spectrum CT. At day 10, the length of the colon was
measured from the cecum to the anus. The stool was collected in 1 ml EnrichmentofTh17cellsviamouseIL-17Secretion Assay
Lennoxbroth,weighed,homogenized,anddilutedserially.Todetermine
NaiveTcellswereisolatedandculturedunderTh17-polarizingconditions,
thebacterialloadinorgans,theliverandspleenwerehomogenizedand
asdescribedabove.Onday4,cellswerestimulatedwithPMA(10ng/ml)
diluted serially. Serial dilutions were added to MacConkey Agar (Roth)
andionomycin(1mM;bothfromSigma-Aldrich)for3hat37˚C.Amouse
andculturedat37˚Cfor24h.CFUwerecountedand,forfeces,bacterial
IL-17SecretionAssay(MiltenyiBiotec)wasusedforenrichmentofIL-
burden was normalized to stool weight. For histology of C. rodentium– 17+ cells. The cells were incubated and enriched according to the sup-
infected mice, whole-colon samples were placed in a shape of a “Swiss
plier’sinstructionmanual,andisolatedcellswereusedtoanalyzecytokine
roll”andfixedin4%Roti-Histofix(Roth),embeddedinparaffin,cutinto
secretion.
8-mmsections,deparaffinized,stainedwithH&E,andscoredinablinded
manner. The histological sections were scored for epithelial hyperplasia Analyses ofcytokine expression
(scorebasedonpercentageabovetheheightofthecontrolwhere0=no
change,1=1–50%,2=51–100%,and3=.100%).Epithelialintegrity ToanalyzethecytokinesecretionofenrichedIL-17–secretingcellsbyPro-
was scored as follows: 0, no change; 1, mild epithelial ulceration and teomeProfilerArray,0.83106cellswerestimulatedwithPMA(10ng/ml)
crypticdestruction;2,moderateepithelialulcerationandcrypticdestruc- andionomycin(1mM;bothfromSigma-Aldrich).After4h,thesupernatant
tion; and 3, severe epithelial ulceration and cryptic destruction. Tissue wastakenandusedundilutedordiluted1:10or1:50forcytokineanalysis
sections were scored for mononuclear cell infiltration as follows: 0, no usingtheProteomeProfilerArray(R&DSystems).Thearraymembranewas
change;1,mild;2,moderate;and3,severe. activatedandthereafterincubatedwiththeTh17supernatant,asdescribedin
TheJournal ofImmunology 3
the user manual. Finally, the chemiluminescence of the membrane was 6 h in medium containing IL-23 (20 ng/ml) with PMA (10 ng/ml) and
measuredusingtheFusionFX-7camera(VilberLourmat),andthevolumeof ionomycin(1mg/ml;bothfromSigma-Aldrich).BrefeldinA(10mg/ml;
bandswasquantifiedwiththeprogramBIO-1D(Peqlab). Sigma-Aldrich)wasaddedforthelast3h.Cellswereprocessedforflow
To analyze the cytokine secretion of Th17 cells by flow cytometry, cytometry,asdescribedabove,andanFc-blockstep(10min,onice)was
0.83106enrichedIL-17–secretingcellswerestimulatedwithPMA(10 included before cell surface staining and the fixation/permeabilization
ng/ml)andionomycin(1mM;bothfromSigma-Aldrich).After4h,the buffers(eBioscience)wereused.
supernatant was used for cytokine analysis with a FlowCytomix kit
(eBioscience).Thesupernatantwasusedundilutedorwasdiluted1:10 Celllysisanddeterminationofprotein concentration
or 1:50. The standard curves and samples were prepared according to
Total-celllysateswereobtainedbylysinginTPNElysisbuffer(13PBS,
themanufacturer’sinstructionsandmeasuredbyflowcytometryinthe
BD LSRII FlowCytometer System or theBDLSRFortessa (BDBio- 300 mM NaCl,2 mMEDTA,1% v/vTriton X-100)supplemented with
1mMPMSF,1mg/mlproteaseinhibitormix(aprotinin,leupeptin,Pepstatin
sciences).
A,chymostatin),and0.4mMsodiumorthovanadate.Incubationfor20min
on ice was followed by centrifugation (14,000 rpm, 4˚C, 15 min). The
Flow cytometricanalyses
proteinconcentrationofthelysatewasdeterminedbyaBCAProteinAssay
Forintracellularcytokinestaining,cellswerestimulatedfor4hwithPMA (ThermoScientific),followingthemanufacturer’sinstructions.
(10ng/ml)andionomycin(1mM;bothfromSigma-Aldrich).BrefeldinA
(10mg/ml;Sigma-Aldrich)wasaddedduringthelast2hofstimulation. Westernblotanalysis
Live/deadstainingwasperformedin100ml13 PBS(Invitrogen)con-
For protein separation via SDS-PAGE, 20–40 mg the protein lysate was
taining one of the LIVE/DEAD dyes from Life Technologies (30 min,
loadedontoagel.Proteinswereseparatedona12%polyacrylamidegel.For
4˚C,inthedark).SurfacemarkerswereincubatedinFACSbuffer(13
furtheranalysis,theproteinswereblottedontoapolyvinylidenedifluoride
PBS,2%w/vBSA,0.01%v/vNaN )at4˚C,for15to20mininthedark,
3 membrane (GE Healthcare) and detected with the following Abs: b-actin
using the following fluorochrome-conjugated Abs: CD3–allophycocya-
(AC-74;Sigma-Aldrich),c-Rel(290512;R&DSystems),ERK(9102;Cell
nin–eFluor780(17A2;eBioscience),CD3-FITC(145-2C11;BD),CD3-
SignalingTechnology);IkB (rabbit;madein-house);IkBa(C-21;Santa D
PECy7 (145-2C11; eBioscience), CD4–Alexa Fluor 488 (RM4-5; BD), Cruz),p38(9212;CellSignNalSingTechnology),p50/105(E381;Epitomics), ow
CD4-allophycocyanin (RM4-5; eBioscience), CD4-FITC (RM4-5; p52/100 (4882; Cell Signaling Technology), p65 (C-20; Santa Cruz Bio- n
eBioscience),CD4–PacificBlue(RM4-5;BioLegend),CD4-PE(RM4-5; technology),p-Akt(4060;CellSignalingTechnology),p-ERK(4370;Cell loa
BD), CD4–PerCP–Cy5.5 (RM4-5; eBioscience), CD8-allophycocyanin d
Signaling Technology), p-IkBa (2859; Cell Signaling Technology), p-p38 e
(53-6.7; BD), CD8-FITC (53-6.7; BD), CD25-allophycocyanin (PC61; d
BD),andCD62L-PerCPCy5.5(MEL-14;eBioscience).Subsequently,the (T9e2c1h1n;olCogeyll);Spig-TnyalrionsgineTe(c4hGn1o0lo;gkyi)n,dpg–iSftAoPfKP-JrNofK.B(.92S5c1h;ravCeenll, OSitgton-avloinng- fro
cells were fixed and permeabilized using the FoxP3 Staining Buffer Set m
(Miltenyi Biotec) for 30 min (4˚C, in the dark). Intracellular cytokine GuerickeUniversity),andRelB(C-19;SantaCruzBiotechnology). h
FstIaTinCing(XwMasGp1e.2rf)o,rmIFeNd-ign–1P0E0(mXlM13G1p.2er)m, eIaFbNil-igz–atPioEnCybu7ff(eXrMusGin1g.2IF),Na-gnd– Chromatinimmunoprecipitation ttp://w
IL-17A–allophycocyanin (eBio17B7; all from eBioscience) Abs (4˚C, for For chromatin immunoprecipitation (ChIP) analyses, Th17 cells were w
30mininthedark).LabeledcellswereanalyzedwithaBDLSRIIFlow generated and stimulated with PMA/ionomycin for 4 h, as described w
CytometerSystemorBDLSRFortessa(BDBiosciences). above. ChIP was performed with a ChIP-IT Express Enzymatic kit .jim
ForflowcytometricanalysisuponC.rodentiuminfection,cellsfromthe (Active Motif), using a modified procedure to achieve ChIP without m
spleenandmesentericlymphnodes,aswellasLPLs,wererestimulatedfor cross-linking.Hence,proceduralstepstoremoveparaformaldehydeand u
n
o
l.o
rg
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
5
, 2
0
1
9
FIGURE1. IkB deficiencyresultsinimpairedTh17developmentduringgutinflammation.(A–E)TransfercolitiswasinducedinRAG12/2miceby
NS
adoptivetransferoffreshlyisolatedCD4+CD252TcellsfromIkB +/+(n=35)orIkB 2/2(n=36)mice.MiceinjectedwithPBS(n=17)servedas
NS NS
controls.(A)Thehistologicalscoreisthesumofindividualscoresofinflammatorycellinfiltrationandtissuedamage.(B–E)Lymphocytesofthecolonwere
isolated,andtheexpressionofsurfacemarkersandintracellularcytokineswasanalyzedbyflowcytometry.(B)Representativedotplots.FrequencyofIL-
17A+(C),IL-17A+/IFN-g+(D),andIFN-g+cells(E)withintheCD4+Tcells.(F)Transfercolitiswastriggeredasdescribedabove.Whereindicated,GFP+
TregsofIkB 2/2Foxp3DTR-eGFPorIkB +/+Foxp3DTR-eGFPreportermicewerecotransferred.IFN-g+lymphocytesofthecolonwereanalyzedbyflow
NS NS
cytometry.Datawerepooledfromtwo(F)orfour(A–E)independentexperiments.Horizontallinesrepresentthemean;errorbarsshowSEM.*p,0.05,
**p,0.01,***p,0.001,****p,0.0001,two-tailedMann–WhitneyUtest.
4 IkB REGULATESTh17DIFFERENTIATION
NS
to reverse cross-linking were not performed. Rabbit polyclonal IkB Statistics
NS
Abwasused;afterelutionoftheprecipitatedDNA,IL-10wasamplified
byreal-timePCR. GraphPadPrismsoftware(GraphPad)wasusedforallstatisticalanalyses.
Todeterminestatisticalsignificance,thetwo-tailedMann-WhitneyUtestor
Real-timePCR Studentttestwasused;errorbarsrepresenttheSEM.
cDNA was used as a template for real-time PCR using SYBR Green Results
(Roche). Ubiquitin C (UBC) was used as a housekeeping gene for nor- IkB triggersTh17developmentduringgutinflammation
malization. Measurements were run in duplicates in the LightCycler 480 NS
system using the following primers: GM-CSF forward (fwd), 59-GCATG- In response to intestinal infections and inflammation, Th17 cells
TAGAGGCCATCAAAGA-39andGM-CSFreverse(rev),59-CGGGTCTG- accumulateinthegutandsurroundinglymphoidtissues(20,21).
CACACATGTTA-39; IFN-g fwd, 59-ATCTGGAGGAACTGGCAAAA-39
TounveilwhetherIkB deficiencyaffectsThcelldifferentiation,
andIFN-grev,59-TTCAAGACTTCAAAGAGTCTGAGGTA-39;IkB fwd, NS
59-GGGCTCTTTTCCCATTCTCT-39andIkB rev,59-GGACACANASTCC- weinducedchroniccolitisthatispromotedbyTh1andTh17,as
AGCCTGTCT-39; IL-2 fwd, 59-CCTGAGCNASGGATGGAGAATTACA-39 wellasBcells(22),inIkB 2/2andwild-typemicebyrepeated
NS
and IL-2 rev, 59-TCCAGAACATGCCGCAGAG-39; IL-10 fwd, 59-TGCC- DSSfeeding.AlthoughDSS-fedIkB 2/2andwild-typemicedid
NS
AAGCCTTATCGGAAATG-39 and IL-10 rev, 59-CCCAGGGAATTCAAA-
not display marked histological differences, there was inherent
TGCTCC-39; IL-17a fwd, 59-CAGGGAGAGCTTCATCTGTGT-39 and IL- colon damage in the nontreated IkB 2/2 mice that was not
17a rev, 59-GCTGAGCTTTGAGGGATGAT-39; IL-17f fwd, 59-CAAAAC- NS
CAGGGCATTTCTGT-39 and IL-17f rev, 59-ATGGTGCTGTCTTCCTGA- present in wild-type mice (Supplemental Fig. 1A–D). Most im-
CC-39;IL-22fwd,59-TCATCGGGGAGAAACTGTTC-39andIL-22rev,59- portantly,thefrequencyofIL-17A+TcellswasdecreasedinLPLs
CATGTAGGGCTGGAACCTGT-39;MIP-1afwd,59-ATGAAGGTCTCCA- of IkB 2/2 mice compared with wild-type mice (Supplemental
CCACTG-39 and MIP-1a rev, 59-GCATTCAGTTCCAGGTCA-39; RORgt NS
Fig.1E,1F).Interestingly,Th17frequenciesalreadywerereduced
fwd,59-TGCAAGACTCATCGACAAGG-39andRORgtrev,59-AGGGGA-
TTCAACATCAGTGC-39; and UBC fwd, 59-AAGAGAATCCACAAGG- in nontreated, IkBNS2/2 animals compared with respectivewild- Do
AATTGAATG-39andUBCrev,59-CAACAGGACCTGCTGAACACTG-39. typemice (Supplemental Fig.1E, 1F). w
n
lo
a
d
e
d
fro
m
h
ttp
://w
w
w
.jim
m
u
n
o
l.o
rg
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
5
, 2
0
1
9
FIGURE2. ImpairedTh1andTh17differentiationintheabsenceofIkB .CD4+CD252CD62L+naiveTcellswereisolatedbyflowcytometryand
NS
culturedunderTh1-polarizing,Th17-polarizing,ornonpolarizing(Th0)conditions.CellswereanalyzedwithorwithoutPMA/ionomycin(P/I)(A,B,E,and
F)oranti-CD3/anti-CD28stimulation(4h)(CandD).(AandC)TheinductionofIkB /NfkbidmRNAwasanalyzedbyreal-timePCR.Therelative
NS
expressionofNfkbidwascalculatedbynormalizationtounstimulatedTcells.(BandD)WesternblotanalysesofIkB andb-actinproteinexpression.(D)
NS
Thesingleblot(rightpanel)representstheproteinexpressionofIkB -deficientTh17cells.(E)TheexpressionofIFN-gandIL-17Awasanalyzedbyflow
NS
cytometryinwild-typeandIkB 2/2TcellsuponTh1andTh17polarization.Representativedotplotsareshown.(F)Statisticalanalysisofthefrequencies
NS
ofinvitro–differentiatedIL-17A2IFN-g+(Th1)andIL-17A+IFN-g2(Th17)cells.Datashownarerepresentativeofatleastthree(A–D)orfour(F)in-
dependentexperiments.ErrorbarsshowSEM.*p,0.05,**p,0.01,two-tailedMann-WhitneyUtest.
TheJournal ofImmunology 5
Next, we investigated whether Th17 differentiation also is im- Th17-polarizedcellsuponrestimulationwithPMAandionomycin
paired in T cell–based transfer colitis. Rag12/2 mice receiving (Fig.2A).Inaddition,highIkB expressionwasobservedatthe
NS
IkB 2/2Tcellsexhibitedsignificantlymorecolondamagethan proteinlevelinreactivatedTcells(Fig.2B).Similarfindingswere
NS
did wild-type controls (Fig. 1A). Importantly, the induction of obtainedwhenTcellswererestimulatedwithanti-CD3andanti-
bothIL-17A+IFN-g2(Fig.1B,1C)andIL-17A+IFN-g+(Fig.1D) CD28 Abs (Fig. 2C, 2D). Th1 cells exhibited a higher IkB
NS
TcellswascompletelybluntedinIkB 2/2mice.Incontrast,the proteinexpressionthandidTh17cells.Notably,thenonactivated
NS
frequency of IL-17A2IFN-g+ T cells (Fig. 1E) was slightly, but Th17cellsdisplayedhigherIkB expressioncomparedwiththe
NS
significantly, increased in IkB 2/2 mice, demonstrating the im- othernonactivatedcells(Fig.2B,2D),substantiatingapivotalrole
NS
portanceofIkB fortheformationofTh17cellsintheinflamed forIkB specificallyinthisTcellsubset.Indeed,duringinvitro
NS NS
gut.Previously,wereportedareducednumberofTregsinIkB 2/2 priming we monitored severely reduced frequencies of both IL-
NS
mice(17).ToanalyzewhethertheincreasedfrequencyofTh1cells 17A+ Th17 cells and IFN-g+ Th1 cells in the absence of IkB
NS
in IkB -deficient animals arose from reduced numbers of Tregs, (Fig.2E,2F).WeconcludethatIkB expressionisnotrestricted
NS NS
TregsfromIkB 2/2Foxp3DTR-eGFPorIkB +/+Foxp3DTR-eGFPre- toasingleThcellsubsetbutforcesthedifferentiationofTh17and
NS NS
porter mice were cotransferred (Fig. 1F). The cotransfer of Tregs Th1cellsinvitro.
hadnoeffectontheincreasedTh1cellfrequencyinIkB 2/2mice,
NS ProliferationdefectinIkB -deficientTh17cells
regardlessofwhethertheTregswereIkB deficientorproficient. NS
NS
Insummary,bothcolitismodelsrevealedarequirementforIkB ToidentifythereasonforthereducedfrequencyofTh17andTh1
NS
inTh17celldifferentiationduringgutinflammation. cells induced by IkBNS deficiency, we first analyzed Th cell
proliferation in vitro. Although all T cells proliferated equally
IkBNSdrives Th17andTh1developmentinvitro during the first 2 d of culture, IkBNS2/2 T cells primed for both D
TodeterminewhetherIkBNSisdifferentiallyexpressedinThcell Th17 and Th1 differentiation proliferated much less compared ow
subsets, we analyzed IkBNS (Nfkbid) expression at the mRNA withthewild-typecellsinthefollowingdays(SupplementalFig. nlo
level by real-time PCR (Fig. 2). We detected comparable Nfkbid 2).Todeterminewhetherwild-typecellscouldcompensateforthe ad
mRNA expression in unprimed T cells (Th0) and in Th1- and proliferation defect of IkB 2/2 cells when cocultured, we dif- ed
NS fro
m
h
ttp
://w
w
w
.jim
m
u
n
o
l.o
rg
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
5
, 2
0
1
9
FIGURE3. IkB -deficientTcellsdisplayaproliferationdefectduringThcelldifferentiationinvitro.CD4+CD252CD62L+naiveTcellswereisolated
NS
byflowcytometryandculturedunderTh1-polarizing,Th17-polarizing,ornonpolarizing(Th0)conditions.CellswereanalyzedafterPMA/ionomycin(P/I,
4h)stimulation.NaiveIkB +/+(CD90.1+)andIkB 2/2(CD90.2+)Tcellsweremixedataratioof1:1,labeledwithCFSE,andculturedunderTh1-
NS NS
polarizing,Th17-polarizing,ornonpolarizing(Th0)conditions.Proliferationofeachgenotypewasanalyzedbyflowcytometry.(A)Representativedot
plotsofviableIkB +/+(CD90.1+)andIkB 2/2(CD90.2+)cells.(B)RepresentativegraphsofCSFEintensityofIFN-g+orIL-17A+cells(pregatedto
NS NS
CD90.1+orCD90.2+cells).(C)Statisticalanalysisofthedivisionindexofwild-typeandIkB 2/2Tcells.ErrorbarsshowtheSEMoffourindependent
NS
experiments.*p,0.05,two-tailedMann–WhitneyUtest.
6 IkB REGULATESTh17DIFFERENTIATION
NS
ferentiated IkB 2/2 (CD90.2+) T cells in the presence of con- (encodingRORgt)inIkB 2/2andwild-typeTh1andTh17cells
NS NS
genicwild-type(CD90.1+)cells,bothstainedwithCFSE.Again, by real-time PCR. Despite defective Th17 differentiation of
the frequencies of IkB 2/2 Th0, Th1, and Th17 cells showed IkB 2/2 cells, Rorc expression was comparable in wild-type
NS NS
a50%reductioncomparedwithwild-typecells,suggestingacell- and IkB 2/2 Th17 cells (Fig. 4A). Next, we analyzed whether
NS
intrinsic effect of IkB on T cell differentiation (Fig. 3A). The IkB 2/2 Th17 cells display an altered cytokine expression pro-
NS NS
CFSE profile and mean number of divisions/cell (division index) file. Coculturing wild-type and IkB 2/2 T cells under Th17-
NS
revealedenhancedproliferationofwild-typeTh0,Th1,andTh17 polarizing conditions, we observed, in addition to the prolifera-
cells compared with IkB 2/2 cells (Fig. 3B, 3C). Remarkably, tion defect (Fig. 3), a significantly decreased mean fluorescence
NS
the proliferation defect caused by IkB deficiency was most intensity (MFI) of IL-17A inIkB 2/2 cells(Fig. 4B, 4C), indi-
NS NS
pronouncedinTh17-differentiatedcells.Ofnote,theproliferation cating a role for IkB in Th17 cell function. To determine
NS
defect was not due to impaired proximal TCR signaling because whether IkB also impairs the expression of other cytokines,
NS
tyrosinephosphorylation;theactivationofthekinasesAkt,ERK, acytokinearraymembranewasperformed.Weenrichedinvitro–
and p38; expression of the NF-kB family members c-Rel, NF- generated wild-type and IkB 2/2 Th17 cells to obtain compa-
NS
kB1, NF-kB2, p65, and RelB; and the expression and phosphor- rable numbers of IL-17A–secreting cells. The results revealed
ylationstatus ofIkBawereunaltered (Supplemental Fig.3). reduced secretion of IL-2, IL-10, GM-CSF (Csf2), and MIP-1a
(Ccl3) by IkB 2/2 Th17 cells (Fig. 4D, Supplemental Fig. 4).
IkB regulatesTh17cytokine expression NS
NS Using a bead-based assay and flow cytometry, the reduced se-
Because RORgt is an essential transcription factor for Th17 dif- cretion of the cytokines IL-2, IL-10, and MIP-1a in IkB 2/2
NS
ferentiationandfunction(6),weanalyzedtheexpressionofRorc Th17 cells was verified (Fig. 4E). Nevertheless, secretion of the
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://w
w
w
.jim
m
u
n
o
l.o
rg
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
5
, 2
0
1
9
FIGURE4. ExpressionofTh17-specificcytokinesisimpairedbyIkB deficiency.CD4+CD252CD62L+naiveTcellswereisolatedbyflowcytometry
NS
andculturedunderTh1-polarizing,Th17-polarizing,ornonpolarizing(Th0)conditions.(A)TcellswerestimulatedwithPMA/ionomycin(P/I)for4hor
wereleftuntreated.Subsequently,theexpressionofRorcwasmeasuredbyreal-timePCR,andthefoldinductionwascalculatedbynormalizationtoUBC.
(B)CFSE-labeledCD4+CD252CD62L+naiveIkB +/+(CD90.1+)andIkB 2/2(CD90.2+)Tcellswereculturedata1:1ratiounderTh17-polarizing
NS NS
conditions.Onday4,IL-17Aexpressionwasanalyzedbyflowcytometry.MFIofIL-17Aforeveryproliferationcyclewasanalyzed.Representativedot
plotsshowingdifferentMFIofIL-17Awithinfiveproliferationsteps(G1–G5).(C)ThefoldreductionintheMFIofIL-17AinIkB 2/2cellscompared
NS
withIkB +/+cellswasdeterminedwithinthefiveproliferationstepsshownin(B).(D)IsolatednaiveTcellswereculturedunderTh17-primingconditions.
NS
IL-17A–expressingcellswereenrichedviaacytokine-secretionassay.RelativelevelsofcytokinesinthesupernatantofstimulatedIkB +/+andIkB 2/2
NS NS
Th17cellswereassessedusingacytokinearraymembrane.TheexpressionofIL-2,IL-10,MIP-1a,andGM-CSFinIkB 2/2cellsrelativetowild-type
NS
Th17cellsisshown.(E)Supernatantswerepreparedasin(D),andcytokinesweremeasuredbyabead-basedflowcytometryassay.(F)Isolatednaive
TcellswereculturedunderTh17-primingconditions.Subsequently,RNAwaspreparedforanalysesbyreal-timePCR.ThefoldinductionofIl2,Il10,
Il17a,Il17f,Il22,Ccl3,andCsf2inTh17cellswascalculatedrelativetononstimulatedTh17cellsafternormalizationtoUBC.Errorbarsrepresentthe
SEMoftwo(D),three(A),orfour(CandE)independentexperimentsorthemeanofsevendifferentiationsinfiveindependentexperiments(F).*p,0.05,
**p,0.01,two-tailedMann–WhitneyUtest.
TheJournal ofImmunology 7
Th17 cytokines IL-17A, IL-17F, and IL-22 was not affected by We next studied the impact of IkB deficiency on the host
NS
IkB deficiency. Furthermore, we analyzed the effect of IkB Tcellresponse.WefoundthatthefrequenciesofTh17cellswere
NS NS
on mRNA expression of the respective cytokines in stimulated reduced by ∼50% in the colonic LP and in the spleen (Fig. 6A,
Th17cellsbyreal-timePCR.Inaccordancewiththeproteindata, 6B). Interestingly, the frequency of IL-17A+IFN-g+ double-
we detected reduced mRNA expression of Il10 and Csf2 in positive cells showed a similar reduction in both organs (Fig. 6A,
IkB 2/2Th17cells,whereasIl17aandIl17fexpressionwasnot 6C), whereas the frequency of IFN-g+ Th1 cells was slightly
NS
affected (Fig. 4F).Interestingly,incontrast to IL-22cytokinese- reducedinthespleenofIkB 2/2micebutnotintheotherorgans
NS
cretion, IkB 2/2 Th17 cells showed an increased expression of investigated (Fig. 6A, 6D). In addition, we examined LP cell–
NS
IL-22mRNA.WeconcludethatIkB regulatestheexpressionof derived mRNA levels of the cytokines IFN-g, IL-10, IL-17A,
NS
multiplekeyTh17cytokines. IL-17F,andIL-22byreal-time PCR.Consistentwiththe previous
IkB 2/2 micedisplayimpairedTh17developmentandhigh data,IkBNS deficiency resultedindecreased mRNA expression of
NS Ifng, Il17a, Il17f, and Il10 by LPLs of the colon (Fig. 6E). In
susceptibility toC.rodentiuminfection
contrast with the invitro data, but consistent with the other cyto-
C. rodentium is a noninvasive pathogen that establishes acute kines in vivo, the expression of Il22 mRNA was slightly down-
infections in the murine large intestine (23). The adaptive im- regulatedinIkB 2/2mice(Fig.6E).
NS
mune response plays an important role for the host response to ToassesswhetherIkB deficiencyalsoaffectspathologyofthe
NS
C.rodentiumbecausemicelackingBandTcellsareunabletoclear colon, we investigated cellular infiltration and the level of tissue
the infection (24). C. rodentium induces a strong Th17 response damage in the colon of infected mice. Despite the increased
(25,26).Therefore,weexaminedwhetherIkBNSisrequiredforan bacterial burden, IkBNS2/2 mice exhibited significantly less im-
appropriate hostresponseagainst C.rodentium. munopathology in the large intestine compared with wild-type D
The bacterial burden in feces of IkBNS2/2 mice at day 10 mice (Fig. 6F), suggesting that the defective Th17 immune re- ow
postinfectionwassignificantlyincreasedcomparedwithwild-type sponse in these mice results in less inflammation-induced tissue nlo
mice (Fig. 5A). A closer investigation of colonization using live destruction.Altogether,ourdatademonstratethatIkBNSpromotes ade
ibnotvhivgoeniomtyagpiensguinntdilicdaatyed5a(Fsiigm.i5laBr,i5nCfe)c.tHioonwkeivneert,icalitnhomugichethoef Tinhf1ec7tiodnifsf.erentiation and cytokine expression to fight intestinal d fro
m
bdieoclruemasienebsectewnetesnigdnaaylsf7roamndth1e0,gtuhteosfigwnialld-intytepnesimtyiciencsrteaarsteeddtion IkBNSbindstotheIl10genelocus http
IkBNS2/2 mice, confirming impaired C. rodentium clearance in Theexpressionofseveralcytokinesisregulatedbytheproteinsof ://w
theseanimals(Fig.5B,5C).Inlinewiththisobservation,IkB 2/2 theIkBfamily(16).Forinstance,itwasdescribedthatIkBzand w
NS w
mdisicseemailnsaotiosunfofefrtehdepfraotmhogaennteoletvhaetelidvesrywstaesmsiicgniinfifceacntitolyn,enbheacnacuesde (I2k7B,N2S8)r.egInultahtiestshtuedeyx,pwreescsoionnsisotfenILtl-y6fobuynbdinthdaintgextporeitsssipornoomfoItLer- .jim
in IkBNS2/2micecompared with wild-type mice (Fig. 5D). These 10isaffectedbyIkBNSdeficiencyinvariousinvitroandinvivo mun
findingsindicatethatIkB isrequiredforrestrictingC.rodentium experiments. To investigate whether IkB regulates the expres- o
infectionstothegut. NS sionofIL-10directlybybindingtoitsgeNnSelocus,weperformed l.org
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
5
, 2
0
1
9
FIGURE5. IkB 2/2micearemoresusceptibletoC.rodentiuminfection.IkB +/+orIkB 2/2micewereinfectedorallywith131010C.rodentium
NS NS NS
bacteriaexpressingtheluxoperon.(A)AnalysisofC.rodentiumburdeninstoolofIkB +/+(n=14)orIkB 2/2(n=15)mice.CFU/gofstoolareplotted
NS NS
onalogarithmicscale.(B)ThebacterialloadinIkB +/+(n=3)orIkB 2/2(n=3)miceinfectedwithbioluminescentC.rodentiumwasmeasuredinvivo
NS NS
bybioluminescenceimaging(IVISSpectrumCT).Representativebioluminescenceimagesareshownforthedayofinfection(day0)andfordays5and10
postinfection.(C)StatisticalanalysesofthetotalfluxofbioluminescenceofC.rodentiumduringthecourseoftheexperiment.(D)Thebacterialloadinliver
(IkB +/+n=6,IkB 2/2n=8)andspleen(IkB +/+n=4,IkB 2/2n=9)wasanalyzed10dpostinfection.Horizontallinesin(A),(C),and(D)represent
NS NS NS NS
themean,anderrorbarsshowtheSEM.Datawerepooledfromatleastthreeindependentexperimentswithatleastthreemice/group(AandD)orare
representativeofoneexperimentwiththreemice/group(BandC).*p,0.05,**p,0.01,two-tailedMann–WhitneyUtest(AandD)orStudentttest(C).
8 IkB REGULATESTh17DIFFERENTIATION
NS
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://w
w
w
.jim
m
u
n
o
l.o
rg
aFdImGiUnRistEer6ed. oFraelwlyertoTIhk1B7ce+l/l+sodrevIkeBlop2in/2IkmBiNcSe2./A2nmimicaelsuwpoerneCa.narolydzeendtiu1m0dinpfeocsttiionnfe.cAtiotno.ta(lAo)fC1el3ls1fr0o1m0bthioelucmolionneswceenretCis.orlaotdeedn,tisutaminbeadcwteirtihaAwbass by g/
NS NS u
directedagainstCD3,CD4,IFN-g,andIL-17A,andmeasuredbyflowcytometry.Representativedotplotsareshown.(B–D)Cellsfromcolon,spleen,and es
mesentericlymphnodes(mLN)wereisolated,stained,andmeasuredbyflowcytometryasin(A).FrequencyofIL-17A+(B),IL-17A+/IFN-g+(C),and t o
n
IFN-g+ cells (D) within CD4+CD3+ T cells. (E) The relative expression of Ifng, Il10, Il17a, Il17f, and Il22 in LPLs was measured by real-time PCR J
a
andnormalizedtoUBC.(F)RepresentativeH&Estainingofcolonsection.Scalebar,200mm(upperpanels).Thehistologicalscorewasdeterminedfrom n
u
ttcwhorloeoeninmsdeeiccpteei/ongndrsoeunoptfeI(kxinpBetNorSitm+a/l+eIn(ktnsB=wNS6i+t)h/+oarntIl=keBa9sN,tSItk2wB/2oNm(Sn2ic/=2e/4gn)r=omui1pc1e([FoE)n].)Hdoaoryrwi1ze0orneptoapslotlioinnlfeeedsctfrireoopnmre(sltoehwnreteetrhipenadmneepelae)nn.,dDaeanntdtaeaexrreproerrreibpmareerssnetssnhtwoawtiitvhtehtowefoStEwtoMof.eox*upprem,riimc0ee.0/ng5trs,o*wu*pipth(B,a–tD0le.)0ao1srt, ary 5, 2
0
two-tailedMann–WhitneyUtest. 1
9
nativeChIPexperiments.WedetectedincreasedbindingofIkB ther unveiled that the proliferation defect of IkB 2/2 T cells
NS NS
to the Il10 gene locus in stimulated Th17 cells compared with primed for Th17 differentiation could not be overcome by the
unstimulated Th17 cells and IkB -deficient controls (Fig. 7). presence of wild-type cells, indicating that IkB ’s function is
NS NS
This suggests that IL-10 is a direct IkB target gene and that Tcellintrinsic.
NS
IkB regulates Th17 differentiation, at least in part, by direct Remarkably, we also detected expression of IkB in acutely
NS NS
control ofcytokineexpression. stimulated Th1 and Th0 cells. Overall, IkB expression in Th1
NS
andTh0cellswascomparabletotheexpressionmeasuredinTh17
Discussion cells.DifferencesbetweenmRNAandproteinexpressionmightbe
WerecentlyreportedthatIkB regulatestheinductionofFoxp3 duetoposttranscriptionalcontrolofIkB expressionbyRoquin,
NS NS
expression in Tregs (17). Although IkB 2/2 mice displayed whichwasdescribedpreviouslyincelllines(29).Ofnote,fewer
NS
a severe reduction in immunosuppressive Tregs, we found no Th1 cells were generated in vitro from IkB 2/2 naive cells as
NS
signs of autoimmune disease. Thus, we speculated that IkB a result of impaired proliferation. Importantly, IkB does not
NS NS
promotesthedevelopmentorfunctionofproinflammatoryTcells. affect proximal TCR signaling. Thus, IkB forces proliferation
NS
In this study, we show that IkB additionally controls the pro- furtherdownstreaminthesignalingcascade.Noteworthy,invivo
NS
liferationandcytokineexpressionofdifferentiatedCD4+effector proliferation of IkB 2/2 Tregs is not altered (17). Therefore,
NS
T cells. Although impaired in vitro proliferation of IkB 2/2 IkB acts differentially in Tregs and effector T cells. For in-
NS NS
thymic and peripheral CD4+ and CD8+ cells upon anti-CD3 and stance, IkB is required for development but not function of
NS
anti-CD28 stimulation was reported (18), our data indicate that Tregs (17), whereas in differentiated CD4+ Th cells, it regulates
Th17 cellsare particularly affected byIkB deficiency. We fur- proliferationandeffectorcytokineproduction.Thus,ourfindings
NS
TheJournal ofImmunology 9
InadditiontoIL-17AandIL-17F,whichwereinvolvedinlate
host defense against C. rodentium, the IL-10 cytokine family
member IL-22 is essential in early host protection (26, 38). IL-
222/2miceexhibitincreaseddamagetotheintestinalepithelium,
systemic bacterial burden, and mortality (39). In this study, we
observedanincreasedmRNA,butunalteredprotein,expressionof
IL-22 by IkB 2/2 cells in vitro. Of note, Il22 expression was
NS
reduced invivo upon C. rodentium infection. The reduced IL-22
levels might be sufficient to support protection of the intestinal
epithelium; however, the defects in the expression of IL-22 and
other cytokines wereinsufficientto reduce bacterial burden.
WhydoweseedecreasedhistologicaldamageinC.rodentium–
FCIDG2U52RCED76.2L+IknBaNivSebTindcseltlostwheereILi-s1o0lagteednebylocfluoswincyTtohm17etcrye,llsc.ulCtuDre4d+ infected IkBNS2/2 micedespite theincrease inbacterial burden?
Interestingly, IL-23 also was reported to induce GM-CSF ex-
under Th17-polarizing conditions, and stimulated with PMA/ionomycin
for4h.ChIPwasperformedusinganIkB -specificrabbitpolyclonalAb. pression in Th17 cells, which causes pathogenicity in EAE, the
NS
Representative quantitative PCR analysis of amplification of IL-10 is murinemodelformultiplesclerosis(9,10).Thus,thereductionin
shown.IkB 2/2cellsservedascontrol.Themeanoftwoexperimentsis GM-CSFproductionbyIkB 2/2Th17cellsmayaccountforthe
NS NS
shown,errorbarsdisplaytheSEM. reducedtissuedamageinthegut.Alternatively,impairedbacterial
clearanceinconjunctionwithreducedtissuedamagewasreported
provide an explanation for our previous observation that Treg recently (40). In this infection model, Th17 differentiation de- D
reductiondoesnotcauseanautoimmunephenotypeinIkBNS2/2 pended on the presence of Tregs and the consumption of IL-2. ow
n
mice (17). Thus,itappearsthatTh17cellsduringC.rodentiuminfectionare lo
a
We(17)andotherinvestigators(30)previouslydescribedsevere notonlyinvolvedinpathogenclearance(i.e.,beneficial),theyalso d
DpaSthSo-ilnodguiccaeldchaanndgetsrainnstfheerccoololintisi,nIrkesBpNeSc2tiv/2elmy.icIenasthaerecsuurlrteonft cauInseatdisdsiutieondatmoapgroeinanflda,mtmhearteofroyreo,rcpanathboegpeantihcoTgehn1i7c.cells, pro- ed fro
m
study, chronic DSS colitis led to a reduced number of IL-17A+ tectiveorsuppressiveTh17cellshavebeendescribed(21,41).For h
FTuhr1t7hercmelolsre,inusIikngBNaS2T/2celml–idceepecnodmepnatrterdanswfeitrhcowliitlids-tmypoedeml,iwcee. ainnsdtaInLc-e6,IwLe-1re0–neoxtppreasthsionggeTnihc1(74c1e)l.lTshtheartefwoerree,IpLri-m10edcoweixthprTeGssFio-bn ttp://w
demonstratedthatalmostnoTh17cellsorIL-17A+IFN-g+Tcells by Th17 cells is presumably a self-regulatory mechanism to re- w
w
developedfromtransferredIkBNS2/2Tcells.Hence,IkBNSisnot duce the inflammatory damage of Th17 cells. Although it was .jim
only an important regulator of Th17 priming in vitro, it is also initially believed that Th17 cells produce IL-10 early on in an
m
essential for Th17 and IL-17A+IFN-g+ T cell development in infection (41), it was later shown that Th17 cells migrate from u
n
mthoeugsuetmwoadseilnscorefagsuetdininflaremcmipaietinotns.oFfuIrkthBeNrSm2o/2re,Tticsesullesdaasmaargeesuilnt tshueppsrpelseseinvetofutnhcetiognutainndaILC-C10R6e-xdperpeesnsidoenntthmeraenn(2er1)a.nWdeacfqouuinrde ol.org
ofthereducednumbersofimmunosuppressiveTregs,aspreviously decreased IL-10 expression in IkBNS2/2 Th17 cells both when by/
reported (17). In addition to a lower Treg frequency, the elevated generated invitro and in mice infected with C. rodentium. There- g
frequency of IFN-g+ Th1 cells is likely to be responsible for the fore,IkB iscrucialforthedifferentiationofproinflammatory,as ue
NS s
enhancedpathologicaldamage.AlthoughbothIkBNS2/2Th1and wellassuppressive,Th17cells. t on
Th17 cells show a proliferation defect invitro, Th1 development WeshowedpreviouslythatIkBNSinTregsisessentialtoinduce Ja
was not affected during colon inflammation invivo. Because co- theexpressionofthetranscriptionfactorFoxp3(17).IkBNSbinds nu
a
transfer of Tregs did not affect the enhanced Th1 differentiation, togetherwithp50andc-ReltoCNS3andthepromoterofFoxp3. ry
thismightindicatethepresenceofadditionalsignalsthatpromote BecauseoftheextenuatedexpressionofIL-10invivoandinvitro, 5
Th1developmentinvivo. weanalyzedtheinfluenceofIkBNSinIL-10expressionbyChIP, , 201
Inadditiontotheautoimmunemodelsofcolitis,weaddressed whichrevealedbindingofIkB totheIL-10genelocus.Previous 9
NS
thequestionofwhetherIkB isinstrumentalforTh17responses studies identified the binding of NF-kB to the IL-10 gene locus
NS
duringinfection.Tothisend,weusedC.rodentiumbecausethis (42, 43). Thus, we suggest that IkB binding to the IL-10 gene
NS
murine pathogen induces strong Th17 responses (25, 26, 31). locusismediatedbyp50and/orc-Rel,becauseIkB itselfhasno
NS
C.rodentiumsharesvirulencefactorswithenteropathogenicEscheri- DNA-binding domain. Although IL-10 appears to be directly
chiacoliandenterohemorrhagicE.coliand,thus,isacommonly regulated by IkB , IL-17A, IL-17F, and IL-22 do not contain
NS
used murinemodel to studyimmune responses against thesegut- canonical NF-kB–binding sites. Consequently, we conclude that
tropic pathogens (32). Similar to the colitis models, IkB defi- IkB doesnotregulatethesegenesdirectly;furtherexperiments
NS NS
ciency resultedinimpaired Th17 andIL-17A+IFN-g+ Tcell gen- are needed to clarify how IkB regulates the expression of
NS
erationininfectedmice.BecauseIFN-g,aswellasthep40andthe cytokines inan indirectmanner.
p19 subunit of IL-23, which is known to stabilize the Th17 phe- Several components of the NF-kB signaling pathway are im-
notype(33),arerequiredforC.rodentiumclearance(25,34,35),it portant for Th17 development. Most upstream in the signaling
is conceivable that the lack of IL-17A+ and IFN-g+ T cells in complex,CARMA1deficiency resultsindefectiveTh17celldif-
IkB 2/2miceuponC.rodentiuminfectionisresponsibleforthe ferentiation (44). MALT1 is highly expressed in Th17 cells gen-
NS
increased bacterial burden in these mice. Interestingly, NF-kB1/ eratedinvitro,andMALT12/2Th17cellsproducelessIL-17and
p502/2 mice also are defective in C. rodentium clearance (36), GM-CSF(45).Lossofp65/RelAorc-RelresultsinimpairedTh17
which is in line with the observation that IkB preferentially induction invitro, and c-Rel was shown to interact directly with
NS
interacts with this NF-kB family member (17, 37). Furthermore, theRorc and Il21promoters(46,47).Moreover, theatypical nu-
becauseofthenormalIL-17AandIL-17Fexpressioninvitro,we clear IkB protein IkBz, another nuclear IkB protein with high
speculate that the reduced mRNA expression in vivo arises from sequence similarity to IkB , regulates Th17 proliferation in
NS
thedecreasedfrequencyofTh17andIL-17A+IFN-g+cells. acell-intrinsicway(48).IkBzinteractswithRORaandRORgtto