Inhibition of TGF-β Signaling by IL-15: A New Role for IL-15 in the Loss of Immune Homeostasis in...

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GASTROENTEROLOGY 2007;132:994 –1008

nhibition of TGF-� Signaling by IL-15: A New Role for IL-15 in the Lossf Immune Homeostasis in Celiac Disease

ÉLIKA BENAHMED,*,‡ BERTRAND MERESSE,*,‡ BERTRAND ARNULF,‡,§,� ULLAH BARBE,*,‡

EAN–JACQUES MENTION,*,‡ VIRGINIE VERKARRE,*,‡,¶ MATTHIEU ALLEZ,# CHRISTOPHE CELLIER,*,‡,**LIVIER HERMINE,‡,§,� and NADINE CERF–BENSUSSAN*,‡

INSERM U793, Paris; ‡Université Paris Descartes, Faculté de Médecine René Descartes, Paris; §CNRS UMR 8147, Paris; �Department of Hematology, ¶Departmentf Pathology, AP-HP, Hôpital Necker-Enfants Malades, Paris; #Department of Gastroenterology, AP-HP, Hôpital Saint Louis, Paris; and **Department of
astroenterology, AP-HP, Hôpital Européen Georges Pompidou, Paris, France
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ackground & Aims: Interleukin (IL)-15 deliversignals that drive chronic inflammation in severaliseases, including celiac disease. Smad3–trans-orming growth factor-beta (TGF-�) signaling isnstrumental to counteract proinflammatory sig-als and maintain immune homeostasis. Our goalas been to investigate why the proinflammatoryffects of IL-15 cannot be efficiently controlled byGF-� in celiac disease. Methods: The impact of

L-15 on TGF-� signaling in T cells and in thentestinal mucosa of celiac disease patients was an-lyzed by combining cell and organ cultures, im-unohistochemistry, flow cytometry, real-time

olymerase chain reaction, electromobility gelhift, and Western blot. Results: IL-15 impairedmad3-dependent TGF-� signaling in human

lymphocytes downstream from Smad3 nuclearranslocation. IL-15-mediated inhibition was asso-iated with a long-lasting activation of c-jun-N-erminal kinase and reversed by c-jun antisenseligonucleotides, consistent with the demonstrated

nhibitory effect of phospho-c-jun on the forma-ion of Smad3–DNA complexes. In active celiac dis-ase, intestinal lymphocytes showed impaired TGF-�–mad3-dependent transcriptional responses and up-egulation of phospho-c-jun. Anti-IL-15 antibody and-jun antisense both downmodulated phospho-c-junxpression and restored TGF-�–Smad-dependentranscription in biopsies of active celiac disease. c-junntisense decreased interferon gamma transcription.onclusions: Impairment of TGF-�-mediated signal-

ng by IL-15 might promote and sustain intestinal in-ammation in celiac disease. More generally, our datarovide a new rationale for the potent proinflammatoryffects of IL-15, and further support the concept thatL-15 is a meaningful therapeutic target in inflamma-ory diseases associated with irreducible elevation of
L-15.
nterleukin (IL)-15, a cytokine produced by many celltypes, plays pleiotropic functions at the interface be-

ween innate and adaptive immunity. IL-15 is critical forhe differentiation and/or homeostasis of several murinennate immune cell subsets, including natural killerNK), NK/T, and CD8�� intraepithelial lymphocytesIEL), as well as for the generation and maintenance ofpecific memory CD8 TCR�� cells. In addition, IL-15lays redundant functions with other cytokines to pro-ote maturation of dendritic cells, proliferation of T andcells, cytotoxicity of NK and CD8� T cells, and pro-

uction of proinflammatory cytokines.1 IL-15 may thuslay an important role in the triggering and maintenancef immune responses to invading pathogens and in tu-or immunosurveillance. Yet, due to its potent anti-

poptotic and proinflammatory properties, its over ex-ression at different sites in transgenic mice has severeeleterious consequences, causing benign and malignantroliferation of NK and CD8 T cells, multiorgan lym-hocyte infiltration, and/or severe inflammatory lesionsf the skin or the small intestine.2,3 Multifaceted regula-ory checkpoints, particularly at the level of IL-15 mes-age translation, allow a tight control of IL-15 expres-ion.4 Yet, disorders of IL-15 have been observed inssociation with several proinflammatory or autoim-une-related diseases in humans, including psoriasis and

heumatoid arthritis (where IL-15 was recently shown toe a relatable therapeutic target),5,6 and celiac disease.

Celiac disease (CD) is a small intestinal enteropathynduced by cereal-derived prolamins (gluten) in geneti-ally susceptible individuals. In the current view of theathogenesis of CD, adaptive immunity plays a key role,ccounting for the interplay between the triggering envi-

Abbreviations used in this paper: CD, celiac disease; HLA, humaneukocyte antigen; IEL, intraepithelial lymphocytes; IL, interleukin; JNK,-jun-N-terminal kinase; LPL, lamina propria lymphocytes; NK, naturaliller; PAI, plasminogen activator inhibitor; PBMC, peripheral bloodononuclear cells; SDS, sodium dodecyl sulfate; TGIF, TG-interacting

actor; TNF-alpha, tumor necrosis factor-�.© 2007 by the AGA Institute

0016-5085/07/$32.00
doi:10.1053/j.gastro.2006.12.025

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March 2007 IL-15 INHIBITION OF TGF-� SIGNALING 995

onmental factor, prolamins, and HLA-DQ2/8 haplo-ypes, the major genetic risk factor. Due to their contentn proline and glutamine, some gluten peptides can beeamidated by tissue transglutaminase, the autoantibodyarget, and adopt a configuration that enables their bind-ng into the peptide pocket of HLA-DQ2/8 molecules.7

luten peptides can subsequently be presented to laminaropria CD4� T cells, triggering their activation and theelease of interferon gamma (IFN-�).8 Although this at-ractive scheme has considerably improved our under-tanding of CD pathogenesis, the fact that only a smallubset of HLA-DQ2/8 individuals develop CD points todditional factors mandatory for disease development.e and others have provided evidence of the complemen-

ary role of IL-15. IL-15 is induced in the intestinalucosa of CD patients by a peptide distinct from T cell

pitopes, the peptide 31– 43/49 common to the N-termi-us of �-gliadins.9,10 IL-15 orchestrates within the epi-helium an abnormal immune response that induces thexpansion of IEL and their cytotoxicity against entero-ytes via interaction of the NKG2D innate receptor withts major histocompatibility complex class I-related epi-helial ligands.9,11–13 IL-15 may also act on dendritic cellsnd enhance presentation of gliadin epitopes to CD4� Tells.10 However, it remains unclear why the potent im-unoregulatory mechanisms that control intestinal im-une responses to intraluminal antigens cannot prevent

he inappropriate activation of lamina propria lympho-ytes (LPL) and IEL in celiac patients. Notably, transgenicice expressing human CD4 and HLA-DQ8 fail to de-

elop enteropathy upon oral challenge with gliadins, de-pite a strong specific CD4� T cell response.14 A strikingeature of this mouse model is the high production ofransforming growth factor-beta (TGF-�1) induced byluten exposure, suggesting that this cytokine may avoidroinflammatory intestinal responses to gluten.TGF-�1 is a multifunctional cytokine that plays a

ritical role in controlling autoimmune conditions, asell as intestinal inflammatory responses.15 TGF-�1 islso a key cytokine in the establishment of oral toler-nce.16 TGF-�1 binds to a complex of transmembraneerine–threonine kinases type I and type II receptors thatrigger several intracellular pathways. The main pathwayntails phosphorylation of receptor-regulated Smad2nd Smad3, which then associate with the common me-iator Smad4 and translocate into the nucleus where themad2/3/4 complexes act as transcription factors regu-

ating gene expression.17 This pathway is instrumentalor the regulatory effects of TGF-�1, particularly in the

ucosal compartments. Thus, disruption of Smad3 re-ults in inflammatory T-cell infiltrates almost exclusivelyocalized to mucosal surfaces and chronic intestinal in-ammation.18 In active CD, large amounts of TGF-�1ave been observed in the intestinal mucosa,19 pleading
gainst a quantitative defect, but reminiscent of the sit- h
ation in other human inflammatory bowel diseases. Inhe latter diseases, TGF-�1 is present but the Smad3ignaling pathway is severely impaired due to up-regula-ion of the inhibitor Smad7 by as-yet unidentified mu-osal proinflammatory factors.20 –22

The massive increase of the proinflammatory cytokineL-15 in CD led us to investigate whether and how IL-15

ight interfere with the Smad signaling pathway ofGF-�. We then sought evidence that the inhibitoryathway elicited by IL-15 is operative in CD, and mayepresent a novel mechanism by which IL-15 contributeso the loss of intestinal homeostasis, and more generallyromotes chronic inflammation.

Materials and MethodsCD Patients and ControlsPeripheral lymphocytes were from healthy volun-

eers. Histologically normal small intestinal samples wererom 16 adults (age, 33– 80 years; mean, 55 years) under-oing intestinal surgery for morbid obesity or pancreaticancer. Duodenal biopsies were from 25 active CD adultsage, 18 –70 years; mean age, 39 years) with partial toubtotal villous atrophy and positive serology for antien-omysium antibodies. This study was approved by the

ocal ethics committee.

Lymphocyte Isolation and Cell CulturePeripheral blood mononuclear cells (PBMC) were

solated on Ficoll-Hypaque gradient and lymphocyte sub-ets were separated using magnetic beads (CD3 mi-robeads, CD4� or CD8� T cell isolation kits II, Miltenyiiotec, Paris, France). Purity ranged from 85% to 98%. IELnd LPLs were isolated from endoscopic or surgical sampless described.23,24 Yield from biopsies was 0.4–0.6 � 106 forEL and 0.6–0.8 � 106 for LPL. Contamination by livepithelial cells evaluated after 2-hour incubation at 37°Cas �5%. Cells were cultured in medium11 supplemented orot with human recombinant cytokines IL-2, IL-15, TGF-�1

R&D Systems, Abingdon, Scotland), or of humanized an-ihuman tumor necrosis factor alpha (TNF-�) (Remicade,chering-Plough, Hérouville Saint Clair, France). TGF-�1as used in all studies at 10 ng/mL, as this concentrationas optimal to induce Smad3-dependent activation and tolock lymphocyte proliferation25 (and not shown). Forroliferation studies, lymphocytes were cultured in6-well plates (105/well) and uptake of [3H]thymidineAmersham Biosciences, Saclay, France) was measured 18ours after adding 0.4 �Ci/well. For c-jun antisense treat-ent, 25 �g/mL phosphorothioate single-stranded oli-

onucleotides to c-jun (antisense 5=- TTC CAT CTT TGCGT CAT-3=; sense 5=-ATG ACT GCA AAG ATG GAA-3=)

Invitrogen, Cergy Pontoise, France) were added to lym-hocyte cultures 24 hours after stimulation and every 24
ours. For organ culture, duodenal biopsies were cul-

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996 BENAHMED ET AL GASTROENTEROLOGY Vol. 132, No. 3

ured in 95% oxygen–5% carbon dioxide at 37°C for 24 to0 hours on a stainless steel mesh in an organ cultureish (BD Biosciences, Le Pont de Claix, France) contain-

ng DMEM-F12 supplemented with 5% SVF, 1% HEPES,0 �g/mL gentamycine, 2.5 �g/mL amphotericin B (In-itrogen) added or not with 50 �g/mL of sense or anti-ense phospho-c-jun oligonucleotides, 25 �g/mL oflocking antihuman IL-15 mouse mAbs or control IgG1

sotype (R&D Systems) and/or 10 ng/mL TGF-�1.

Flow CytometryLymphocytes (105) were incubated with phyco-

rythrin-, fluorescein isothiocyanate-, APC-Cy-chrome-onjugated mAbs to human, CD3, CD4, CD8, CD45, (BDiosciences, Le Pont de Claix, France), TGF-�RII, or con-

rol isotypes (R&D Systems) for 30 minutes at 4°C. Forntracellular phospho-c-jun detection, cells first stainedith fluorescein isothiocyanate and APC-conjugatedAbs to CD3, CD4, or CD8, were fixed, permeabilized

sing DAKO intrastain kit (Dakocytomation, Trappes,rance), and labeled with 10 �g/mL phycoerythrin-con-

ugated mAb KM1 to human phospho-c-jun (Santa Cruziotechnology, Santa Cruz, CA) or control isotype (R&Dystems). For intracellular TNF-� labeling, cells werereated with Golgistop (BD Biosciences) for the last 6ours of culture. They were then fixed and permeabilizedsing BD Cytoperm/cytofix plus kit and labeled withhycoerythrin-conjugated mouse antihuman TNF-� orontrol isotype (BD Biosciences) according to manu-acturer’s instructions. Analyses were performed with aD-LSR using the CELLQuest software (BD Bio-ciences).

ImmunochemistryDeparaffinized sections (5 �m) of duodenal for-

ol fixed biopsies were incubated overnight with 10g/mL of mouse mAb against human phospho-c-jun oruman TGF-�1 or control mouse IgG1 (R&D Systems),r with a 1:2000 dilution of anti-phospho-Smad2/3 an-ibody, a polyclonal rabbit antibody that detects Smad2nly when dually phosphorylated at serine residues 463nd 465, and that can react with phospho-Smad3 but notther Smad-related proteins (Cell Signaling Technology,everly, MA). Labeling was revealed by the R.T.U Vec-

astain universal Elite ABC kit using either diaminoben-idine or VIP (Vector Laboratories, Burlingame, CA). Totudy nuclear translocation of Smad3, cytospins of pe-ipheral CD3� T cells cultured in medium added or notith IL-15 for 5 days and stimulated overnight with 10g/mL of TGF-�, were fixed with 4% paraformaldehyde,ermeabilized with 0.1% Triton X-100 (Sigma, St. Louis,O), incubated with 5 �g/mL of rabbit antihuman

mad3 (Zymed Laboratories, South San Francisco, CA)or 2 hours, and revealed by the R.T.U Vectastain univer-
al Elite ABC kit using VIP and methyl green counter- p
tain. Percentages of cells containing nuclear Smad3 wereetermined at �40 on a Leitz Dialux 20 microscope.

Western-Blot AnalysisWhole-cell extracts were obtained from intestinal

iopsies of healthy controls and active CD patients usinghe Nuclear extract kit (Active motif, Rixensart, Belgium).roteins (20 �g) were separated on a 10% sodium dodecylulfate (SDS)-PAGE gel, transferred to nitrocellulose

embranes, and labeled with monoclonal antibodygainst human TGF-�1 (R&D Systems), or polyclonalabbit antibodies against human Smad7 (Santa Cruziotechnology) and phospho Smad2/3 (Cell Signalingechnology) followed by horseradish peroxidase-conju-ated antimouse or antirabbit antibodies (Cell Signalingechnology). Membranes were then stripped and ana-

yzed with monoclonal antibody against human �-actinSanta-Cruz Biotechnology) and horseradish peroxidase-onjugated antimouse antibody. Visualization was per-ormed using the enhanced chemiluminescence systemECL plus, Amersham Biosciences).

Electromobility Shift AssayElectromobility shift assay was performed with

uclear extracts from lymphocytes treated or notith TGF-� (10 ng/mL) for 45 minutes using double-

tranded oligonucleotide probes plasminogen activa-or inhibitor (PAI) probe: 5=-TCG AGA GCC AGA CAAGA GCC AGA CAA GCA GCC AGA CAC-3=,BE probe, 5=-CTCTATCAATTGGTCTAGACTTAACC-GA-3=) end-labeled with [�-32P] dCTP as described.25

rotein–DNA complexes were resolved in a 5% polyacryl-mide gel. Binding specificity was checked using a 50olar excess of nonradiolabeled PAI-1 promoter as a

pecific competitor.

Real-Time Reverse-Transcription PolymeraseChain ReactionTotal RNAs extracted with RNeasy Mini Kit (Qia-

en, Courtaboeuf, France) from intestinal tissues or lym-hocytes, were reverse transcribed as described.11 TGF-�-

nteracting factor (TGIF) and IFN-� mRNAs wereuantified by real-time polymerase chain reaction (PCR)sing SYBR Green PCR Master Mix (Applied Biosystems)nd 300 nmol/L of the corresponding primers, and dataormalized referring to expression of glyceraldehyde-3-hosphate dehydrogenase. Primers for IFN-� and glyceraldehyde--phosphate dehydrogenase have been previously published.11

pper and lower primers for TGIF were, respectively, 5=-GCAAACACACCTGTCTACGCTAC-3= and 5=-GGCGGG-AATTGTGAACTGAT-3=. TTP (Tristetraprolin) and Smad7RNAs were quantified by real-time PCR using availablessay-on-demand and Taqman PCR Master Mix (Appliediosystems) and data normalized referring to expressionf ribosomal Protein, Large, PO. For PCR, 40 cycles were
erformed as followed: denaturation at 95°C for 15 sec-

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nds and annealing and extension at 60°C for 1 minute,sing an ABI PRISM 7700 sequence detection system

software version 1.6). TGF-�-dependent transcriptionas monitored in biopsies using Smad7 and TTP genes,nd Smad7 and TGIF genes in isolated lymphocytes.

GST-c-Jun(1-79) Binding/ProteinKinase AssayFicoll-isolated human PBMC were stimulated

ith 10 ng/mL of IL-15, 300 U/ml IL-2, or medium alone.ells were lysed in 20 mmol/L Tris–HCl (pH 7.5), 150mol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA,

% Triton X-100, 25 mmol/L sodium pyrophosphate,mmol/L �-glycerophosphate, 1 mmol/L Na3VO4,

�g/mL leupeptin, 1 mmol/L phenylmethylsulfonyl flu-ride. Protein extracts (200 �g) were incubated overnightt 4°C with GST-c-Jun (1-79) (a kind gift from J. Rain-eaud, Chatenay-Malabris) immobilized to glutathione-garose (10 �L of packed beads per sample containing 10g of protein). After washing, the c-jun-N-terminal ki-ase (JNK)-GST-c-Jun (1–79)–agarose complexes were in-ubated for 1 hour at 30°C in 40 �L of kinase bufferontaining 25 mmol/L Tris–HCl (pH 7.5), 5 mmol/L-glycerophosphate, 2 mmol/L DTT, 0.1 mmol/La3VO4, 10 mmol/L MgCl2, 20 �mol/L cold ATP, and�Ci [-32P] ATP. Reactions were stopped by adding SDS

ample buffer and boiling. Samples were loaded andesolved on 10% SDS-polyacrylamide gels. Radioactivityncorporated into the GST fusion proteins was visualizedsing a STORM 840 phospho-imager (Molecularynamics, Sunnyvale, CA).

Statistical AnalysisValues obtained in the different study groups were

ompared by the nonparametric Mann–Whitney U test.tatistical significance was assigned to a value of P � .05.

ResultsIL-15 Inhibits Smad3-Dependent TGF-�Signaling in Human T LymphocytesTGF-�1 plays a role in the negative regulation of

he immune response in part by inhibiting normal T-cellroliferation after stimulation. Notably, TGF-�1, via ac-ivation of the Smad pathway, efficiently inhibits theroliferative response of T cells to IL-2,26 a cytokine thathares its receptor � and � chains with IL-15.1 In agree-

ent with previous reports,25,27 TGF-�1 inhibited IL-2nduced proliferation of PBMC (Figure 1A). The inhibi-ory effect of TGF-�1, absent during the early phase oftimulation, progressively increased after 48 hours andecame maximal at 96 hours (59% � 6% inhibition, P �

05). In contrast, TGF-�1 had no detectable effect onL-15-induced proliferation at all time points tested (P �05) (Figure 1A). Furthermore, although TGF-� inhibited
he IL-2-induced proliferative response of IEL (90% � 5% w
D8�) and LPL (68% � 6% CD4�) from healthy con-rols, it failed to block IL-15-induced proliferation inoth intestinal T-cell subsets (Figure 1B). Although theseesults might reflect the lack of impact of TGF-� on theL-15 signaling pathway leading to proliferation, theylso raised the possibility that IL-15 inhibited Smad-ediated TGF-� signaling.Following activation by TGF-�, Smad2/3/4 proteins

ssemble into a complex that translocates into the nu-leus, binds to the promoters of TGF-�1-target genes,nd induces their transcription. The inhibitory effect ofL-15 on Smad-dependent TGF-�-signaling was firsttudied by investigating the impact of IL-15 on the bind-ng of Smad complexes to the promoters of TGF-�-targetenes, using an electromobility shift assay and a probeontaining a 3-CAGAC box derived from the PAI pro-oter, as described.28 IL-15 pretreatment of PBMCs for

4 hours impaired the formation of Smad3–DNA com-lexes in response to a 45-minute stimulation withGF-�. A comparable inhibitory effect was observed afterdays (Figure 1C), indicating a long-lasting effect of

L-15 compatible with the lack of inhibition of TGF-�n IL-15-induced proliferation at 96 hours. Similar re-ults were obtained with another synthetic probe thatontains a palindromic Smad3 specific target sequenceAGATCTG (data not shown).The inhibitory effect of IL-15 on Smad-mediated

GF-� signaling in T lymphocytes was further tested byonitoring the transcription of TGIF, a target gene ofGF-� with a promoter activated by Smads proteins.29

his gene was chosen as expressed in T cells and notodulated directly by IL-15 in peripheral or intestinal T

ells (Figure 4C and supplemental Figure 1 [See supple-ental material online at www.gastrojournal.org]). TGF-

-dependent gene transcription was studied after a-hour stimulation with TGF-�, using real-time RT-PCR.

1-, 2-, 6-, and 24-hour pretreatment with IL-15 inhib-ted TGF-�-induced transcription of TGIF in CD3� andD8� peripheral T cells (Figure 1D and E). The inhibi-

ory effect of IL-15 persisted after a 5-day incubationn peripheral T cells, particularly in the CD8� subsetFigure 1E), a finding perhaps ascribable to the knownreferential effect of IL-15 on CD8� peripheral T cells.30

oticeably, IL-15 also reduced TGF-� induction of TGIFn IEL and LPL by approximately 50% (P � .05) on day 4Figure 1E). By comparison, IL-2 exerted a transient in-ibitory effect on Smad3-dependent transcription thatas detected after a 24-hour incubation, but was much

ess pronounced at 96 hours. The distinct effects of IL-2nd IL-15 on Smad3-dependent transcription at late timeoints were compatible with the distinct effects of TGF-�n IL-2 and IL-15-induced T-cell proliferation (FigureB). These results led us to investigate the mechanism by
hich IL-15 inhibits Smad-dependent TGF-�-signaling.
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IL-15 Exerts Its Inhibitory Effect on TGF-�Signaling Downstream of NuclearTranslocation of Smad3Several checkpoints regulate the Smad-dependent

GF-� signaling pathway. First, surface expression ofGF-� receptor II (TGF-� RII) can be downmodulated by

nflammatory cytokines.31 No significant variation ofGF-� RII levels was noticed, however, upon IL-15 stim-lation (Figure 2A).A second important checkpoint lies at the level of

mad2 and Smad3 phosphorylation by the kinase activ-ty of the TGF-� RII. The inhibitory factor, Smad7, not

igure 1. IL-15 inhibits TGF-� signaling in human T-cell lymphocytes. (An the absence or presence of TGF-�1 (10 ng/mL). Proliferative responsor 24 hours to 96 hours (results representative of 5 independent experimf 3 to 4 independent experiments). Bars indicate means � SD. *P � .0he presence or not of IL-15 (10 ng/mL) for 24 hours (upper panel) or 96uclear extracts were analyzed by electromobility shift assay usingonradiolabeled PAI-1 promoter was added as a specific competitor (coultured in medium added or not with IL-15 (10 ng/mL) for 1, 2, 6, and 2

evels were quantified by real-time PCR. Results are expressed in arbindicate that inhibition is significant at 1,2,6, and 24 hours). (E) Peripherultured in medium added or not with IL-15 (10 ng/mL) for 24 hours anRNA levels were quantified as in D. Results are representative of 3 in

nly promotes the degradation of the TGF-� receptor c

ut also competitively blocks Smad2 and Smad3 phos-horylation and the subsequent signaling cascade (re-iewed in Javelaud et al17). Because Smad7 is a target genef the Smad3–TGF-� pathway, its primary role may be torovide a feedback mechanism modulating TGF-� sig-aling. Smad7 transcription can also be induced by sev-ral proinflammatory cytokines including IFN-�,32 IL-1�,nd TNF-�,33 an effect that underlies the inhibitory effectf these cytokines on TGF-� signaling. Smad7 mRNAxpression was therefore monitored by real-time quanti-ative RT-PCR in peripheral T lymphocytes cultured for 2ours to 96 hours with IL-15 or TGF-� as a positive

ymphocytes were stimulated with IL-2 (300 IU/mL) or IL-15 (10 ng/mL)ere assessed by [3H] thymidine uptake in triplicate. (A) PBMC cultured). (B) PBMC, IEL, and LPL from controls stimulated for 96 hours (resultsen compared with the condition without TGF-�. (C) PBMC cultured in

rs (lower panel) were stimulated by TGF-�1 (10 ng/mL) for 45 minutes.-labeled probe derived from PAI-1 promoter. Fifty molar excess ofobe). (D) Peripheral CD3� and CD8� lymphocytes from controls werers and then stimulated with 10 ng/mL TGF-�1 for 2 hours. TGIF mRNAunits and are representative of 3 independent experiments. (Asterisks3�, CD4�, and CD8� lymphocytes, IEL, and LPL from controls were6 hours and then stimulated with 10 ng/mL TGF-�1 for 2 hours. TGIF

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ivating effect on Smad7 transcription, IL-15 had noetectable direct impact on Smad7 RNA levels neither ineripheral (Figure 2B) nor in intestinal T cells (Supple-ental Figure 1 [See supplemental material online atww.gastrojournal.org]). IL-15, however, inhibited TGF--induced transcription of Smad7 in peripheral and in-

estinal T cells (Figure 2C), confirming results obtainedith TGIF (Figure 1C), showing that IL-15 impairsmad3-dependent transcription. Altogether these find-

ngs suggested that IL-15 does not require Smad7 tolock the TGF-�-Smad3 signaling cascade but acts afterhe steps regulated by Smad7. Therefore, we investigatedhether IL-15 acts upstream or downstream from Smad3uclear translocation. As shown in Figure 2D, TGF-�

nduced a massive nuclear translocation of Smad3 ineripheral T lymphocytes either untreated or cultured in

igure 2. IL-15 acts downstream from Smad3 nuclear translocation. (AL-15 (10 ng/mL). Membrane expression of TGF-� RII was monitored bB) Messenger RNA expression of Smad7 was monitored by real-time P10 ng/mL) or TGF-�1 (10 ng/mL). Results are representative of 2 indepEL, and LPL from controls were cultured in medium added or not wit0 ng/mL TGF-�1 for 2 hours. Smad7 mRNA levels were quantifiedxperiments. (D) Immunocytochemical staining of Smad3 in peripheraulture in the presence or not of IL-15 (10 ng/mL). Percentage of cellsxperiments, TGF-�-induced nuclear translocation of Smad3 was com

85%–99%). Nuclear translocation was observed in �2% of lymphocyL-15, likely due to the induction of an endogenous production of TGF-�nlarged blast cells, while T cells cultured in control medium remain sm

he presence of IL-15, indicating that IL-15 blockade a

akes place downstream from Smad3 nuclear transloca-ion.

IL-15 Inhibition of TGF-� Signaling in TLymphocytes Is Mediated by Phospho-c-junUp-regulationRecent work has demonstrated that the JNK path-

ay can repress TGF-� signaling following Smad3 nu-lear translocation via the inhibitory effect of phospho--jun on the formation of Smad–DNA complexes.25,34

revious studies in the Ba/F3 cell line have shown thatL-2 can activate JNK through recruitment of Shc to theL-2R� chain that is shared by IL-2 and IL-15 receptors.35

sing a sensitive JNK assay, activation of JNK was de-ected in PBMC 30 minutes after stimulation with IL-15Figure 3A) and IL-2 (data not shown). The signal, strong

C were cultured for 24 hours to 96 hours in medium added or not withcytometry. Results are representative of 3 independent experiments.PBMC cultured for 2 hours to 96 hours in the presence or not of IL-15

nt experiments. (C) Peripheral CD3�, CD4�, and CD8� lymphocytes,5 (10 ng/mL) for 24 hours and/or 96 hours and then stimulated with

al-time PCR. Results expressed are representative of 3 independent� T cells stimulated overnight with TGF-�1 (10 ng/mL) after a 5-daynuclear staining was determined among 500 cells. In 3 independentle in lymphocytes cultured in medium alone or in the presence of IL-15ultured in medium alone but in 25%–35% of lymphocytes cultured inlatter cells (data not shown). Note that IL-15-activated T cells becomed round.

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8 hours and 96 hours in the presence of IL-15 (FigureA) but, as previously observed,25 decreased at 48 hours

n the presence of IL-2. Intracellular expression of phos-ho-c-jun was next monitored by flow cytometry in pe-ipheral and intestinal lymphocytes cultured or not inhe presence of IL-2 or IL-15 for 1 to 10 days. Stainingas specifically absent or markedly inhibited when stim-lation was performed in the presence of c-jun antisenseligonucleotide (Figure 4A) or of SP600, a specific inhib-

tor of JNK (data not shown). In PBMC, induction ofhospho-c-jun by IL-2 and IL-15, visible at 24 hours (data

igure 3. IL-15 induces a long-lasting expression of phospho-c-junultured with IL-15 (10 ng/mL) or IL-2 (300 IU/mL) for 30 minutes to 96ST-c-jun as described in Materials and Methods, and proteins were se

unning an identically loaded SDS-polyacrylamide gel that was stained bere cultured with IL-15 (10 ng/mL) or IL-2 (300 IU/mL) for 10 days. Intra

B) Histogram analysis of intracellular staining of phospho-c-jun in the lynstimulated PBL (data not shown). (C) Representation of the increasesults were pooled from 3 independent experiments. Bars indicate meulture is compared in different subsets of peripheral and intestinal lymp

ot shown), was more obvious at 48 hours (Figure 3B). t

-jun phosphorylation remained high in peripheral lym-hocytes after 5 or 10 days in the presence of IL-15, while

t markedly decreased in cells stimulated by IL-2 (FigureB and C). Simultaneous membrane labeling with anti-D3, -CD4, and/or -CD8 antibodies revealed higherhospho-c-jun up-regulation in CD8� than in CD4�eripheral T lymphocytes (Figure 3D). Phospho-c-jun up-egulation was even more striking in IEL and LPL: by 96ours, phospho-c-jun median fluorescence was 5-fold in-reased in both subsets in response to IL-15, but only-fold increased in response to IL-2 (Figure 3D). In LPL,

ripheral and intestinal lymphocytes. (A) Peripheral lymphocytes werers. JNK kinase assay was performed on cell extracts precipitated withed by 10% SDS-PAGE (upper panel). Loading control was obtained byomassie blue (lower panel). (B–D) Peripheral and intestinal lymphocytesar staining of phospho-c-jun (p-c-jun) was monitored by flow cytometry.cyte gate at the indicated time points. Isotype control was identical tohospho-c-jun median fluorescence in PBMC stimulated for 10 days.

SD. (D) Increase of phospho-c-jun median fluorescence after a 96-hourtes. Results are the mean value (�SD) of 3 independent experiments.

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D8� subsets (data not shown). The higher induction ofhospho-c-jun in intestinal T cells might be related toheir activation state,36 because T-cell receptor ligationromotes expression of JNK.37

C-jun antisense treatment was used to investigatehether blockade of TGF-� signaling by IL-15 in T lym-hocytes depends on up-regulation of phospho-c-jun.hus, c-jun antisense but not sense oligonucleotide effi-iently inhibited phospho-c-jun induction in T lympho-ytes stimulated by IL-15 (Figure 4A). The capacity of-jun antisense to restore the inhibitory effect of TGF-�n IL-15-induced proliferation was tested by adding theligonucleotides 24 hours after initiation of the culture.ndeed, preliminary experiments indicated that JNK ac-ivation was necessary to initiate IL-15 and IL-2-inducedroliferation. In contrast, after 24 hours, cytokine-in-uced proliferation was only reduced by approximately0% in the presence of c-jun antisense, allowing to inves-igate the specific effect of c-jun blockade on TGF-�ignaling (Figure 4B). In these conditions, adding c-jun

igure 4. Antisense oligonucleotides for c-jun restore TGF-� effecresence of cytokines for 96 hours. Sense or antisense c-jun oligonuclenalysis of intracellular phospho-c-jun (p-c-jun) expression in PBMC atresence of the indicated reagents. Oligonucleotides were added at 24

�SD) and are representative of 3 independent experiments. (C) TGIFD8� lymphocytes stimulated or not with TGF-�1 (10 ng/mL) for 2 horbitrary units, are from 2 independent experiments. *P � .05 when co

ntisense but not sense oligonucleotides restored the a

nhibitory effect of TGF-� on IL-15-induced proliferationFigure 4B). This effect was not associated with any in-reased apoptosis (data not shown). Furthermore, c-junntisense treatment restored a normal level of TGIF andmad7 transcription in response to TGF-� in peripheralD8� T lymphocytes cultured with IL-15 for 5 days

Figure 4C). These data demonstrate that IL-15 preventsGF-� signaling in T lymphocytes by up-regulation ofhospho-c-jun.Previous studies have shown that TNF-� can prevent

GF-� signaling via JNK-mediated c-jun phosphoryla-ion.34,38 Because this cytokine can be induced by IL-15 in

cells activated by the anti-CD3 antibody,39 we testedhether the effect of IL-15 on phospho-c-jun up-regula-

ion depended or not on TNF-� production in lympho-ytes. Phospho-c-jun induction by IL-15 in CD3� periph-ral and intestinal T lymphocytes was not modified in theresence of a blocking anti-TNF-� antibody (Supplemen-al Figure 1A, [See supplemental material online at www.astrojournal.org] and not shown). In addition, IL-15

lymphocytes cultured with IL-15. Lymphocytes were cultured in thes (25 �g/mL) were added every 24 hours to the cultures. (A) Histogramurs. (B) Proliferative responses of PBMC after a 96-hour culture in the

rs. Results are expressed as mean cpm of 3H thymidine incorporationmad7 mRNA induction was quantified by real-time PCR in peripheralfter a 5-day culture in the indicated conditions. Results, expressed ined with the condition without IL-15.

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ll tested T lymphocyte subsets (Supplemental Figure 1BSee supplemental material online at www.gastrojournal.rg]).

TGF-� Signaling Is Impaired in the Mucosaof Patients With Active CD Secondary toPhospho-c-jun Up-Regulation by IL-15Confirming a previous report,19 immunohisto-

hemical staining of intestinal biopsies revealed intenseGF-� labeling in epithelium and lamina propria ofatients with active CD, comparable with controls (Fig-re 5A). Immunoblotting confirmed that amounts ofGF-� were comparable in intestinal biopsies from pa-

ients with active CD and from controls (Figure 5A).GF-� signaling in intestinal biopsies was first assessedy measuring the transcription levels of TTP (tristetrapro-

in), a TGF-� target gene under the control of the Smad3athway,40 which is easily detectable in tissue specimens.ontrasting with the substantial in situ synthesis of

igure 5. Intestinal lymphocytes from active CD have defective Smadmmunohistochemistry (left panel) and immunoblotting (right panel) in duf the immunoblots are shown in the upper right panel for 2 active Censitometry were comparable between patients and controls (bars indiy real-time PCR in duodenal samples of 13 active CD and 8 controls. (hours with TGF-�1 (10 ng/mL). TGIF and Smad7 mRNA levels were

ndicate the median values. *P � .05 compared with controls.

GF-� protein, transcription levels of TTP were approx-mately decreased 3-fold in duodenal biopsies from pa-ients with active CD compared with controls (FigureB). Impaired response to TGF-� was confirmed in iso-

ated intestinal T lymphocytes using TGIF and Smad7, 2eliable probes to monitor TGF-�/Smad3-induced tran-cription in T cells. Although incubation in the presencef TGF-� for 2 hours induced transcription of TGIF andmad7 in IEL and LPL isolated from normal controls, itad no effect on the transcription of these 2 genes in IELnd LPL from active CD (Figure 5C).

Impairment of TGF-� signaling despite strong TGF-�xpression has been observed in other inflammatoryowel diseases and ascribed to Smad7 up-regulation.20,21

mad7 was first assessed at the transcription level.mad7 mRNA levels were significantly less in the biopsiesf patients with active CD compared with controls (Fig-re 6A). This result is compatible with inhibition of

ndent response to TGF-�. (A) Expression of TGF-�1 was analyzed byal biopsies of 5 patients with active CD (ACD) and of 5 controls. Resultstients and 2 controls. The ratios of TGF-�1 to �-actin assessed by

he median value). (B) Messenger RNA expression of TTP was quantifiedand LPL were isolated from active CD and controls and stimulated fortified by real-time PCR. Results are expressed in arbitrary units. Bars

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mad3-dependent transcription in the intestine of pa-ients with active CD (Figure 5B and C). Smad7 is, how-ver, not only regulated at the transcriptional but also athe posttranscriptional levels by antagonist mechanismshat modulate its degradation by the proteasome.41 Pro-ein levels were therefore assessed by immunoblotting.omparable amounts of Smad7 protein were detected in

he intestinal biopsies of patients with active CD and inontrols (Figure 6B). To define a possible impact ofmad7 on TGF-� signaling in active CD, the levels ofmad2/3 phosphorylation were compared in patientsith active CD and controls. No difference was demon-

trated by immunoblotting with a polyclonal antibodyhat detects phosphorylated Smad2/3 (Figure 6C). Fur-hermore, immunostaining of duodenal biopsies fromatients with active CD with the same antibody revealedxtensive nuclear labeling (Figure 6D). The latter resultsndicated that the initial steps of TGF-� signaling weressentially preserved in patients with active CD, pleadinggainst a major inhibitory effect of Smad7 on Smad3-ependent transcription and suggesting that blockade ofGF-� signaling in CD takes place downstream nuclear

ranslocation of Smad2/3.The latter finding, compatible with an inhibitory effect

f IL-15, a cytokine strongly expressed in the mucosa ofatients with active CD11 (and not shown), led us to

nvestigate phospho-c-jun expression in active CD. Im-unohistochemical staining of intestinal biopsies

howed phospho-c-jun labeling in a small number ofuclei in the villous epithelium of histologically normal

igure 6. The early steps of TGF-�-mediated signaling are not impairedy real-time PCR in duodenal samples of 13 active CD (ACD) and 8 coontrols. (B) Expression of Smad7 was analyzed by immunoblotting in to �-actin assessed by densitometry (right panel) were comparable betwesults of the immunoblot are shown in the left panel for 4 active CD pa

mmunoblotting in total extracts from 4 active CD patients and 4 controlsanel) were comparable between patients and controls. (D) Phosphoiopsies of 4 patients with active CD. Staining is shown at 100� (left p

amina propria and epithelium.

ontrols, while in active CD patients, almost all IEL, LPL, a

nd most epithelial cells were intensively positive withoth nuclear and cytoplasmic staining (Figure 7A). Up-egulation of phospho-c-jun was confirmed in freshlysolated intestinal lymphocytes by flow cytometry: whileEL and LPL from controls were negative, phospho-c-junas strongly expressed in IEL and LPL from patients withctive CD (Figure 7B). The role of phospho-c-jun overex-ression in the impairment of Smad3-dependent tran-cription in active CD was then assessed in 24-hour organultures by antagonizing c-jun expression with antisenseligonucleotides. c-jun antisense oligonucleotide abol-

shed phospho-c-jun staining (Figure 8A). Concurrent tonhibition of phospho-c-jun expression, c-jun-antisensenduced a small but reproducible increase in both TTPnd Smad7 mRNA levels (P � .05) (Figure 8B). Compa-able effects were observed with and without addition ofxogenous TGF-� (Figure 8B). Notably, increase inmad3-dependent transcription was associated with aecrease in IFN-� mRNA expression, pointing to a pos-ible restoration of the regulatory function of TGF-�. Anffect via JNK blockade of TCR-42,43 and NKG2D-medi-ted12 activation could not, however, be excluded.

Therefore, we next investigated whether IL-15 partici-ated in the up-regulation of phospho-c-jun in CD using4-hour organ cultures. In 4 patients with active CD,ddition of anti-IL-15 antibody markedly reduced phos-ho-c-jun staining, while control isotype and anti-TNFntibody had no effect (Figure 8A, and not shown),ointing out to the prominent role of IL-15 in the in-uction of phospho-c-jun. We then tested the capacity of

u in active CD. (A) Messenger RNA expression of Smad7 was quantifiedand expressed as arbitrary units (AU). *P � .05 when compared withxtracts from 5 active CD patients and 4 controls. The ratios of Smad7patients and controls (bars indicate the median value of arbitrary units).and 3 controls. (C) Expression of phospho-Smad2/3 was analyzed byratios of phospho-Smad2/3 to �-actin assessed by densitometry (rightd2/3 distribution was studied by immunohistochemistry in intestinaland 200� (right panel) magnifications. Arrows show positive nuclei in

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ion in intestinal biopsies of patients with CD. There wasmarked increase in TTP and Smad7 mRNA levels after a0-hour culture in the presence of anti-IL-15 antibodyFigure 8B, lower panel). Altogether, these data providetrong evidence that, in active CD, IL-15-induced phos-ho-c-jun is involved in the local down-regulation ofGF-� signaling.

DiscussionOur data, showing that IL-15 impedes Smad-de-

endent signaling of TGF-�, provide a new rationale forhe potent proinflammatory effect of this cytokine that

ay be central to the pathogenesis of CD.The inhibitory effect of IL-15 on TGF-�1 signaling inlymphocytes was demonstrated by the negative impact

f this cytokine on the formation of Smad–DNA com-lexes and on Smad3-dependent transcription. Consis-ent with these results and a previous report,27 TGF-�as unable to inhibit IL-15-induced lymphocyte prolifer-tion. Our results indicate that IL-15 acts late in themad cascade via a mechanism that implicates JNK ac-ivation. First, IL-15 did not modify TGF-� RII expres-ion or Smad7 transcription, 2 checkpoints important toontrol the initiation of the cascade. Second, IL-15 did

igure 7. Phospho-c-jun is up-regulated in intestinal lymphocytes in acontrols were stained with antiphospho-c-jun antibody (10 �g/mL).agnification (right panel). (B) IEL and LPL were isolated from active CDy flow cytometry (results representative of 3–4 experiments).

ot prevent Smad3 nuclear translocation. Third, IL-15 m

nduced a long lasting activation of JNK and up-regula-ion of phospho-c-jun. Previous studies have demon-trated that JNK activation impaired TGF-� signalingecause phospho-c-jun can, within the nucleus, inhibitmad3 binding to DNA.25,34 That IL-15 inhibits themad–TGF-� pathway via the latter mechanism wasemonstrated using c-jun antisense oligonucleotides be-ause they restored the effects of TGF-� on transcriptionnd cytokine-induced proliferation in lymphocytes. IL-15elongs to the 4 �-helix bundle cytokine family andhares with the closely related IL-2 cytokine, the receptor

and �c chains. The 2 chains form 1 signaling moduleble to recruit several pathways including JAK1/3-TAT3/5, PI3– kinase–AKT and RAS–MAP kinase cas-ades.1 As previously reported,25 IL-2 had only a brief andodest effect on the induction of phospho-c-jun, which

aralleled transient refractoriness to TGF-� antiprolifera-ive effects. How IL-15 but not IL-2 can induce a longasting induction of phospho-c-jun remains to be de-ned. One possibility might be that IL-15 but not IL-2

nduces another cytokine that can activate JNK and takehe relay of IL-15. One obvious candidate was TNF-�.34,38

et, our data indicate that the prolonged effect of IL-15as not associated with the induction of TNF-� (Supple-

eliac disease. (A) Small intestinal sections from 4 active CD (ACD) andnohistochemical staining is shown at 100� (left panel) and at 400�controls. Intracellular staining of phospho-c-jun (p-c-jun) was studied

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ww.gastrojournal.org]). Although another as yet un-nown relay of IL-15 is not excluded, it is interesting tobserve that the private � chain of the IL-15 receptor canecruit the tyrosine kinase syk44 and TRAF2,45 2 proteinsble to potentiate JNK activation.46,47 Yet, the contribu-ion of IL-15 R� chain to IL-15 signaling in lymphocytesemains controversial.48 Further studies, in progress, willim to decipher the mechanisms of JNK activation inesponse to IL-15.

A delicate balance between inflammatory immune re-ponses and tolerance is necessary to permit the evictionf pathogens while avoiding protracted inflammationnd preserving immune homeostasis. Smad3–TGF-� sig-aling plays a central role in tipping the balance towardolerance.15,18 The inhibitory effect of IL-15 on this path-ay might invert the balance and promote protective

mmune responses, in particular, against intracellularathogens.49 Conversely, persistent expression of IL-15,bserved in several autoimmune or chronic inflammatoryiseases,50 may durably impair the regulatory functionsf TGF-� and alter immune homeostasis. Our data in CD

igure 8. Effect of c-jun antisense and anti-IL-15 antibody ex vivo inultured for 24–36 hours with medium containing sense or antisense couse IgG1 (20 �g/mL) in the absence or presence of 10 ng/mL exog

ochemical staining of organ cultures (OC) with antiphospho-c-jun antixpression in duplicate biopsies after incubation in the presence of the inf 3 to 4 independent experiments. *P � .05 when compared with the

upport this hypothesis. s

A molecular basis to CD pathogenesis has been sup-lied by deciphering the specific interactions between theeptide pocket of HLA-DQ2/8 molecules and gliadin-erived T-cell epitopes.8 However, why loss of toleranceo gluten and triggering of the intestinal gliadin-specificD4� T cell response only occurs in a small subset ofatients with the at-risk HLA remain unsolved. Data inransgenic mice expressing both human CD4 and HLA-Q8 molecules support a role for TGF-� in preventing

ntestinal inflammation in response to oral challenge byliadin peptides.14 Our results, showing that Smad–TGF--dependent transcription is decreased in the intestinalucosa of active CD patients and cannot be up-regulated

n isolated intestinal lymphocytes or in biopsies by exog-nous TGF-�, indicate that Smad-dependent TGF-� sig-aling is impaired in active CD, and may therefore fail toaintain tolerance to gluten. Inhibition of Smad3 sig-

aling has been previously demonstrated in intestinalnflammatory bowel diseases,21,22 as well as in Helicobacterylori-induced gastritis.20 In the latter conditions, as-yetnidentified mucosal factors up-regulate Smad7 expres-

enal biopsies from patients with active CD. Duodenal biopsies wereligonucleotides (50 �g/mL), monoclonal anti-IL-15 antibody or controls TGF-� added 3 hours before the end of the culture. (A) Immunohis-(B) Quantification by real-time PCR of TTP, Smad7, or IFN-� mRNAs

ed reagents. Results, expressed as relative induction, are means (�SD)ol condition.

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ctive CD, however, Smad7 was not up-regulated, andmounts of phosphorylated Smad2/3, as those of TGF-�,ere comparable in the mucosa of CD and controls.ltogether, these results pleaded against the prominent

ole of Smad7, and rather pointed to an inhibitory mech-nism operating downstream Smad2/3 nuclear translo-ation and therefore independent of Smad7 expression.

We have shown that chronic exposure to gluten in CDatients is associated with a massive up-regulation ofL-15 in the intestinal mucosa.11 Consistent with theong-lasting in vitro induction of phospho-c-jun by IL-15,hospho-c-jun was markedly increased in the intestinalucosa and T lymphocytes of patients with active CD,

nd downmodulated in duodenal tissue explants treatedith blocking anti-IL-15 antibody. Furthermore, both

-jun antisense oligonucleotide and anti-IL-15 antibodyncreased TGF-�-dependent transcription in the biopsiesf patients with active CD. Although of small amplitude,he changes in transcription detectable in organ cultureere reproducible for the 2 genes tested. The effect of thentisense was comparable with and without addition ofxogenous TGF-�, indicating that sufficient amounts ofndogenous TGF-� were present in the biopsies of activeD to induce signaling after downmodulation of phos-ho-c-jun. Restoration of TTP transcription might be

nstrumental to downmodulate the local inflammatoryeaction. Indeed, TTP is a protein that binds the AU-richegions in the 3=UTR of mRNA of various proinflamma-ory molecules, including TNF-�, IL-2, cyclooxygenase 2,L-1�, and matrix metalloproteases to promote their de-ay and prevent their translation.51,52 In line with thisypothesis, TTP�/� mice develop an autoimmune syn-rome characterized by arthritis, dermatitis, and autoanti-ody formation.53 Noticeably, restoration of Smad3-depen-ent transcription by c-jun antisense oligonucleotide wasssociated with a decrease in IFN-� mRNA expression. Thisnding, coherent with the known role of Smad3 in theontrol of IFN-� production,15 points to a possible res-oration of the regulatory function of TGF-�. An effect ofNK blockade on TCR-42,43 and/or NKG2D-mediated12

ctivation cannot be excluded. Yet, as shown in Figure 8,hospho-c-jun expression was almost entirely abolishedy the blocking anti-IL-15 antibody, suggesting a prom-

nent role of IL-15 in the in situ activation of JNK in CD.Previous studies have substantiated several contribu-

ions of IL-15 to the pathogenesis of CD, includingctivation of lamina propria dendritic cells,10 inductionf IEL cytotoxicity against enterocytes via innate immuneeceptors,9,11–13 loss of IEL homeostasis, and emergencef T-cell lymphomas.11 Noticeably, 2 very recent reportsave demonstrated that complete ablation of TGF-� sig-aling in T cells engendered an aggressive, autoimmuneisease associated with uncontrolled activation ofH154,55 and cytolytic differentiation programs in CD4�nd CD8� peripheral T cells, and strikingly with the
ppearance of highly cytotoxic CD8 T cells bearing NK
eceptors,55 the latter finding reminiscent of CD.24,56

herefore, our current results suggest an additional sce-ario where the abnormal and chronic induction of IL-15y the 31– 43/49 peptide of �-gliadins9,10 also alters local

mmune regulation by TGF-�, thereby favoring and per-etuating gliadin-specific CD4� T-cell response in lam-

na propria and IEL activation.57 Furthermore, inhibitionf Smad3-dependent TGF-� signaling may synergizeith the potent antiapoptotic effects of IL-15 to promote

ntestinal T lymphomagenesis,58,59 a rare but character-stic complication of CD.60 Studies are in progress toemonstrate more directly the consequences of Smad3lockade on the functions of LPL and IEL in CD.

In conclusion, our results delineate a novel mechanismnderlying the deleterious effects of chronic overexpres-ion of IL-15. IL-15 was identified as a potential thera-eutic target in certain human T-cell malignancies and ineveral inflammatory and autoimmune disorders.61 A re-ent report showed that targeting IL-15 with a human-zed anti-IL-15 antibody improved patients with rheuma-oid arthritis with minimal side effects.5 Our finding ofhe interplay between IL-15 and the TGF-�–Smads path-ay further supports the concept that IL-15 is a mean-

ngful therapeutic target in inflammatory diseases asso-iated with irreducible elevation of IL-15.

Supplementary Data

Supplementary data associated with this articlean be found, in the online version, at doi:10.1053/j.gastro.006.12.025.

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Received November 15, 2005. Accepted November 27, 2006.Address requests for reprints to: Nadine Cerf-Bensussan, MD, PhD,

NSERM U793, Faculté de Médecine René Descartes-Paris 5, 156 ruee Vaugirard, 75730 Paris Cedex 15, France. e-mail: [email protected];ax: (33) 1-40-61-56-38.

M. Ben Ahmed was supported by INSERM, IRMAD, and AFDIAGAssociation Française des Intolérants au Gluten). This work was spon-ored by INSERM, by ARC Grant 4616, Canceropole Ile de France, andy La Fondation Princesse Grace de Monaco.The authors are grateful to members of the GERMC and particularly

o Pr. M. Lehman (Hôpital Saint Louis), Dr. T. Matysiak-Budnik, Dr. D.amarque (Hôpital Hôtel Dieu), Pr. Chaussade (Hôpital Cochin), andr. Cugnenc (Hôpital Georges Pompidou) for providing material and

nformation from their patients. The authors thank Dr. D. Buzoni-Gatelor helpful discussions and Mr. G. Pivert for technical support with
istology.

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