of May 26, 2018.This information is current as

Caspase-1, NALP-3, and IPAFAntigen-Induced Arthritis Is Independent of Inflammatory Role of ASC in

BussoWim B. van den Berg, Alexander Kai-Lik So and NathalieNarayan, Roberto Salvi, Virginie Pétrilli, Jurg Tschopp, Laeticia Kolly, Mahir Karababa, Leo A. B. Joosten, Sharmal

http://www.jimmunol.org/content/183/6/4003doi: 10.4049/jimmunol.0802173August 2009;

2009; 183:4003-4012; Prepublished online 28J Immunol 

Referenceshttp://www.jimmunol.org/content/183/6/4003.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Inflammatory Role of ASC in Antigen-Induced Arthritis IsIndependent of Caspase-1, NALP-3, and IPAF1

Laeticia Kolly,2* Mahir Karababa,2* Leo A. B. Joosten,§ Sharmal Narayan,* Roberto Salvi,*†

Virginie Petrilli,‡ Jurg Tschopp,‡ Wim B. van den Berg,§ Alexander Kai-Lik So,* and Nathalie Busso3*

Because IL-1� plays an important role in inflammation in human and murine arthritis, we investigated the contribution of theinflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype ofASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC�/� mice showed reduced severity of AIA, decreasedlevels of synovial IL-1�, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 didnot show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classicalNALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellularprocesses, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph nodeand spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-�, whereas IL-10production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in Tcell proliferation in ASC�/� T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantlydecreased in ASC�/� mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, theseresults strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses butnot via its implication in inflammasome formation. The Journal of Immunology, 2009, 183: 4003–4012.

T he IL-1� cytokine has been shown to be an importantproinflammatory cytokine in human and murine arthritis (1,2). In rheumatoid arthritis, therapies directed against IL-1�

have been shown to be clinically beneficial (3), and IL-1� plays acritical role in monosodium urate-induced inflammation in gout (4). Inmurine models of arthritis, IL-1� has been demonstrated to mediatesynovial inflammation as well as cartilage destruction (5).

IL-1� is produced as an inactive cytoplasmic precursor (pro-IL-1�) that is processed to the mature active p17 form by caspase-1(6, 7). Caspase-1 is itself produced as a proenzyme and its activityis regulated by an activation step. The recent identification of anintracellular multiprotein complex called the inflammasome pro-vides new insights into the regulation of caspase-1 activity andIL-1� processing (8–10). The core components of the inflamma-some are proteins of the nucleotide-binding oligomerization do-main (NOD)-like receptor (NLR)4 family. This large family has at

least 22 members and is distinguished by their nucleotide-bindingoligomerization domain (NACHT) and leucine rich repeat domain.NLRs are capable of binding caspase-1 either directly or throughthe adaptor molecule ASC, also known as TMS1, CARD5, orPYCARD (11). Activation of the inflammasome complex bypathogen- or danger-associated molecular patterns such as bacte-rial muramyl dipeptides, viral RNAs, monosodium urate crystals,and ATP converts procaspase-1 into its active form, which in turnconverts the pro-forms of IL-1�, IL-18, and IL-33 into their activeforms (see review Ref. 12). To date, three different inflammasomeplatforms that are capable of processing IL-1� have been de-scribed: the Nacht, LRR, and PYD-containing protein NALP-1inflammasome, the NALP-3 inflammasome, and the ICE proteaseactivating factor IPAF inflammasome (13).

Evidence that the inflammasome is crucial for the regulation ofinflammatory processes in man stems from the identification thatmutations in the NALP-3 gene forms the basis of hereditary au-toinflammatory disorders (14) and the role of NALP-3 in monoso-dium urate crystal-induced release of IL-1� (10). Therapeutic in-hibition of IL-1 is rapidly effective in these disorders (15, 16),further implicating the role of IL-1� in Muckle-Wells syndromeand in gout.

To date, much of the attention has focused around the inflam-matory properties of ASC, which interacts with NALP3 as wellas other NLR proteins, including NALP-1, NALP-2, NALP-6,NALP-12, and IPAF (reviewed in Refs. 17, 18). ASC has beenshown to act as a dual regulator for NF-�B activation, either en-hancing or suppressing NF-�B induction depending on whichpathways for NF-�B activation are stimulated (19). Studies inASC�/� mice have revealed its in vivo role in several physiolog-ical and pathological settings (20–24). Most noticeably, ASC�/�

mice are extremely susceptible to infection with Francisella tula-rensis, succumbing to increased bacterial burdens and mortalitycompared with ASC wild-type mice (ASC�/�) (23). Furthermore,ASC is required to mount protective T and B cell immunity in

*Service of Rheumatology, Departement de l’Appareil Locomoteur, †Service of Endo-crinology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, and‡Department of Biochemistry, University of Lausanne, Lausanne, Switzerland; and§Rheumatology Research and Advanced Therapeutics, Department of Rheumatology,Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Received for publication July 7, 2008. Accepted for publication July 10, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant K-32K1-116460 from the Fonds National Suissede la recherche Scientifique and by the Jean and Linette Warnery Foundation.2 L.K. and M.K. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Nathalie Busso, Service ofRheumatology, Laboratory of Rheumatology, Centre Hospitalier UniversitaireVaudois Lausanne, Nestle 05-5029, 1011 Lausanne, Switzerland. E-mail address:Nathalie.Busso@chuv.ch4 Abbreviations used in this paper: NLR, NOD-like receptor; AIA, Ag-Induced ar-thritis; mBSA, methylated BSA; SAA, serum amyloid A; LNC, lymph node cell;EGFP, enhanced GFP.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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response to influenza virus infection (25). Finally, it has been re-cently shown that bone marrow-associated ASC plays a criticalrole in neointimal formation after vascular injury in mice (25). Allthese ASC-mediated effects are at least partly dependent on IL-1�.Besides its role in regulating inflammation, ASC has also beenshown to interact with apoptotic pathway proteins caspase-8 (26)and Bax (27). Indeed, ASC was first described as an intracellularprotein that aggregated in HL-60 cells undergoing apoptosis (28).ASC has also been reported to be suppressed by methylation-in-duced gene silencing in �40% of human breast cancers (29), thusimplicating ASC in tumor suppression. Finally, oligomerization ofASC is associated with pyroptosome assembly which is mediatedby homotypic interactions with its pyrin domain (30) and thusimplicating ASC in pyroptosis, a novel form of programmed celldeath linked with antimicrobial responses during inflammation andin contrast to apoptosis requires the function of caspase-1 (31).

Ag-induced arthritis (AIA) is a commonly used murine arthritismodel, recapitulating some of the histological and functional al-terations of rheumatoid arthritis. In this model, arthritis occurs inthe injected knee joint, after immunization and subsequent intra-articular challenge with methylated BSA (mBSA), with the con-tralateral placebo-injected knee serving as nonarthritic control. Inadoptive transfer experiments, both immune lymphoid cells and toa much lesser extent, serum transferred the capacity to developarthritis in C57BL/6 mice (32). In addition to the initial immunecomplex formation, chronicity is due to the presence of largeamounts of Ag sequestered in the joint and T cell-mediated de-layed hypersensitivity (see review in Ref. 33). Histologically, agranulocyte-rich exudate is observed in the knee joint space, aswell as, a thickening of the synovial layer with T cell-enrichedmononuclear infiltrates and early proteoglycan loss.

Given the central role of ASC in inflammasome activation and theimportance of IL-1� in inflammatory arthritis, we decided to explorethe AIA phenotype in ASC-deficient mice (ASC�/� and ASC�/�), aswell as mice genetically deficient for the inflammasome componentsNALP-3, IPAF, and caspase-1 and to analyze the mechanisms of jointinflammation in these animals. Our results indicate that ASC regulatesinflammation and IL-1� processing independently of the classicalcaspase-1 containing inflammasomes.

Materials and MethodsAnimals

All knockout mice used in the study were backcrossed into the C57BL/6 back-ground for at least 9 generations. ASC�/� (22), NALP3�/� (10), IPAF�/�

(34), caspase-1 (35), and IL-1��/�, a gift of Dr. J. Mudgett (Merck, Rahway,NJ) mice were compared with their wild-type littermates in all the experimentsdescribed. All mice were 8–12 wk of age at the time of immunization. Insti-tutional approval was obtained for these experiments.

Induction of AIA

AIA was induced as previously described (36). Briefly, arthritis was in-duced in immunized mice by intra-articular injection of 100 �g of mBSAin 10 �l of sterile PBS into the right knee, the left knee being injected with10 �l of sterile PBS alone. Institutional approval was obtained for theseexperiments.

Isotopic quantification of joint inflammation

Joint inflammation was measured by 99mTechnetium (99mTc) uptake in theknee joint, as previously described (36). The ratio of 99mTc uptake in theinflamed arthritic knee vs 99mTc uptake in the contralateral control kneewas calculated. A ratio higher than 1:1 indicated joint inflammation.

Histological examination

At least five mice per group were sacrificed, and the knees were dis-sected and fixed in 10% buffered formalin for 4 days. Fixed tissues weredecalcified for 2 wk in 15% EDTA, dehydrated, and embedded in par-affin. Sagittal sections (6 �m) of the whole knee joint were stained with

safranin-O and counterstained with fast green/iron hematoxylin. Histo-logical sections were graded by two independent observers unaware ofanimal genotype or treatment. Different parameters (synovial thickness,infiltrate, cartilage proteoglycan loss as reflected by reduced safranin-Ostaining intensity) were scored on a scale of 0 –3 in proportion toseverity.

Immunohistochemistry

Deparaffinized and rehydrated sections of arthritic murine knee joints wereincubated for 30 min at room temperature with 5% BSA and 20% normalserum. Endogenous peroxidase activity was blocked with 3% H2O2 for 10min. Slides were then overlaid with rabbit polyclonal anti-ASC Ab(AL117; Alexis Biochemicals) for 1 h at room temperature. Bound Ab wasvisualized using the avidin-biotin-HRP complex (Vectastain Elite ABC kit;Vector Laboratories). The color was developed by 3,3�-diaminobenzidine(Sigma-Aldrich) containing 0.01% H2O2. After extensive washing in wa-ter, slides were counterstained with Papanicolaou (Merck). Lymphocyte,macrophage, and neutrophil infiltrations into mouse knee synovium weredetected using anti-CD3, anti-Mac-2, or anti-myeloperoxydase primaryAbs, respectively. For double-staining immunohistochemistry of AIA sy-novial tissues, Abs against CD3, Mac-2, B220, and ICAM-1 (all fromSigma-Aldrich) were detected by HPR-conjugated secondary Abs and VIPas substrate (red staining). Rabbit polyclonal anti-ASC Abs were detectedusing HRP-conjugated secondary Abs and SG substrate (gray staining). Forapoptotic cells, the ApopTag kit (S7100) was used as described by themanufacturer (Chemicon International). Immunohistochemistry positivestaining was evaluated after magnification through a microscope (Olym-pus), scanning using a color video camera (Intas), and analysis using Nu-ance image analysis software (Intas).

In vivo lymph node cell (LNC) proliferation

Mice were injected i.p. with 0.2 ml of 5 mg/ml of BrdU (Sigma-Aldrich)1 h before sacrifice. At the end of the experiment, inguinal lymph nodeswere dissected and embedded in paraffin. Lymph node paraffin sectionswere subjected to BrdU immunohistochemistry. Briefly, after peroxidaseblockade with 3% H2O2 sections were treated with Retrievagen A (BDBiosciences) for 10 min at 89°C, and incubated for 60 min. with a mouseanti-BrdU biotinylated primary Ab (anti-BrdU; Zymed Laboratories) atroom temperature. Proliferating cells, having incorporated BrdU hadbrown-stained nuclei. Quantification of stained LNCs was performed bymorphometry with the Nuances software as described.

In vitro LNC or spleen cell proliferation assay and cytokineproduction

Mice were sacrificed according to the experimental protocol at day 3 or 7after the onset of arthritis. Inguinal lymph nodes and spleen were removed,and single cell suspensions were incubated in DMEM supplemented with5 � 10�5 M 2-ME, 1% penicillin, 1% streptomycin, 1% HEPES and 10%FCS. Spleen or LNC (2 � 105/200 �l/well) were plated in 96-well flat-bottom plates and stimulated with 50 �g/ml mBSA (Sigma-Aldrich) or 4�g/ml Con A. The cells were incubated at 37°C in 5% CO2 for 1, 3, or 5days (24 h with Con A), with 0.5 �Ci/well of [3H]thymidine added incultures for the last 18 h. The cells were harvested and [3H]thymidineuptake was measured using a beta scintillation counter. Splenocytessupernatants were collected after 1, 3, and 5 days of culture. IFN-� andIL-10 concentrations were detected by ELISA using reagent from eBio-science for IFN-� and R&D Systems for IL-10 following the manufac-turer’s protocol. Assay sensitivities were 10 and 80 pg/ml for IFN-� andIL-10, respectively.

In vitro T cell proliferation

Mice were sacrificed, spleen removed, and T cells enriched from spleno-cyte suspensions by negative selection using the EasySep mouse T cellenrichment kit (StemCell Technologies). T cell content of the enrichedfraction was typically over 95%. T cells (2 � 105/200 �l/well) were cul-tured in 96-well plates previously coated with anti-mouse CD3 (clone 145-2C11; eBioscience) alone or in conjunction with anti-CD28 (0.75 �g/ml,clone 37-51; eBioscience). After 1–4 days in culture, cells were pulsedovernight with 0.5 �Ci/well of [3H]thymidine and harvested before scin-tillation counting.

Serum amyloid A (SAA) measurements

Sera were collected at day 7 of AIA. SAA levels were determined using adirect ELISA according to the manufacturer’s instructions (Biosource).

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Serum levels of anti-mBSA Abs

Anti-mBSA IgG levels were measured by ELISA as previously described(34). Briefly, 96-well plates (Maxisorp-Nunc; Life Technologies) werecoated overnight at 4°C with 1% BSA in PBS. After four washes with TBSwith Tween 20 (50 mmol/L Tris (pH 7.4), 140 mmol/L NaCl, containing0.05% Tween 20), 100 �l of serum, serially diluted in 1% gelatin/PBS(final dilutions 1/100, 1/200) were incubated for 2 h at room temperature.Wells were washed four times and subsequently 100 �l/well of alkalinephosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich) diluted1/500 in TBS with Tween 20 was added for 30 min. After four washes withTBS with Tween 20, color was developed with 100 �l/well of p-nitrophe-nylphosphate (Sigma-Aldrich) and the reaction stopped by adding 25 �l/well of 3 mol/L NaOH. Plate reading was performed at 405 nm and resultscalculated according to a standard curve with a reference serum.

Knee joint tissue protein extracts preparation and cytokinelevels

At day 3 and 7 after the onset of the arthritis, mice were killed and kneeswere frozen. Knees were cut into little pieces and homogenized in proteinbuffer (50 mM Tris-HCl (pH 7.4), 110 mM NaCl, 10 mM EDTA (pH 8),0.1% Nonidet P-40) containing Roche complete protease inhibitors usingan Ultratorrax T8 homogenizer (IKA). The homogenates were centrifugedat 15,000 � g for 15 min at 4°C, and the supernatants stored at �80°C.Cytokine content was evaluated using a DuoSet ELISA kit for determina-tion of IL-1� (eBioscience) and IL-6 and TNF-� (R&D Systems). Resultswere normalized for total protein content using the Micro BCA kit accord-ing to the manufacturer’s instructions (Pierce).

Statistics

Data are expressed as mean value � SEM. Statistical analysis was calcu-lated by the two-tailed unpaired t test using the Prism software (v.4; Graph-Pad). Differences were considered statistically significant for a value ofp � 0.05.

ResultsOverexpression of a soluble mouse IL-1RII or IL-1� deficiencyreduces the severity of AIA

To determine whether IL-1� plays a role in AIA, we studied theeffects of IL-1� blockade during AIA induction. An adenoviralvector expressing a functional soluble murine IL-1R (mIL-1RII-

Ig) fusion protein (37) was i.v. injected 2 days before intra-artic-ular injection of mBSA. As a control, an adenoviral enhanced GFP(Ad-EGFP) construct was used. The mIL-1RII-Ig-treated miceshowed a significant decrease in arthritis severity as measured by99mTc uptake when compared with the Ad-EGFP control group(day 1: 1.32 � 0.04 vs 1.45 � 0.04, p � 0.02; day 3: 1.14 � 0.02vs 1.47 � 0.02, p � 0.001; day 7: 1.11 � 0.04 vs 1.3 � 0.04, p �0.002) (Fig. 1a). Decrease in 99mTc uptake correlated with a sig-nificant reduction of histological scoring of synovial inflammation(Fig. 1b) and a decrease in proteoglycan loss at day 7 (Fig. 1c).Similar results were obtained when AIA was induced in IL-1��/�

mice. A significant reduction in 99mTc uptake at days 2 and 7 afterthe onset of arthritis was observed compared with wild-typecontrols (Fig. 1d), and histological scoring showed significantreductions in synovial infiltrate (Fig. 1e) and in proteoglycanloss scores (Fig. 1f). Taken together, these data show that in-terruption of the IL-1� pathway leads to a reduction of jointinflammation in AIA.

Expression of ASC during AIA

As IL-1� is an important cytokine in the inflammatory phenotypeof AIA, we studied the expression of ASC, a central component ofthe IL-1�-producing inflammasome. We found by RT-PCR anal-ysis increased levels of ASC in arthritic joints compared with non-arthritic naive joints (Fig. 2a). This result was confirmed at theprotein level by Western blot and densitometric analyses. Expres-sion of ASC in the arthritic knee joint was significantly increasedat day 3 of AIA compared with nonarthritic naive knee joints (Fig.2, b and c). By ASC immunohistochemistry, we found markedstaining of synovial tissue (Fig. 2d, right). Ab specificity was con-firmed by the absence of staining when using synovial tissues fromASC-deficient mice (Fig. 2d, left). Colocalization studies usingcell-specific markers demonstrated ASC expression in all the celltypes analyzed: neutrophils (myeloperoxydase-positive cells),macrophages (Mac-2-positive cells), T cells (CD3�), and B cells

FIGURE 1. IL-1 blockade preventsthe development of AIA. a, C57BL/6mice were i.v. injected 2 days beforeonset of AIA with an adenoviral con-struct containing either EGFP as a con-trol (Ad-EGFP, n � 7) or mouse solu-ble IL-1RII-Ig fusion protein (Ad-msIL-1RII-IgG, n � 11) and jointinflammation measured by externalgamma counting of 99mTc uptake ondifferent days after Ag challenge intothe right knee. At day 7 after the onsetof the arthritis mice from a and d weresacrificed and subjected to histopatho-logical infiltrate (b and e) or proteogly-can loss scoring (c and f) using an arbi-trary scale as described in Materialsand Methods. d, AIA was induced inIL-1�-deficient mice and their corre-sponding wild-type littermates and jointinflammation measured by externalgamma counting of 99mTc uptake. Re-sults are represented by mean � SEMof at least n � 7 mice per group. Sta-tistical significance between groups wastested by using the two-tailed unpaired ttest. �, p � 0.05; ��, p � 0.01; ���, p �0.001 were considered significant.

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(B220�), and endothelial cells (ICAM-1-positive) (results notshown).

ASC deficiency attenuates AIA

To determine the role of ASC in murine arthritis, we inducedAIA in ASC-deficient mice and compared the degree of jointinflammation with ASC�/� and ASC�/� littermates. ASC�/�

mice developed severe arthritis characterized by a high 99mTcuptake ratio, whereas the loss of one or two copies of ASC geneled to a dose-dependent reduction in 99mTc ratio (Fig. 3a). How-ever, only the ASC�/� group showed a significant decreaseover time when compared with ASC�/� and ASC�/� groups.These observations correlated with a significant decrease in theacute phase protein SAA level in ASC�/� mice compared withASC�/� mice (22.1 � 4.6 arbitrary units in ASC�/� vs 61.2 �

19.4 in ASC�/�, p � 0.03) (Fig. 3b). Moreover, synovial IL-1�was also significantly reduced (17.73 pg/mg of protein � 5.33in ASC�/� vs 58.08 pg/mg � 14.36 in ASC�/�), whereas sy-novial levels of IL-6 and TNF-� were similar in both groups(Fig. 3c). Histological examination (Fig. 4a) and scoring ofarthritic knee joints from ASC�/� and ASC�/� mice sacrificedat day 7 after the onset of arthritis revealed that the synovialthickness was significantly decreased in ASC�/� mice com-pared with ASC�/� (1.29 � 0.18 in ASC�/� vs 1.93 � 0.16 inASC�/�, p � 0.02 (Fig. 4b), whereas proteoglycan loss wassimilar in both mice groups (Fig. 4c). ASC deficiency did notinfluence the composition of the inflammatory cell recruitment,as the proportion of synovial neutrophils (Fig. 4d) and macrophages(Fig. 4e) from ASC�/� and ASC�/� mice was similar. Moreover,the frequency of apoptotic cells was not changed between ASC�/�

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FIGURE 2. ASC expression is in-creased in arthritic joints. a, ASC wasdetected in wild-type mice by RT-PCR in synovial tissues from naive orfrom arthritic joints at day 3 of AIA.b, ASC expression by Western blot ofsynovial knee tissue extracts from na-ive ASC�/� (1), wild-type (2), andfrom arthritic joints at day 3 (3) andday 7 (4). As expected, no ASC is de-tectable in AIA joints from ASC�/�

mice. c, Increased levels of ASC werequantified in arthritic joints by densi-tometric analysis. Results are repre-sented by mean � SEM of at leastn � 4 mice per group. �, p � 0.05. d,ASC immunohistochemistry in AIAsynovial membranes from ASC�/�

and ASC�/� mice. Lining and sublin-ing layers showed staining. Within theinflammatory infiltrate, some cellswere positive. In contrast, no ASCstaining was detected in ASC�/�

tissues.

FIGURE 3. ASC deficiency reduces local and sys-temic inflammation in AIA. a, Time course of knee jointinflammation in ASC�/�, ASC�/�, and ASC�/� micewith AIA. Joint inflammation was measured by gammacounting of 99mTc uptake on different days after onset ofarthritis. Results are expressed as the ratio of 99mTc up-take in the arthritic knee joint vs the nonarthritic kneejoint. Results represent mean � SEM of 17 ASC�/�, 18ASC�/�, and 14 ASC�/� mice. b, SAA levels in seracollected at day 7 after the onset of arthritis were mea-sured by ELISA and expressed in arbitrary units (A.U.).Results are the mean � SEM of seven ASC�/� andseven ASC�/� mice. c, IL-1�, IL-6, and TNF-� levelsin synovial knee tissue extracts at day 3 after arthritisonset were determined by ELISA. Results represent themean of nine ASC�/� and seven ASC�/� samples. �,p � 0.05 and ��, p � 0.01; ASC�/� compared withASC�/� or ASC�/� samples.

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and ASC�/� synovial membranes (Fig. 4f), thus ruling out a roleof ASC in apoptosis of synoviocytes.

No contribution of NALP-3 and IPAF, two other inflammasomecomponents to the development of AIA

We next investigated which signal-sensing protein could be asso-ciated to synovial ASC. Expression of different inflammasomecomponents was assessed in arthritic joint tissue vs nonarthriticnaive tissue by RT-PCR. Among the 15 NALPs, only NALP-1,NALP-3, NALP-10, and NALP-12 could potentially associatewith ASC (38). Expression of all these NALPs was increased in

arthritic joints (Fig. 5a). IPAF, another candidate interacting withASC through CARD-CARD homotypic interactions, was also in-duced in arthritic tissues (Fig. 5a). ASC�/� and ASC�/� micewere used together with NALP3�/�, and IPAF�/� mice to eval-uate the effect of individual inflammasome deficiencies on arthritisseverity in AIA. In agreement with the results of Fig. 3a, at day 1and 3 after the onset of the arthritis, ASC�/� mice group showeda significant decrease in joint inflammation as compared with theASC�/� control group (Fig. 5b). In contrast, NALP-3 and IPAFdeficiency had no effect on 99mTc uptake, which was similar to thatof ASC�/� control group (Fig. 5b). Consistent with this latter

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loss

FIGURE 4. ASC deficiency is as-sociated with reduced histological in-flammation in AIA. a, Representativehistology of knee joint sections fromAIA mice stained with safranin-O at7 days after arthritis induction. Notethe difference of thickness of synovialmembrane (sy), which is thicker inASC�/� than in ASC�/� mice. Notealso the similar proteoglycan stainingof the cartilage (ca) in the two groups.b and c, Histological grading of ar-thritic knee joints. Mice were sacri-ficed at day 7 of AIA. Infiltrate andproteoglycan loss were scored histo-logically using an arbitrary scale from0 to 3. A total of n � 14 mice pergroup were analyzed. d–f, Morpho-metric analysis of myeloperoxydase(d), MAC-2 (e), and apoptotic cell (f)immunohistological staining per-formed as described in Materials andMethods. Each data point correspondsto the analysis of a different micro-scopic field. Immunohistology wasperformed on synovial tissues from atleast n � 4 mice per group.

FIGURE 5. Effect of NALP-3 andIPAF deficiency in AIA. a, Differentcomponents of the inflammasome com-plex were detected by RT-PCR in sy-novial tissues from naive or from AIAarthritic joints at day 3 of AIA. b, AIAwas induced in ASC-, NALP-3-, andIPAF-deficient mice, all in C57BL/6background. As a control group for thedeficient mice, corresponding wild-typelittermates from ASC heterozygousbreeding were used. Joint inflammationwas measured by external gammacounting of 99mTc uptake at days 1 and3 of arthritis. Results represent themean � SEM of six ASC�/�, eightASC�/�, ten NALP-3�/�, and fiveIPAF�/� mice. ��, p � 0.01 and ���,p � 0.001 compared with ASC�/� ateach time point.

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result, we found similar histological scoring between wild-typemice, NALP-3- and IPAF-deficient mice (results not shown).Thus, these results show that IPAF and NALP-3 are not contrib-uting elements in inflammation induction in AIA.

Effect of ASC is independent of caspase-1 activation in AIA

As activation of caspase-1 is mediated via the inflammasome, wenext investigated whether ASC can be involved in caspase-1 ac-tivation. The presence of active caspase-1 in tissue extracts of in-flamed joints from ASC�/� and ASC�/� mice was examined byWestern blotting. Active caspase-1 (10 kD) was detectable in allAIA arthritic knee joints from both mice strains, whereas no activecaspase-1 could be detected in caspase-1-deficient mice (Fig. 6a).Additionally, no difference was found in active caspase-1 levelsbetween ASC�/� and ASC�/� mice, as determined by densito-metric analysis of Western blots (Fig. 6b). To confirm that theeffects observed in ASC-deficient mice were indeed independentof caspase-1 and therefore cannot be recapitulated by caspase-1deficiency, we analyzed the phenotype of caspase-1 deficient miceduring AIA. No significant differences in 99mTc uptake were ob-served at days 2 and 7 after the onset of arthritis (Fig. 6c), andhistological scores of synovial infiltrate and proteoglycan losswere comparable between caspase-1-deficient mice and wild-typemice (Fig. 6, d and e). Taken together, these data clearly demon-strate that during AIA, there must be a caspase-1-independentIL-1� processing mechanism and that impact of ASC on AIA isindependent of caspase-1.

ASC deficiency inhibits cellular immune responses

As AIA is a Th1-dependent model of arthritis, we assessedwhether the loss of ASC can alter the immune response againstmBSA, the model Ag in AIA and thus explaining the observedphenotype. We investigated the humoral and cell-mediated im-mune responses from ASC�/� and ASC�/� mice in AIA. Serumlevels of total anti-mBSA Abs measured by ELISA in mice at day7 after the onset of arthritis were similar in ASC�/� (n � 14) and

ASC�/� (n � 10) groups (day 7: 0.96 � 0.21 in ASC�/� vs1.01 � 0.41 in ASC�/�, p � 0.86). Moreover, IgG1 and IgG2alevels, implicated in Th2 and Th1 response, respectively, wereequivalent in both groups (IgG1 levels: 1.32 � 0.31 in ASC�/� vs1.28 � 0.39 in ASC�/�, p � 0.93; IgG2a levels: 1.07 � 0.3 inASC�/� vs 0.94 � 0.36 in ASC�/�, p � 0.79). These resultsdemonstrate that ASC deficiency has no effect on humoral immuneresponses. In contrast, proliferation of LNC (data not shown) andsplenocytes (Fig. 7a) in the presence of the specific Ag, mBSAwas impaired in ASC�/� cells compared with ASC�/� cells iso-lated from AIA mice. In similar conditions, caspase-1-deficientLNC had the same proliferation index upon mBSA restimulationas wild-type LNC (results not shown), indicating that in this con-text caspase-1 deficiency does not reproduce the phenotype ob-served in ASC deficiency. The observed reduction in the prolifer-ative capacity of ASC�/� immune cells is Ag-specific as thenonspecific mitogenic Con A stimulation induced the same fold ofproliferation in cells from ASC�/� and ASC�/� AIA mice (37 �2 for ASC�/� vs 39 � 3 for ASC�/� cells). To explain the pro-liferation defect, we measured the levels of Th1 and Th2 cytokinesby ELISA in the supernatant of mBSA-induced ASC�/� andASC�/� splenocytes over 5 days (Fig. 7c). When compared withwild-type splenocytes, ASC�/� splenocytes secreted significantlyreduced levels of the Th1 cytokine IFN-� (at days 3 and 5),whereas the level of IL-10, a Th2 cytokine, was significantlyhigher in ASC�/� cultures at days 1 and 3. As ASC has beenreported to play a role in apoptotic processes, we tested whetherthe difference of proliferation between splenocytes from ASC�/�

and ASC�/� AIA mice could be at least in part, accounted for byapoptosis. Splenocytes from ASC�/� and ASC�/� AIA mice wereisolated and stimulated with 50 �g/ml mBSA. Apoptosis was as-sessed by flow cytometeric quantification of Annexin V staining.As shown in Fig. 7b, the proportion of splenic apoptotic cells werecomparable between ASC�/� and ASC�/� AIA mice thus dem-onstrating that proliferation reduction in ASC�/� splenocytes can-not be accounted for by a difference in apoptosis. These in vitro

FIGURE 6. ASC effects arecaspase-1-independent. a, At day 7 af-ter the onset of arthritis in AIA, micewere sacrificed, protein extracts wereprepared from arthritic knees ofASC�/�, and ASC�/� mice were ana-lyzed by Western blot using an Ab rec-ognizing the p10 active caspase-1. As acontrol, tissue extract from a caspase-1-deficient mouse (casp1�/�) is shown. b,Densitometric analysis from fiveASC�/� and five ASC�/� tissue ex-tracts analyzed by Western blot analy-sis. c, AIA was induced in caspase-1-deficient mice and their correspondingwild-type littermates and joint inflam-mation measured by external gammacounting of 99mTc uptake at days 2 and7 of arthritis. d and e, At day 7 after theonset of arthritis, mice were sacrificedand subjected to histopathological scor-ing using an arbitrary scale as describedin Materials and Methods. No statisticaldifference is found between differentgroups.

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results were supported by in vivo observations. Proliferation wasmarkedly decreased in ASC-deficient mice compared with wild-type littermates, (Fig. 7d), as shown by decreased BrdU-positivecells in lymph nodes from BrdU-injected mice. By contrast, apo-ptosis was similar in ASC-deficient mice and wild-type littermates,as demonstrated by in situ detection of apoptosis in lymph nodetissues using the Apoptag kit, which can detect single apoptoticcells (Fig. 7e).

Reduced Ag-specific splenocyte proliferation could result fromimpaired T cell signaling or impaired Ag presentation in ASC�/�

mice. To test whether CD3-TCR signaling is reduced in ASC-deficient mice, we purified T cells from naive ASC�/� andASC�/� spleens and incubated cells in the presence of increasedamount of immobilized anti-mouse CD3 mAb for 2 days and thenpulsed them with [3H]thymidine. As shown in Fig. 8a, TCR-CD3ligation induced a dose-dependent increase in T cell proliferationin both ASC�/� and ASC�/� T cells. However, the proliferationindex was significantly reduced in ASC�/� cells compared withwild-type cells. To ensure that the reduced proliferative potentialin ASC�/� T cells observed after 2 days in Fig. 8a was not simplya delay in proliferation, we performed kinetics of T cell prolifer-ation upon CD3 ligation, in the presence or absence of the co-stimulatory CD28 ligation (Fig. 8b). At all time points, prolifera-tion of ASC�/� T cells was significantly impaired (at days 1 and

2 with anti-CD3 stimulation alone or in conjunction with anti-CD28, at days 3 and 4 with stimulation with anti-CD3 plus anti-CD28). To test whether this reduced ASC�/� T cell proliferationwas associated to a skewing in cytokine profile, we measuredIFN-� and IL-10 secretion in the supernatants from T cell culturesactivated using CD3 with or without CD28 ligation (Fig. 8c). Cy-tokine analysis revealed that ASC�/� T cells produced less IFN-�and more IL-10 when activated by anti-CD3 (either alone or incombination with anti-CD28). Maximal IFN-� and IL-10 re-sponses were observed at 72 h after initial stimulation by ASC�/�

and ASC�/� T cells, respectively.To test whether ASC deficiency could also impact on APCs, we

purified macrophages and dendritic cells from ASC�/� andASC�/� bone marrow and activated them with 1 �g/ml LPS. Theactivation markers analyzed by FACS (MHC class II, CD80,CD86) were similarly expressed on cells from wild-type or ASC-deficient mice (results not shown), thus ruling out an involvementof these cells in the reduced splenocyte proliferative response ob-served previously. Altogether, these data demonstrate that inhibi-tion of immune cell proliferation by ASC deficiency was not dueto apoptosis but rather attributed to a shift toward more IL-10production by activated T cells and implies that the immunomodu-latory effect of ASC is mediated almost exclusively by T cells.

FIGURE 7. ASC effects on in vitro and in vivo cell-mediated immune response. Proliferation of splenocytes from ASC�/� and ASC�/� mice in responseto 50 �g/ml mBSA for 1–5 days (a) was measured by [3H]thymidine incorporation. The proliferation index was calculated as the ratio of cpm from activatedover cpm from nonactivated cells. Data represent mean of quadruplicate cultures. b, Apoptosis was quantified by FACS analysis as the percentage ofAnnexin V-FITC-labeled cells from mBSA (50 �g/ml) induced ASC�/� and ASC�/� splenocytes. c, IFN-� and IL-10 levels in ASC�/� and ASC�/�

splenocyte supernatants from AIA mice, cultured with 50 �g/ml mBSA for 1–5 days, were assessed by specific ELISAs. d, Incorporation of BrdU into DNAof mitotic cells. BrdU was injected i.p. 1 h before sacrifice, and detected by immunohistochemistry on lymph node sections. The mean number ofBrdU-positive cells (referred as proliferative cells) was determined as a percentage of positive cells in the analyzed fields as described below. e, In situdetection of apoptosis in lymph node tissues was conducted using the Apoptag kit, which can detect a single apoptotic cell. The mean number of apoptoticcells over the total number of cells was calculated from 25 fields (five different fields/mouse, using n � 5 mice per group) using the image analysis softwareNuances. �, p � 0.05; ��, p � 0.01; ���, p � 0.001, ASC�/� vs ASC�/� cells.

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DiscussionIL-1� is the prototypic proinflammatory cytokine and its par-ticipation in inflammatory arthritis has been amply demon-strated in animal models such as collagen-induced arthritis andin human rheumatoid arthritis. It is therefore of interest to un-derstand the mechanisms which regulate IL-1� secretion in ar-thritis. Caspase-1 represents the central effector protein thatdrives IL-1�-mediated inflammation. The recent emergence ofthe inflammasome complex as a caspase-1 activator and its rolein hereditary autoinflammatory syndromes and in gout raisesthe question of whether this complex is equally relevant inIL-1� production in arthritic joints. The observed increase inthe expression of ASC, as well as other key inflammasome com-plex components in arthritic joints, does indicate that all thecomponents needed to form functional inflammasome com-plexes are present within the joint. To address the role the in-flammasome plays in arthritis pathogenesis, we assessed theimpact of genetic deletion of different inflammasome elementson the arthritis phenotype in AIA, an established murine modelof immune-mediated inflammatory arthritis.

Despite demonstrating that AIA is IL-1�-dependent, we weresurprised to find that only ASC deficiency affected arthritis sever-ity, whereas caspase-1, NALP-3, and IPAF deficiencies had no

apparent impact on arthritic phenotype. These results indicate thatthe NALP-3 or IPAF inflammasomes are not essential for the in-flammatory effects associated with AIA. The fact that caspase-1�/� mice did not show a major reduction in arthritis severity (ascompared with IL-1��/�) suggests that caspase-1 is not critical forprocessing of pro-IL-1� in this model either. Although we reportreduced levels of IL-1� in synovial tissues of ASC�/� mice asmeasured by an ELISA, which detected both pro- and active-IL-1�, we could not discriminate the pro-IL-1� (p35) form and theactive-IL-1� (p17) form, as the levels of IL-1� in synovial sampleswere too low for detection by immunoblotting in repeated exper-iments (data not shown). The most likely explanation for theseresults is that reduced IL-1� levels in the synovial extracts reflectthe overall reduced cellular inflammatory activity, made up prin-cipally of pro-IL-1�, and not IL-1� processing capacity. Further-more, in ASC�/� mice, active caspase-1 was expressed in thesynovium at comparable levels to wild-type littermate controls,indicating that ASC and caspase-1 expression are regulated inde-pendently, and that alternative ASC-independent pathways existthat facilitate procaspase-1 activation. One possible molecule isIPAF, a member of the NLR family of proteins, which collaborateswith caspase-1 in the absence of ASC (34, 39). Taken together,these findings show that ASC plays a role in immune mediated

FIGURE 8. ASC effects on T cell activation. a, ASC Western blot was performed with splenic T cell extracts from naive ASC�/� (lane 1) or ASC�/�

(lane 2) spleens, or with tissue extracts from naive ASC�/� (lane 3) and ASC�/� (lane 4) synovial tissues (inset). Alternatively, splenic T cells from naiveASC�/� and ASC�/� were incubated in the presence of increase amount of immobilized anti-mouse CD3 mAb for 2 days and then pulsed with [3H]thy-midine. �, p � 0.05; ��, p � 0.01; ���, p � 0.001, ASC�/� vs ASC�/�. b, Spleen-derived T cells from naive ASC�/� and ASC�/� mice were incubatedwith anti-mouse CD3 mAb alone or with anti-CD3 plus anti-CD28 (each Ab at 0.75 �g/ml) for 1–4 days and then pulsed with [3H]thymidine. For a andb, the proliferation index was calculated as the ratio of cpm from activated over cpm from nonactivated cells. c, IFN-� and IL-10 levels in ASC�/� andASC�/� T cells activated with anti-CD3 alone or anti-CD3 in conjunction with anti-CD28 for 1–4 days were assessed in supernatants by specific ELISAs.For b and c, �, p � 0.05; ��, p � 0.01; ���, p � 0.001 in ASC�/� vs ASC�/� cells stimulated with anti-CD3 (brackets) or stimulated with anti-CD3 plusanti-CD28 (no brackets).

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inflammation, but acts independently of its associated inflamma-some partner proteins such as caspase-1 and the NLRs IPAF andNALP-3.

The absence of an obvious influence of the NALP-3 inflamma-some on arthritis induction does not exclude a role for other in-flammasome complexes in AIA. NALP-1 has been reported toform an inflammasome in response to pathogen-associated molec-ular pattern molecules such as bacterial peptides, in conjunctionwith ASC and caspase-1 (40). Unfortunately, no NALP-1 deficientmice are currently available to enable us to investigate this hy-pothesis. It is also possible that another caspase is involved in AIAassociated inflammation. Caspase-11 (equivalent to humancaspase-5) is induced by LPS and in cooperation with caspase-1, iscapable of processing IL-1� in mice (38). We have shown thatcaspase-11 is also present in arthritic AIA joints (results notshown). However, the fact that the caspase-1 knockout mice didnot differ from wild-type mice with respect to their arthritis phe-notype makes it improbable that NALP-1 and caspase-11 are di-rectly involved.

Reduced Ag-specific spleen cell proliferation could result fromimpaired T cell signaling or impaired Ag presentation in ASC�/�

mice. This latter explanation seems very unlikely as we detected asimilar number of dendritic cells in both arthritic ASC�/� andASC�/� LNCs, as well as comparable expression of MHC class II,CD80, and CD86 (results not shown). In addition, LPS-inducedactivation profiles of dendritic cells and macrophages generatedfrom ASC�/� and ASC�/� bone marrow was similar (results notshown), thus ruling out the involvement of these cells in the ob-served reduction in the proliferative capacity of ASC�/� spleno-cytes. By contrast, anti-CD3 with or without anti-CD28 inducedproliferation of purified T cells from ASC�/� mice was impairedand associated with a reduced IFN-� production with a concomi-tant increase IL-10 secretion, which mirrors the results obtainedwith mBSA-stimulated AIA ASC�/� splenocytes. It has been pre-viously shown that ASC is expressed in leukocytes, including neu-trophils and T cells (41), and is capable of regulating NF-�B ac-tivation in myeloid cells (19). We have excluded an effect of ASCon lymphoid cell apoptosis, but its capacity to oligomerize andinteract with signaling pathway proteins may explain the T celleffects we observed.

It is interesting to note that whereas ASC deficiency reducedjoint inflammation in AIA, in another model, which is essentiallybased on transfer of arthritogenic serum of K/BxN mice (due toautoantibodies recognizing glucose-6-phosphate isomerase, and inwhich there is no T cell dependence), we observe a marginal andnonsignificant reduction of the severity of arthritis in ASC�/�

mice (L. Kolly, S. Narayan, J. Tschopp, A. So, and N. Busso;manuscript in preparation). It is clear that in the AIA and K/BxNserum transfer arthritis models the relative contribution of immunecomplex-mediated pathology vs T cell-driven arthritis is very dif-ferent. Whereas passive K/BxN arthritis is exclusively based onimmune complex formation (42), AIA is essentially T cell medi-ated, with a relatively small contribution of anti-mBSA Abs (32).Therefore, as expected from the different mechanisms underlyingthese two models, in a T cell-dependent model of arthritis such asAIA, impaired T cell responses observed in ASC�/� mice can leadto reduced arthritis severity, whereas in a T cell-independentmodel (K/BxN), these responses will not impact on the arthritisphenotype.

Because AIA is characterized as a Th1-dependent experimentalmodel of joint disease (43, 44), the demonstration of a suppressiveIL-10 orientated T cell response in ASC�/� mice could be a pos-sible link to the observed reduced inflammatory status in theseanimals, although we cannot directly correlate this altered T cell

phenotype in ASC�/� mice with their milder disease outcome.Our results are in agreement with previous studies that have shownthat the development of Th1-mediated experimental arthritis iscritically linked with the balance of cytokines produced by Th1/Th2 cells (45–47). Th1 responses appear most predominant at thetime of arthritis onset and progressively decline during diseaseprogression and subsequent remission, which in turn correlateswith increased IL-10 levels. Collectively, these results suggest thatTh2 polarization of the immune response during Ag-induced mod-els of arthritis may act to blunt disease severity. Because IL-10 hasnot only been associated to Th2 cells but also implicated in reg-ulatory T cell function, it is also possible that IL-10-producingregulatory T cells preferentially differentiate and respond over Th1and Th2 cells in ASC�/� mice upon immune assault. On the basisof this hypothesis, recruitment of more regulatory T cells into in-flamed ASC�/� arthritic joints would lead to reduced AIA severityin these mice. Indeed, it has previously been shown that adoptivetransfer of CD4�CD25� regulatory T cells at the time of AIAinduction decreases disease severity and is associated with prefer-ential accumulation of CD4�CD25� cells in inflamed arthriticjoints (48).

Our results undeniably demonstrate that impaired IFN-� pro-duction and a reciprocal up-regulation of IL-10 occurred inASC�/� splenocytes and T cells, upon antigenic stimulus or TCRligation, respectively. In this context, we were most surprised toobserve that Ag-specific IgG levels (total, IgG1 and IgG2a) werenot affected in ASC�/� mice as predicted during a Th1 to Th2 shift(49, 50). We currently have no explanation for this discrepancy,but such discrepancy has already been observed in other experi-mental situations (51–54).

In conclusion, we have demonstrated that in AIA, componentsof the inflammasome implicated in hereditary autoinflammatorydiseases and in gout do not play a major role in disease pathogen-esis. However, ASC deficiency attenuated joint inflammation andAg-specific T cell reactivity. Furthermore, our data highlight thatthe inhibition of immune cell proliferation by ASC deficiency wasnot due to apoptosis but rather to the shift toward more IL-10production by activated T cells, thus suggesting that the immuno-modulatory effect of ASC is mediated almost exclusively by Tcells. These findings reveal a novel function of ASC in the regu-lation of inflammation and immune responses, which will be in-vestigated in subsequent studies.

AcknowledgmentsWe thank the Nuclear Medicine Department of the Centre Hospitalier Uni-versitaire Vaudois Lausanne (Dr. Angelika Bischof-Delaloye) for kindlyproviding 99mTc, Vishva Dixit for the ASC- and IPAF-deficient mice, andThibaut de Smedt for helpful discussions. We are indebted to VeroniqueChobaz and Monica Azevedo for excellent technical support.

DisclosuresThe authors have no financial conflict of interest.

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