Demyelination Mice from Autoimmune-Mediated ...

Paracaspase MALT1 Deficiency Protects Mice from Autoimmune-Mediated Demyelination This information is current as of November 2, 2015.

Conor Mc Guire, Peter Wieghofer, Lynn Elton, David Muylaert, Marco Prinz, Rudi Beyaert and Geert van Loo

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2013; 190:2896-2903; Prepublished online 11 February 2013; doi: 10.4049/jimmunol.1201351 http://www.jimmunol.org/content/190/6/2896

The Journal of Immunology

Paracaspase MALT1 Deficiency Protects Mice from Autoimmune-Mediated Demyelination Conor Mc Guire,*,† Peter Wieghofer,‡ Lynn Elton,*,† David Muylaert,*,† Marco Prinz,‡,x Rudi Beyaert,*,† and Geert van Loo*,†

E

xperimental autoimmune encephalomyelitis (EAE) is the main animal model for multiple sclerosis (MS), the most common chronic inflammatory demyelinating disease of the CNS (1, 2). Both MS and EAE are characterized by an autoreactive immune response that targets the CNS, causing typical pathological hallmarks, such as demyelination, oligodendrocyte cell death, and neurodegeneration. Encephalitogenic Th1/Th17– polarized T cells play a central role in this immune response; *Department for Molecular Biomedical Research, Unit of Molecular Signal Transduction in Inflammation, VIB, B-9052 Ghent, Belgium; †Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium; ‡Department of Neuropathology, University of Freiburg, D-79106 Freiburg, Germany; and xBIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79106 Freiburg, Germany 1

R.B. and G.v.L. share senior authorship.

Received for publication May 14, 2012. Accepted for publication January 10, 2013. This work was supported by research grants from the Interuniversity Attraction Poles Program (IAP6/18 and IAP7), the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Belgian Foundation against Cancer, the Strategic Basis Research Program of the Instituut voor Innovatie door Wetenschap en Technologie, the Hercules Foundation, and the Concerted Research Actions and Ghent Researchers On Unfolded Proteins in Inflammatory Disease Multidisciplinary Research Partnership of Ghent University. C.M. and D.M. were supported as a Ph.D. and postdoctoral fellow, respectively, by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, and L.E. was supported as a Ph.D. fellow by the Instituut voor Innovatie door Wetenschap en Technologie. G.v.L. was supported by a Fonds voor Wetenschappelijk OnderzoekVlaanderen Odysseus grant and by grants from the Geneeskundige Stichting Koningin Elisabeth and the Charcot Foundation. C.M. performed the experiments, analyzed data, and prepared the figures; P.W., L.E., D.M., and M.P. contributed to experimental work and/or provided assistance; and R.B. and G.v.L. supervised the overall research. C.M., R.B., and G.v.L. wrote the manuscript. Address correspondence and reprint requests to Dr. Geert van Loo and Dr. Rudi Beyaert, Department for Molecular Biomedical Research, VIB and Ghent University, Technologiepark 927, B-9052 Ghent, Belgium. E-mail addresses: geert.vanloo@ dmbr.vib-ugent.be (G.v.L) and [email protected] (R.B.) The online version of this article contains supplemental material. Abbreviations used in this article: APP, amyloid precursor protein; CBM, CARMA1/ B cell lymphoma-10/MALT1; CYLD, cylindromatosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; Treg, regulatory T cell. Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201351

although the trigger for this autoimmune disease has not been elucidated, considerable efforts have been made in clarifying the molecular mechanisms behind autoreactive T cell activation. The transcription factor NF-kB plays a central role in the activation and proliferation of T cells. Upon stimulation of the TCR, protein kinase C u–mediated phosphorylation of the CARD-containing protein CARMA1 (also known as CARD11) results in the recruitment of B cell lymphoma-10 (BCL10) and MALT1 (3). This CARMA1/BCL10/MALT1 (CBM) complex subsequently recruits TRAF2 and TRAF6, allowing further downstream signaling and leading to nuclear translocation and activation of NF-kB. Genetargeting strategies showed that MALT1 is indispensable for NF-kB activation downstream of TCR stimulation, resulting in an absence of T cell activation and proliferation in MALT1-deficient T cells (4, 5). Besides acting as a scaffold mediating TCR signaling, MALT1 has proteolytic activity. Indeed, recent findings demonstrated that A20 (6), BCL10 (7), RelB (8), and cylindromatosis (CYLD) (9) are substrates of MALT1. Although the adaptor function of MALT1 is indispensible for T cell activation, its proteolytic activity is considered critical for a full-blown NF-kB response, shaping the extent of T cell activation (3). Because of its essential role in T and B cell activation, MALT1 is considered an important therapeutic target in autoimmunity. However, its role in the development of autoimmune disease has not been reported. In this study, we sought to address the role of MALT1 in the generation of autoreactive T cells in the context of EAE. For this, we induced EAE in mice deficient in MALT1 (MALT12/2) and in wild-type and heterozygous littermate control mice (MALT1+/+ and MALT1 2/+). MALT1 2/2 mice were completely protected from EAE, which was reflected in the absence of immune cell infiltration, demyelination, and axonal damage in the spinal cord. Furthermore, splenocytes from MALT12/2 mice failed to produce an autoreactive T cell response and failed to induce autoimmune inflammation upon transfer in wild-type mice. Finally, cleavage of the MALT1 substrates A20 and CYLD were shown in wild-type T cells from mice with EAE. Collectively, these data demonstrate a crucial role for MALT1 in T cell activation and in the early


The paracaspase MALT 1 is a major player in lymphocyte activation and proliferation. MALT1 mediates Ag-induced signaling to the transcription factor NF-kB by functioning both as a scaffold protein and cysteine protease. We studied the role of MALT1 in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. MALT1-knockout mice did not develop any clinical symptoms of EAE. In addition, lymphocyte and macrophage infiltration into the spinal cord was absent in MALT1-knockout mice, as were demyelination and proinflammatory gene expression. Adoptive transfer experiments showed that MALT1 deficiency in splenocytes is sufficient for EAE resistance. Moreover, autoreactive T cell activation was severely impaired in MALT1-deficient T cells, suggesting the inability of MALT1-deficient effector T cells to induce demyelinating inflammation in the CNS. Finally, the MALT1 substrates A20 and CYLD were completely processed in wild-type T cells during EAE, which was partially impaired in MALT1-deficient T cells, suggesting a contribution of MALT1 proteolytic activity in T cell activation and EAE development. Together, our data indicate that MALT1 may be an interesting therapeutic target in the treatment of multiple sclerosis. The Journal of Immunology, 2013, 190: 2896–2903.

The Journal of Immunology priming phase of EAE, suggesting that targeting MALT1 might be an important therapeutic strategy to treat MS.

Materials and Methods Animals MALT12/2 mice (5), backcrossed to C57BL/6 mice, were kindly provided by Dr. Tak W. Mak (Ontario Cancer Institute, Toronto, ON, Canada). MALT12/+ mice were intercrossed to generate MALT1+/+, MALT12/+, and MALT12/2 offspring. Mice were housed in individually ventilated cages in either specific pathogen–free or conventional animal facilities. All animal experiments were performed according to institutional, national, and European animal regulations. Animal protocols were approved by the ethics committee of Ghent University.

Induction and assessment of EAE

Histological analysis Mice were transcardially perfused with PBS containing 5 IU/ml heparin (De Pannemaeker, Ghent, Belgium), followed by perfusion with 4% paraformaldehyde. Spinal cords were dissected, dehydrated, and embedded in paraffin blocks. Sections of 2 mm were stained with H&E, Luxol fast blue (Solvent Blue 38, practical grade; Sigma Genosys) for assessment of demyelination, and Abs against CD3 (clone CD3-12; Serotec), Mac-3 (clone CD107b, M3/84; Becton Dickinson Biosciences), B220 (clone RA3-6B2; Becton Dickinson Biosciences), or amyloid precursor protein (APP; clone 22C11; Millipore). Sections were rehydrated and incubated in 10 mM citrate buffer for 5 min at 94˚C. Nonspecific binding was blocked by incubating sections in 0.1 M PBS containing 10% FCS and 1% Triton X-100 for 30 min. Primary Abs were incubated overnight at 4˚C. Histological quantification was described previously (11).

T cell recall assay T cell recall responses were assessed in splenocytes isolated from mice 10 d after immunization with MOG35–55 peptide. After erythrocyte lysis using ACK lysis buffer, splenocytes were cultured in flat-bottom 96-well plates at a density of 7 3 105/well in DMEM supplemented with 5% FCS, L-glutamine, nonessential amino acids, and antibiotics. Cells were incubated for 48 h in the presence of 1, 10, or 30 mg/ml MOG35–55 peptide. After 48 h, supernatant was collected, and concentrations of IL-2 and IFN-g were measured by ELISA (eBioscience). IL-10 was quantified using a Bio-Plex Pro kit on the Bio-Plex 200 system (both from Bio-Rad), according to the manufacturer’s instructions. For analysis of cell proliferation, cultures were pulsed with 0.5 mCi [3H]thymidine/well (1 mCi/ml [3H]TdR; GE Healthcare) during the last 18 h. Cells were harvested onto glass fiber filter membranes using a 96-well plate cell harvester (IH110-96; Inotech), and thymidine incorporation was measured by scintillation counting (MicroBeta Plus 1450 reader; PerkinElmer).

T cell isolation and Western blot CD4+ T cells were isolated from splenocytes using a MACS CD4+ T cell isolation kit II (Miltenyi Biotec), according to the manufacturer’s instructions. The purity of CD4+ T cells was analyzed on a FACS LSRII apparatus using CD3-PE (clone 145-2C11; BD Pharmingen) and CD4FITC (clone GK1.5; BD Pharmingen). For Western blots, CD4+ T cells were lysed in 50 mM HEPES (pH 7.6), 250 mM NaCl, 5 mM EDTA, and 0.5% (v/v) Nonidet P-40, including phosphatase and protease inhibitors. The proteins were separated by SDS-PAGE, followed by semidry immunoblotting and detection via ECL (PerkinElmer) for analysis. The Abs used were anti-A20 (clone A12, sc-166692; Santa Cruz Biotechnology), antiCYLD (clone E10; Santa Cruz Biotechnology), and anti-actin (clone MP 6472J; MP Biomedicals).

Flow cytometry Splenocytes and thymocytes were isolated in PBS containing 0.5% BSA (Sigma-Aldrich). Cells were stained with Aqua Live/Dead (Life Technologies), anti-CD16/CD32 (clone 2.4G2; Fc Block; Becton Dickinson Biosciences), anti–CD3-eFluor 450 (clone 145-2C11; eBioscience), anti– CD4-FITC (clone GK1.5; Pharmingen), anti–CD8-PE-Cy7 (clone 53-6.7; eBioscience), anti–CD25-allophycocyanin-Cy7 (clone PC61; BD Biosciences), anti–CD44-allophycocyanin (clone IM7; eBioscience), anti– CD25-PerCP-Cy5.5 (clone PC61; BD Biosciences), and anti–CTLA4-

Quantitative real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen) and an Aurum Total RNA Isolation Mini Kit (Bio-Rad), according to manufacturer’s instructions. Synthesis of cDNA was performed using an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s instructions. A total of 10 ng of cDNA was used for quantitative PCR in a total volume of 10 ml with LightCycler 480 SYBR Green I Master Mix (Roche) and specific primers on a LightCycler 480 (Roche). Real-time PCR reactions were performed in triplicates. The following mouse-specific primers were used: HPRT forward, 59-AGTGTTGGATACAGGCCAGAC-39, HPRT reverse, 59-CGTGATTCAAATCCCTGAAGT-39; TNF forward, 59-ACCCTGGTATGAGCCCATATAC-39, TNF reverse, 59-ACACCCATTCCCTTCACAGAG-39; IL-1b forward, 59-CACCTCACAAGCAGAGCACAAG-39, IL-1b reverse, 59-GCATTAGAAACAGTCCAGCCCATAC-39; IFN-g forward, 59-GCCAAGCGGCTGACTGA-39, IFN-g reverse, 59-TCAGTGAAGTAAAGGTACAAGCTACAATCT-39; IL-6 forward, 59-GAGGATACCACTCCCAACAGACC-39, IL-6 reverse, 59-AAGTGCATCATCGTTGTTCATACA-39; MCP1 forward, 59-GCATCTGCCCTAAGGTCTTCA-39, MCP1 reverse, 59-TGCTTGAGGTGGTTGTGGAA-39; RANTES forward, 59-CGTCAAGGAGTATTTCTACAC-39, RANTES reverse, 59-GGTCAGAATCAAGAAACCCT-39; TGF-b forward, 59-GCTGAACCAAGGAGACGGAATA-39, TGF-b reverse 59-GAGTTTGTTATCTTTGCTGTCACAAGA-39; and IP-10 forward, 59-GTCACATCAGCTGCTACTC-39, IP-10 reverse, 59GTGGTTAAGTTCGTGCTTAC-39.

FIGURE 1. MALT1 is critical for the induction of EAE. EAE was induced in male MALT1+/+ (n = 4), MALT12/+ (n = 9), and MALT12/2 (n = 5) littermates. (A) Clinical symptoms. (B) Loss in body weight. Results are displayed as mean 6 SEM and are representative of three independent experiments. *p , 0.05.


Mouse myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Sigma Genosys. Ten- to 15-wk-old male mice were immunized s.c. with an emulsion of 200 mg MOG35–55 peptide in 200 ml sterile PBS and an equal volume of CFA (Sigma-Aldrich) supplemented with 10 mg/ml Mycobacterium tuberculosis H37RA (Becton Dickinson Bioscience). Mice also received 50 ng pertussis toxin (Sigma-Aldrich) in 200 ml sterile PBS at the time of immunization and 48 h later. For passive induction of EAE, spleens from immunized mice were isolated 10 d postimmunization. Splenocytes were cultured for 48 h in RPMI 1640 supplemented with 10% FBS, sodium pyruvate, L-glutamine, nonessential amino acids, antibiotics, 30 mM MOG35–55 peptide, and 10 ng/ml recombinant mouse IL-23 (eBioscience). After 48 h, splenocytes were washed and resuspended in PBS. A total of 2 3 107 live splenocytes was injected i.v. into recipient mice, which were sublethally irradiated (400 rad) 1 d prior to splenocyte injection. Clinical signs of disease were scored, as described previously (10), on a scale of 0 to 6, with 0.5 points for immediate clinical findings as follows: 0, normal; 1, weakness of tail; 2, complete loss of tail tonicity; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, forelimb paralysis or moribund; and 6, death. To eliminate any diagnostic bias, mice were scored blindly.

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MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS Table I.

Clinical features of MOG-induced EAE in MALT1+/+, MALT12/+, and MALT12/2 littermate mice

Genotype +/+

MALT1 MALT12/+ MALT12/2

Incidence (%)

Day of Disease Onset (mean 6 SEM)

Maximal Clinical Score (mean 6 SEM)

Minimal % Body Weight

4/4 (100) 7/9 (78) 0/5 (0)

15.3 6 0.6 15.0 6 0.5 –

4.1 6 0.1 3.7 6 0.5 1.0 6 0.1

79.6 6 3.4 79.3 6 2.8 97.1 6 0.4

Disease incidence, day of onset, maximal clinical score, and minimal percentage body weight. Results are displayed as mean 6 SEM. –, No disease onset.

allophycocyanin (clone UC10-4B9; eBioscience) for 30 min at 4˚C. Prior to staining for Foxp3 with anti–Foxp3-PE (clone FJK-16s), cells were fixed and permeabilized using the Anti-Mouse/Rat Foxp3 Staining Set PE (both from eBioscience), according to the manufacturer’s instructions. Measurements were performed on a BD LSR II cytometer (BD Biosciences), and data were analyzed using FACSDiva Software (BD Biosciences).

Statistical analysis

Results MALT1 ablation completely protects against EAE To determine the importance of MALT1 in the pathogenesis of EAE, we immunized mice lacking MALT1 (MALT12/2) and both heterozygous (MALT12/+) and wild-type (MALT1+/+) control littermates with MOG35–55 peptide and followed disease progression by assessing both clinical disease symptoms as well as body weight. As expected, all MALT+/+ mice developed EAE and followed a typical disease course, starting ∼15 d after immunization and reaching a mean maximal clinical score of 4.1 (Fig. 1A, Table I). MALT2/+ mice developed similar clinical symptoms, with a mean maximal clinical score of 3.7 (Fig. 1A, Table I). No differences in incidence or disease severity were found between wildtype and heterozygous mice (Table I). In contrast, MALT12/2 mice were completely protected and did not develop any clinical sign of EAE over a period of 20 d postimmunization (Fig. 1A, Table I). In addition, although MALT+/+ and MALT2/+ mice showed a decrease in body weight after immunization reflecting disease progression, MALT12/2 mice did not lose any weight (Fig. 1B, Table I). Absence of spinal inflammatory infiltration in MALT12/2 mice To further characterize the lack of clinical manifestations of EAE in MALT12/2 mice, histopathological analysis was performed on

Peripheral autoreactive T cell activation is impaired in MALT12/2 mice T cell activation was shown to be impaired in MALT12/2 mice (4, 5). To test whether this is also the case for the autoreactive T cell compartment during EAE, we analyzed the generation of MOG35–55-autoreactive T cells in MALT12/2 mice. Splenocytes from MALT1+/+, MALT1+/2 and MALT12/2 were isolated 10 d

FIGURE 2. Absence of CNS demyelination, inflammatory cell infiltration, and axonal damage in MALT1-deficient mice. Immunohistochemistry on spinal cord sections from MALT1+/+ (n = 4), MALT12/+ (n = 6), and MALT12/2 (n = 5) mice. Assessment of demyelination on sections from the lumbar spinal cord by Luxol fast blue (blue) staining (A) and axonal damage by APP (brown) immunohistochemistry (B). (C) Staining for infiltrating T cells (CD3, brown), macrophages (Mac-3, brown), and B cells (B220, brown) by immunohistochemistry. (A) Scale bars, 500 mm (top panels) and 200mm (lower panels). (B and C) S bars, 200 mm. (middle panels). Results are representative of two independent experiments.


Results are expressed as mean 6 SEM. Statistical analysis between multiple groups was assessed using one-way ANOVA, followed by the Bonferroni posthoc correction. Statistical analysis between two groups was assessed using a two-tailed Student t test.

spinal cord sections isolated at day 20 postimmunization from MALT+/+, MALT2/+, and MALT12/2 littermate mice. In line with the findings that MALT12/2 mice are protected from EAE, histopathological hallmarks of EAE were virtually absent in MALT12/2 mice compared with control littermates. Large areas of demyelination of the white matter were present in the spinal cord of MALT+/+ and MALT12/+ mice 20 d after immunization (Fig. 2A, Table II). This was accompanied by axonal degeneration as visualized by APP+ aggregates (Fig. 2B, Table II). In contrast, MALT2/2 mice showed no signs of pathology in the spinal cord after immunization (Fig. 2A, 2B, Table II). Furthermore, although inflammatory infiltration of CD3+ T cells, Mac-3+ macrophages, and B220+ B cells was present to a similar degree in spinal cords of MALT+/+ and MALT2/+ mice at 20 d post-EAE induction, these lymphomononuclear infiltrates were completely absent in MALT2/2 mice (Fig. 2C, Table II). Next, we determined the expression of proinflammatory cytokines and chemokines in spinal cord tissue of MALT+/+, MALT2/+, and MALT12/2 mice by quantitative realtime PCR on tissues isolated at day 16 postimmunization. Although all inflammatory cytokines and chemokines tested were clearly upregulated in MALT1+/+ and MALT12/+ mice during EAE, they remained undetectable in MALT2/2 mice (Fig. 3). These results demonstrate that the absence of MALT1 protects mice from EAE by inhibiting inflammatory cell infiltration into the spinal cord, as well as by preventing the expression of key mediators involved in CNS inflammation.


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Table II. Quantification of spinal cord cell infiltrates, demyelination, and axonal damage from histological sections shown in Fig. 2 Genotype

Demyelination (%)

APP+/mm2

CD3+/mm2

B220+/mm2

MAC3+/mm2

MALT1+/+ MALT12/+ MALT12/2

36.3 6 3.9 29.3 6 3.5 0.0 6 0.0

17.9 6 4.1 23.5 6 8.0 0.3 6 0.2

102.3 6 24.4 82.8 6 21.2 1.4 6 0.6

19.0 6 5.8 18.3 6 3.2 0.2 6 0.1

7780.6 6 116.6 7750.1 6 140.8 20.4 6 1.0

Percentage of demyelination and numbers of APP+ depositions, infiltrating T cells (CD3), B cells (B220), and macrophages (MAC3) in MALT1+/+ (n = 4), MALT12/+ (n = 9), and MALT12/2 (n = 5) littermate mice 25 d after EAE induction. Data are mean 6 SEM. For all parameters between MALT1+/+ and MALT12/+ versus MALT12/2, p , 0.05.

deletion might influence regulatory T cells (Tregs). Indeed, we found that naive MALT12/2 mice show a severe reduction in Treg population (Supplemental Fig. 1A, 1B). Moreover, the percentage of activated Tregs is also decreased in these mice, as shown by CTLA4 expression (Supplemental Fig. 1A, 1B). Similar observations were made in splenocytes isolated from MOG35–55-stimulated mice that were restimulated with MOG35–55 in vitro (Supplemental Fig. 1C, 1D). MOG-specific MALT12/2 splenocytes fail to induce EAE in MALT+/+ mice An impaired peripheral immune response can result in protection from EAE because of the inability of autoreactive T cells to cause pathological damage in the spinal cord. To test this hypothesis,

FIGURE 3. Impaired proinflammatory gene expression in CNS of MALT1-deficient mice. Quantitative measurements of the indicated cytokine and chemokine mRNA expression in spinal cord from MALT1+/+ (n = 2), MALT1+/2 (n = 4), and MALT12/2 (n = 5) littermate mice 16 d after immunization. Results are displayed as mean 6 SEM and are representative of two independent experiments. *p , 0.05, **p , 0.01.


after immunization, after which their in vitro response to secondary exposure to MOG35–55 peptide was analyzed. Autoreactive T cell responses to secondary MOG35–55 stimulation were comparable between MALT1+/+ and MALT12/+ mice, as assessed by proliferation and IL-2 and IFN-g production (Fig. 4A–C). In contrast, however, T cells from MALT12/2 mice were impaired in generating an immune response to secondary MOG35–55 stimulation (Fig. 4A–C). These results show that peripheral T cell activation is severely impaired in MALT12/2 mice and suggest that the protective phenotype of MALT12/2 mice in EAE results from this impaired autoreactive immune response. Interestingly, MALT12/2 splenocytes also produced less IL-10 after in vitro stimulation with MOG35–55 (Fig. 4D). These findings suggest that, in addition to affecting effector T cells, MALT1

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MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS

a series of adoptive-transfer EAE experiments was performed. Splenocytes from MALT1+/+ and MALT12/2 mice were isolated 10 d postimmunization and restimulated in vitro with MOG35–55 peptide and IL-23, after which donor splenocytes were injected into either MALT1+/+ or MALT12/2 recipient mice that were sublethally irradiated 1 d prior to injection to deplete hematopoietic cells. As expected, donor MALT+/+ splenocytes induced EAE in recipient MALT1+/+ mice, reflected in both clinical disease progression and body weight loss (Fig. 5, Table III, Supplemental Fig. 2). Similarly, splenocytes from MALT1+/+ donor mice induced EAE in acceptor MALT12/2 mice to a comparable degree. In line with findings that MALT12/2 splenocytes fail to induce an effective immune response in vitro, MALT2/2 spleno-

cytes also fail to induce clinical manifestations of EAE in both MALT1+/+ and MALT2/2 mice (Fig. 5, Table III, Supplemental Fig. 2). Collectively, these results show that MALT12/2 mice do not develop EAE because of an impaired peripheral immune response. A20 and CYLD processing is MALT1 dependent in T cells during EAE Recently, it was demonstrated that MALT1 not only functions as an adaptor protein, it also acts as a protease capable of cleaving a number of proteins, including A20 and CYLD, which both negatively regulate TCR signaling to NF-kB (6–9, 12). To investigate MALT1’s proteolytic activity during the course of EAE,

FIGURE 5. MOG-specific MALT12/2 splenocytes fail to induce EAE in MALT+/+ mice. Splenocytes from MOG-immunized MALT1+/+ and MALT12/2 donor mice were injected into sublethally irradiated MALT1+/+ and MALT12/2 recipient mice. Clinical disease progression (A) and loss of body weight (B) were scored. Results are displayed as mean 6 SEM and are representative of two independent experiments. **p , 0.01, ***p , 0.001, MALT1+/+ → MALT1+/+ versus MALT2/2 → MALT1+/+.


FIGURE 4. Peripheral T cell activation is impaired in MALT-deficient mice. Splenocytes from MOG peptide–immunized control (MALT1+/+ and MALT12/+) and MALT12/2 mice were cultured and stimulated with the indicated concentrations of MOG peptide. (A and B) Culture supernatants were collected 48 h after MOG peptide stimulation and assayed for IL-2 and IFN-g by ELISA. Results are shown as means 6 SEM and are representative of two independent experiments. (C) T cell proliferation was assessed by [3H]thymidine incorporation. Results are expressed as cpm of triplicate cultures and are representative of two independent experiments. (D) Culture supernatant was collected from splenocytes 48 h after MOG peptide stimulation and assayed for IL-10 by Bio-Plex. Results are shown as means 6 SEM. **p , 0.01, ***p , 0.001.


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Table III. Quantification of spinal cord cell infiltrates, demyelination, and axonal damage from histological sections shown in Supplemental Fig. 2 Transfer

Demyelination (%)

APP+/mm2

CD3+/mm2

MAC3+/mm2

MALT1+/+ → MALT1+/+ MALT1+/+ → MALT12/2 MALT12/2 → MALT1+/+ MALT12/2 → MALT12/2

26.1 6 3.3 45.2 6 14.3 0.0 6 0.0 0.0 6 0.0

105.9 6 21.7 100.2 6 33.9 4.3 6 1.5 5.1 6 3.5

192.4 6 75.9 385.0 6 190.8 0.5 6 0.5 0.0 6 0.0

7482.3 6 87.8 7877.8 6 376.9 00.0 6 0.0 20.0 6 0.0

Percentage of demyelination, and numbers of APP+ depositions, infiltrating T cells (CD3), and macrophages (MAC3) in spinal cord sections from MALT1+/+ → MALT1+/+ (n = 5), MALT1+/+ → MALT12/2 (n = 3), MALT12/2 → MALT1+/+ (n = 3), and MALT12/2 → MALT12/2 (n = 3) transfers 26 d after EAE induction. Data are mean 6 SEM. For all parameters between MALT1+/+ → MALT1+/+ versus MALT12/2 → MALT1+/+, p , 0.05.

Discussion MALT1 is an essential signaling protein in TCR-induced NF-kB and JNK activation. Although MALT1 is essential for thymic T cell maturation, reflected by a block in T cell development at the DN3 to DN4 transition in thymocytes from MALT2/2 mice (Supplemental Fig. 3) (4), deletion of MALT1 has no effect on the total number or distribution of CD4+ and CD8+ T cell populations

FIGURE 6. A20 and CYLD processing in purified T cells from mice with EAE is partially MALT1 dependent. CD4+ T cells were isolated from the spleen of MALT1+/+ and MALT12/2 naive and EAE mice every 2 d from days 14 to 24 postimmunization and assayed for A20 and CYLD expression and processing by Western blot analysis. Full-length A20 and CYLD are indicated by an arrow. Results are representative of two independent experiments.

in the spleen or lymph nodes, but leads to a decrease in the frequency of activated T cells in the periphery. Consistent with these observations, peripheral lymphocytes from MALT1-deficient mice are defective in AgR-mediated T cell activation, proliferation, and IL-2 production (4, 5). Therefore, MALT1 was suggested to be an attractive drug target for the treatment of autoimmune disease. In this study, we investigated the sensitivity of MALT1-deficient mice to the development of EAE as a mouse model for MS. MALT1-deficient mice are completely resistant to myelin autoantigen-induced disease development because of severely reduced peripheral autoantigen-specific T cell responses, consistent with MALT1’s essential role in TCR activation and effector functions in vivo. In addition to the crucial role of MALT1 in mediating an effector T cell response, we demonstrate that MALT1 deficiency leads to a severe impairment in the generation and activation of Tregs. Previous studies demonstrated that Tregs are essential in the dampening of an autoreactive T cell response in the context of EAE (13). However, we believe that, in conditions of MALT1 deficiency, this Treg defect is secondary to the effector T cell defect, resulting in complete protection of MALT1-deficient mice from EAE. Most likely, the protective effect of MALT1 deficiency also reflects its role in TCR-induced NF-kB signaling, because mice lacking the NF-kB isoforms NF-kB1 (p50) or c-Rel and mice specifically lacking IkB kinase b in T cells were reported to be completely resistant to EAE (14–16). One mechanism by which T cell–specific NF-kB responses promote inflammation and T cell– mediated autoimmune pathology is through their control of Th17 cell differentiation, because studies showed that c-Rel regulates the development and severity of EAE by influencing the balance between Th17/Th1 cells and Tregs (17, 18). TCR-induced signaling to NF-kB involves the formation of a CBM scaffolding complex, which subsequently recruits downstream signaling proteins, such as TRAF6, caspase-8, and TAK1, to collaboratively activate the IKK complex (3). Besides functioning as an adaptor protein in the CBM complex, MALT1 exerts proteolytic activity to further promote TCR-induced NF-kB and JNK activation (6–9). In this context, MALT1 was shown to cleave the NF-kB and JNK inhibitory proteins A20 and CYLD in AgR-stimulated T cells in vitro (6, 9), which is believed to contribute to a full-blown NF-kB and JNK response that is essential for T cell function. Interestingly, our data now demonstrate a role for MALT1 in the processing of A20 and CYLD in T cells from mice suffering from EAE, which, to our knowledge, is the first evidence for A20 or CYLD processing in vivo. Surprisingly, although one would expect that not all of the CD4+ T cells in the spleen would be activated MOG35–55-specific T cells, splenic CD4+ T cells from the wild-type EAE mice showed a fully cleaved form of A20 and CYLD. We hypothesize that part of the A20/ CYLD processing comes from T cells that are indirectly activated by Ags other than MOG35–55 and possibly Ags released from dying cells. However, processing of A20 and CYLD was strongly


we analyzed the expression and processing of A20 and CYLD in purified CD4+ T cells at different time points, ranging from 14 to 24 d postimmunization. Full-length A20 and CYLD are present in naive CD4+ T cells of both MALT1+/+ and MALT12/2 mice (Fig. 6). Interestingly, both A20 and CYLD are no longer detectable in MALT1+/+ CD4+ T cells from EAE-diseased mice at days 14–20 postimmunization, most likely reflecting their proteolytic processing. Interestingly, A20 and CYLD expression reappears at 22 and 24 d postimmunization, reflecting their resynthesis. We were not able to clearly detect the 65- and 70-kDa fragments that were previously described to result from MALT1-mediated cleavage of A20 and CYLD, respectively (6, 9). However, a role for MALT1 in the processing of A20 and CYLD in MALT1+/+ CD4+ T cells from EAE-diseased mice is supported by our observation that MALT1 deficiency at least partially prevents the disappearance of full-length A20 and CYLD (Fig. 6). The inability to detect specific A20 and CYLD fragments that are generated by MALT1 is most likely explained by their further processing by other proteases that are activated in CD4+ T cells during EAE. Altogether, these data suggest the activation of MALT1 proteolytic activity in T cells from EAE-diseased mice, leading to the MALT1-mediated processing of two of its known substrates, A20 and CYLD.

2902

Acknowledgments We thank Dr. Tak W. Mak (Ontario Cancer Institute, Toronto, ON, Canada) for donating the MALT1-knockout mouse and K. Barbry (VIB, Ghent, Belgium) for animal care.

Disclosures The authors have no financial conflicts of interest.

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impaired in CD4 T cells derived from MALT1-deficient mice, indicating an important role for MALT1 in the observed A20 and CYLD processing. It should be mentioned that we were unable to detect specific A20 or CYLD fragments that were previously described to be generated by MALT1 in TCR-stimulated cells in vitro (6, 9). Most likely, A20, CYLD, and the fragments generated by MALT1 are also processed by other proteases that are activated in T cells from EAE mice. In this context, increased proteolytic activity in lymphocytes of rats with EAE was described, but the identity of the protease(s) remains unclear (19). In this context it is worth mentioning that CYLD was demonstrated to be processed by caspase-8 (20, 21). In addition, proteasomes were shown to contribute to A20 degradation in activated T cells (12), and a similar proteasome-mediated regulation was suggested for CYLD (22). While revising our manuscript, a similar study describing an essential role for MALT1 in EAE was published (23). In that study, MALT1 deficiency was shown to abolish the expression of the Th17 effector cytokines IL-17 and GM-CSF, which are essential for a pathogenic inflammatory response (23). Moreover, the MALT1 substrate RelB was shown to be cleaved and inactivated in wildtype Th17 cells but not in MALT1-deficient Th17 cells, indicating the importance of MALT1 proteolytic activity for the generation of Th17 cells (23). Altogether, these studies indicate an important role for MALT1 proteolytic activity in the pathophysiology of EAE. Although the specific importance of MALT1-dependent A20, CYLD, and RelB processing in the development of MS remains to be determined, the fact that A20 deficiency in mice is associated with autoimmunity (24–29) and the association between single-nucleotide polymorphisms in the human A20 gene locus and multiple autoimmune diseases, including MS (30–32), suggest a potential role for A20 cleavage. Very recently, small compound MALT1 inhibitors that inhibit T cell activation and suppress the growth of the MALT1-dependent activated B cell subtype of diffuse large B cell lymphoma in vitro and in vivo were described (33, 34). Therefore, it will be of high interest to analyze the effect of such inhibitors on the development of EAE and other T cell–mediated autoimmune pathologies.

MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS

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