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Regulation of Experimental Autoimmune Encephalomyelitis by TPL-2 Srividya Sriskantharajah, Eva Gückel, Niki Tsakiri, Katrin Kierdorf, Christine Brender, Abduelhakem Ben-Addi, Marc This information is current as Veldhoen, Philip N. Tsichlis, Brigitta Stockinger, Anne of September 24, 2021. O'Garra, Marco Prinz, George Kollias and Steven C. Ley J Immunol 2014; 192:3518-3529; Prepublished online 17 March 2014;

doi: 10.4049/jimmunol.1300172 Downloaded from http://www.jimmunol.org/content/192/8/3518

Supplementary http://www.jimmunol.org/content/suppl/2014/03/17/jimmunol.130017 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 53 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/192/8/3518.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Experimental Autoimmune Encephalomyelitis by TPL-2 Kinase

Srividya Sriskantharajah,*,1 Eva Guckel,*€ ,1 Niki Tsakiri,† Katrin Kierdorf,‡ Christine Brender,* Abduelhakem Ben-Addi,* Marc Veldhoen,x Philip N. Tsichlis,{ Brigitta Stockinger,x Anne O’Garra,‖ Marco Prinz,‡ George Kollias,† and Steven C. Ley*

Tumor progression 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental

autoimmune encephalomyelitis (EAE), a Th17 cell–mediated disease model for multiple sclerosis. Nevertheless, TPL-2–deficient Downloaded from mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell–intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to

stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of http://www.jimmunol.org/ TPL-2 may be beneficial in multiple sclerosis therapy. The Journal of Immunology, 2014, 192: 3518–3529.

umor progression locus 2 (TPL-2), also known as COT nase that activates the MAP2 MEK-1/2 (1). During innate and MAP3K8, is expressed in both hematopoietic and immune responses, TPL-2 expression is required for activating T nonhematopoietic cells, and it functions as a MAP3 ki- ERK-1/2 MAPKs in myeloid cells by TLRs, as well as the receptors for TNF and IL-1b (1–4). TPL-2 is critical for the production of TNF during TLR4-induced acute inflammatory responses, and it is *Division of Immune Cell Biology, National Institute for Medical Research, London NW7 1AA, United Kingdom; †Institute for Immunology, Biomedical Sciences Re- also required for the development of TNF-induced Crohn’s-like search Center “Alexander Fleming,” 16672 Vari, Attica, Greece; ‡Department of by guest on September 24, 2021 x inflammatory bowel disease (2, 5). Consequently, TPL-2 is con- Neuropathology, University of Freiburg, D-79106 Freiburg, Germany; Division of Mo- sidered an attractive drug target for TNF-dependent inflammatory lecular Immunology, National Institute for Medical Research, London NW7 1AA, United Kingdom; {Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA diseases (6). ‖ 02111; and Division of Immunoregulation, National Institute for Medical Research, In contrast to its positive role in inflammation driven by innate London NW7 1AA, United Kingdom immune cells, TPL-2 can negatively regulate Th2 cell–driven 1 S.S. and E.G. contributed equally to this work. inflammation. When rechallenged with OVA intranasally, OVA-sen- Received for publication January 16, 2013. Accepted for publication February 18, sitized Map3k82/2 mice express higher levels of IgE and develop 2014. more severe bronchoalveolar inflammation (7). This augmented This work was supported by U.K. Medical Research Council Grants U117584209 (to S.C.L.), U117565642 (to A.O.), and U117512792 (to B.S.) and Arthritis Research inflammation occurs as a consequence of a default increase in Th2 + U.K. Grant 19431 (to S.C.L.), as well as by European Commission programs Inflammation effector cells, resulting from impaired Th1 differentiation of CD4 and Cancer Research in Europe (Contract 223151), Mechanisms to Attack Steering Effectors T cells in Map3k82/2 mice (8), possibly through the removal of of Rheumatoid Syndromes with Innovated Therapy Choices (Contract 223404), and Inno- vative Medicines Initiative Joint Undertaking “Be the Cure” (Contract 115142) (all to G.K.). IFN-g–mediated Th2 inhibition (9). However, it has not been estab- S.S., E.G., N.T., K.K., A.B.-A., and M.V. designed and performed experiments, ana- lished that TPL-2–mediated inhibition of lung inflammation in the lyzed the data, performed statistical analyses, and prepared the figures. C.B. generated OVA lung allergy model actually results from a T cell–intrinsic sig- D270A/D270A 2/2 the Map3k8 mutant mouse strain; P.N.T. provided the Map3k8 mutant naling function. In contrast, the protective role for TPL-2 in pro- mouse strain and helpful discussions; M.V., A.O., B.S., and M.P. contributed key reagents, advised with the experimental design, and discussed the data; G.K. and moting Th1 effector cell differentiation during the immune response S.C.L. designed the study in conjunction with M.V., B.S., and S.S.; and S.C.L., S.S., to Toxoplasma gondii, a Th1 cell–inducing parasite, has been directly and E.G. wrote the manuscript. demonstrated to be mediated by TPL-2 signaling in T cells (8). Address correspondence and reprint requests to Dr. George Kollias or Dr. Steven C. Ley, Th17 cells, a subset of effector CD4+ T cells characterized by Institute for Immunology, Biomedical Sciences Research Center “Alexander Fleming,” 16672 Vari, Attica, Greece (G.K.) or Division of Immune Cell Biology, National Insti- the production of the cytokine IL-17 (10), are important in im- tute for Medical Research, Mill Hill, London NW7 1AA, U.K. (S.C.L.). E-mail ad- mune responses to bacteria and fungi. Additionally, aberrant Th17 dresses: kollias@fleming.gr (G.K.) or [email protected] (S.C.L.) cell activation is causally linked to the development of several The online version of this article contains supplemental material. human autoimmune diseases. In the present study, we investigated Abbreviations used in this article: BM, bone marrow; BMDC, bone marrow–derived the potential requirement for TPL-2 in cytokine-induced Th17 cell dendritic cell; BMDM, bone marrow–derived macrophage; DC, dendritic cell; dLN, draining lymph node; EAE, experimental autoimmune encephalomyelitis; FICZ, 6- differentiation in vitro, and also the role of TPL-2 in experimental formylindolo[3,2-b]carbazole; IKK, IkB kinase; LN, lymph node; MOG, myelin oligo- autoimmune encephalomyelitis (EAE), in which Th17 cells have HI dendrocyte glycoprotein; MS, multiple sclerosis; Mtb , heat-inactivated Mycobacterium an important pathogenic role (11). EAE is an animal model for tuberculosis; PdBu, phorbol dibutyrate; TPL-2, tumor progression locus 2; WT, wild-type. multiple sclerosis (MS), which is induced by immunization with Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 myelin Ags, such as myelin oligodendrocyte glycoprotein (MOG) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300172 The Journal of Immunology 3519

(12). We found that TPL-2 was dispensable for cytokine-induced Laboratories). Mice received 200 ng pertussis toxin (Calbiochem) i.p. differentiation of naive T cells to the Th17 cell lineage in vitro. on day 0 and 2 d after immunization. For passive EAE experiments, 2/2 3 6 Furthermore, TPL-2 was not required for the initial activation and Map3k8 or WT mice were injected i.v. with 30 10 Th17-polarized MOG35–55-specific T cells and i.p. with pertussis toxin (days 0 and 2). To expansion of MOG-specific Th17 cells in the periphery during generate Th17 cell populations, draining lymph node (dLN) cells from EAE. Generation of IFN-g–producing Th1 cells, which may also Map3k82/2 or WT mice were collected 10 d after immunization with 3 6 play a role in EAE (11), was also independent of TPL-2 expres- MOG35–55 peptide plus CFA. Cells were cultured for 3 d (5 10 cells/ml; sion. Nevertheless, TPL-2 was demonstrated to regulate both the 24-well plates) in IMDM (Sigma-Aldrich) supplemented with 10% (v/v) heat-inactivated FCS (LabTech), 5 mM L-glutamine, 50 mM 2-ME, and onset and severity of EAE. Cell transfer experiments established antibiotics (all Life Technologies) in the presence of 10 mg/ml MOG35–55 that TPL-2 regulated the effector phase of EAE in the CNS, func- peptide under Th17-polarizing conditions (20 ng/ml recombinant murine tioning in radiation-resistant stromal cells. Importantly, using a newly IL-23 [R&D Systems] plus 20 ng/ml recombinant murine IL-1b [Invi- generated Map3k8 knock-in mouse strain, we also showed that the trogen]) or Th1-polarizing conditions (20 ng/ml recombinant murine IL-12 development of EAE was dependent on TPL-2 catalytic activity, [R&D Systems] and 1 mg/ml anti–IL-23p19 [eBioscience]). Clinical signs of disease were assigned daily: 0, no symptoms of disease; 1, loss of tail while ruling out any potential function of TPL-2 as scaffolding tonicity; 2, hindlimb weakness/impaired gait; 3, partial hindlimb paralysis; protein in the disease process. Our data therefore suggest that small 4, complete hindlimb paralysis; 5, forelimb paralysis or moribund. molecule inhibitors of TPL-2 might be beneficial therapeutically in MS, the most common inflammatory demyelinating disease of the Generation of bone marrow radiation chimeras CNS (13). Bone marrow cells from Map3k82/2 or WT control mice were depleted of T cells with biotinylated TCRb mAb (H57-597; BD Phamingen) and 3 6 streptavidin-labeled magnetic beads (Dynal/Invitrogen). Five to 10 10 Downloaded from Materials and Methods cells were then transferred by i.v. injection into lethally irradiated (twice Mice 400 rad) Rag12/2 or Tpl22/2Rag12/2 hosts. For mixed bone marrow 2/2 2/2 2/2 2/2 2/2 chimeras, WT or Map3k8 bone marrow cells were mixed with Tcra Map3k8 (2), Tnip2 (14), and Ifnar mice were fully backcrossed 2/2 2/2 2/2 2/2 2/2 or mMT bone marrow cells at a ratio of 1:4, prior to injection into to C57BL/6 mice. Map3k8 Rag1 and Ifnar Map3k8 mice 2/2 were generated by intercrossing the appropriate single knockout mice. For sublethally irradiated (500 rad) Rag1 mice. EAE was induced 6–8 wk the generation of Map3k8D270A/D270A mice a P1-derived artificial chro- after cell transfer. mosome clone (519-N17), including the Map3k8 , was isolated from http://www.jimmunol.org/ an RPC121 P1-derived artificial library (obtained from the Isolation of CNS cells U.K. Mapping Project Resource Centre, Hinxton, U.K.) by Brains and spinal cords were mechanically homogenized and passed through hybridization with an 1.5-kb probe encompassing full-length rat Map3k8 a 70-mm cell strainer (BD Pharmingen). Cells were centrifuged at 400 3 g cDNA. A 6.6-kb ClaI-BamHI fragment containing exon 5, which encodes +/2 for 5 min at 4˚C, and lymphocytes were separated on a 36.5% Percoll D270, was isolated and subcloned into pBluescript SK (Invitrogen) to gradient (GE Healthcare) prior to flow cytometric staining. create pSK-LA (“left arm”). From this plasmid, a 3.2-kb PstI fragment was +/2 subcloned into pBluescript SK to create pSK-LA.PstI. PCR was used to Histological analysis mutate the sequence encoding D270 to alanine. To facilitate screening for the mutation, a BssSI site was introduced next to the 39 of the D270A Spinal cords were removed and fixed in 4% formalin. Paraffin-embedded mutation without altering with the coding sequence. The D270A-containing sections were stained, as previously described (15), with Luxol fast blue,

PstI fragment was subsequently reinserted into pSK-LA to create pSK-LA. anti-CD3 (Serotec), anti–Mac-3, anti-B220 (both BD Pharmingen), and by guest on September 24, 2021 D270A, and from here a 5.2-kb HpaI fragment containing the mutation was amyloid precursor protein (APP; Chemicon). cloned into a unique XhoI cloning site of the pLox-AP1 vector to produce the pLox-AP1-LA (left arm). The pLox-AP1 vector contains a neomycin- T cell recall assay resistance gene flanked by two loxP sites and the HSV thymidine kinase gene, both driven by the PGK1 promoter. A 4.7-kb BamHI-SalI fragment LN cells were isolated from mice 9 d after immunization with MOG35–55 2 3 6 was isolated and subcloned into pBluescript SK+/ to create pSK-RA peptide. Cells were cultured for 24 h in complete medium (2 10 cells/ (“right arm”). From this plasmid, a 4.4-kb HpaI-Bsu36I fragment was ml; 96-well flat-bottom plates) containing 10 mg/ml MOG35–55 peptide, and subcloned into pLox-AP1-LA to create the pLox-AP1-Tpl2D270A target- supernatants were collected for quantification of IL-17 and IFN-g by ing vector (Supplemental Fig. 4D). The vector was linearized with NotI and ELISA. For proliferation assays, LN cells were labeled with CFSE (Mo- transfected into embryonic stem cells (carried out by PolyGene, Rumlang,€ lecular Probes) as described previously (16), and cultured for a total of 72 h. Switzerland). C57BL/6 (CD45.2+, wild-type [WT]), CD45.1 C57BL/6, 2 2 2 2 2 2 ELISA quantitation of cytokines CD45.1 Rag1 / , Tcra / ,andmMT / mice were bred in specific pathogen-free animal facilities of the National Institute for Medical Re- Levels of IL-17 and IFN-g were quantified by ELISA according to the search (London, U.K.) in accordance with the U.K. Home Office regu- manufacturer’s protocol (eBioscience). lations. Six- to– 10-wk-old male mice were used for all experiments. Real-time quantitative PCR Abs Spinal cord tissue was collected in RNAlater stabilization buffer (Qiagen) Abs to TPL-2, IkBa, ERK-1, ERK-2, and actin were purchased from 15 d after MOG35–55 peptide/CFA immunization. Total RNA was isolated Santa Cruz Biotechnology, whereas p-p105 (Ser933), p-p38, and p-ERK from spinal cords, cultured T cells, and primary cultures of microglia and (Thr185/Tyr187) Abs were obtained from Invitrogen. Tubulin mAb was astrocytes (RNeasy kit; Qiagen). After treatment with DNAase I (Invi- provided by Keith Gull (University of Oxford). trogen), cDNA was synthesized (1 mg RNA; SuperScript First Strand Syn- A number of fluorescently labeled Abs for flow cytometry were used thesis System, Invitrogen), and expression of mRNA ws determined using against GM-CSF–PE, Gr1-FITC, CD25-PE, TCRb-PECy5, TCRgd-PE, an ABI Prism 7000 sequence detection system and commercial FAM-labeled streptavidin-PerCP, and streptavidin-PE, which were purchased from probes (Applied Biosystems). is displayed in arbitrary units BD Pharmingen. IL-17A–allophycocyanin, IFN-g–FITC, CD4-FITC, relative to Hprt mRNA (encoding hypoxanthine guanine phosphoribosyl- CD4-PE, F4/80-allophycocyanin, F4/80-PE, biotinylated Gr1, CD25- ). allophycocyanin, CD44-AF450, CD44-FITC, CD45.2-FITC, CD45–Alexa Fluor 450, biotinylated CD45.1, CD11c-PE, CD11b-PE, biotinylated PE, Protein analyses and biotinylated MHC class II were obtained from eBioscience. CD4-PerCP and CD19-Pacific Blue were purchased from BioLegend. CD4-PE/Texas Purified bone marrow–derived macrophages (BMDMs), bone marrow– Red and CD8-PE/Texas Red were obtained from Invitrogen. derived dendritic cells (BMDCs), and T cells were serum-starved for 12 h (1% FCS) to reduce basal ERK activation. BMDMs and BMDCs were Induction and assessment of EAE stimulated with 1 mg/ml heat-inactivated M. tuberculosis (Difco Labora- tories), whereas CD4+ T cells were stimulated with soluble anti-CD3 Active EAE was induced by immunizing mice s.c. with 250 mg MOG35–55 (1 mg/ml; BD Pharmingen) plus anti-CD28 (1 mg/ml; BD Pharmingen). peptide (Cambridge Research Biochemicals), emulsified in CFA, con- Cultured primary microglia and astrocytes were stimulated with LPS (100 taining 250 mg heat-killed Mycobacterium tuberculosis (H37RA; Difco ng/ml; Enzo), recombinant murine TNF (50 ng/ml; R&D Systems), IFN-g 3520 TPL-2 KINASE AND EAE

2 2 (100 ng/ml; R&D Systems), IL-1b (20 ng/ml; PeproTech), and IL-17A T cells from WT and Map3k8 / mice were cultured under Th17- (100 ng/ml; R&D Systems), alone or in the indicated combinations. Cells polarizing conditions (19). Intracellular staining revealed that were washed once in PBS before lysis in buffer A (50 mM Tris [pH 7.5], TPL-2 deficiency did not significantly reduce the fraction of IL- 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na VO , 3 4 17A+ cells (Fig. 1A, 1B). The amount of IL-17A in culture 100 nM okadaic acid; Calbiochem, 2 mM Na4P2O7 plus protease inhib- 2 2 itors) containing 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. supernatants was also equivalent between WT and Map3k8 / Centrifuged lysates were mixed with an equal volume of 23 Laemmli cells (Fig. 1C). Similarly, TPL-2 expression was not required for sample buffer, resolved by SDS-PAGE, and immunoblotted. Protein con- production of IL-17A+ cells or IL-17 protein in culture super- centration in lysates was determined by Bradford assay (Bio-Rad). natants when either IL-1b or the aryl hydrocarbon receptor ligand Flow cytometry 6-formylindolo[3,2-b]carbazole (FICZ) (18, 21) were added to the Single-cell suspensions were obtained from LNs, spleen, brain, or spinal culture medium (Fig. 1). cords of mice via gentle homogenization through nylon mesh filters (70 mm; Quantitative real-time PCR was used to determine whether BD Pharmingen). Cell concentrations were determined using a Casy counter TPL-2 regulated the induction of other Th17 signature cytokines (Scharfe Instrument Systems). Erythrocytes in spleen samples were lysed and transcription factors (22). Steady-state levels of Il17a, Il17f, prior to staining. and Il21 mRNAs were equivalent in Map3k82/2 and WT CD4+ For analysis of surface markers, cells were stained with the indicated Abs in PBS (2% [w/v] BSA). For intracellular cytokine staining, cells were T cells in each of the conditions tested (Fig. 1D). The abun- restimulated for 4 h with phorbol dibutyrate (PdBu; 0.5 mg/ml; Sigma- dance of Il22 mRNA in IL-1b or FICZ containing cultures was Aldrich), ionomycin (0.5 mg/ml; Sigma-Aldrich), and brefeldin A (1 mg/ also similar between Map3k82/2 and WT CD4+ cell cultures (18). ml; GolgiPlug; BD Pharmingen), or with MOG35–55 peptide for 12 h, The orphan nuclear receptor retinoic acid–related orphan receptor adding brefeldin A for the last 4 h of culture. Cells were stained for surface Ags as indicated, fixed for 15 min in 4% (v/v) paraformaldehyde (Sigma- (ROR)gt, which is selectively expressed in Th17 cells, induces the Downloaded from Aldrich), and permeabilized with 0.1% (v/v) Nonidet P-40 for 4 min. In- development of Th17 cells, in conjunction with RORa (23, 24). tracellular Abs were added in PBS containing 0.01% (v/v) sodium azide Expression of mRNAs encoding RORgtandRORa was comparable and 24G2 cell supernatant to block Fc receptor binding. Four- and seven- in Map3k82/2 and WT CD4+ T cells (Fig. 1D). TPL-2, therefore, color cytometric staining was analyzed on FACSCalibur and Cyan instru- was not essential for in vitro differentiation of CD4+ T cells to the ments (Becton Dickinson), respectively. Data analysis was performed with FlowJo v8.5 software (Tree Star). Th17 cell lineage. http://www.jimmunol.org/ Cell culture and purification TPL-2 regulates the onset and severity of EAE Macrophages and myeloid DCs were generated from BM stem cells as To investigate the physiological role of TPL-2 in Th17 cell devel- described previously (17), with purities of $95% for BMDM (F4/80+) opment and function, we tested the susceptibility of Map3k82/2 and BMDC (CD11c+) populations. For biochemical analyses, CD4+ + mice to EAE, a Th17 cell-mediated animal model for MS (11). T cells were purified ($95% CD4 ) from single-cell suspensions pre- 2/2 pared from LNs by negative selection as described (16). For the iso- Map3k8 mice and control C57BL/6 WT mice were immunized + lation of naive T cells, CD4 T cells were prepared from pooled LNs with MOG35–55, and disease progression was monitored (13). 2 2 and spleens by negative selection, as described above. Cells were then Despite 100% incidence, Map3k8 / mice showed a delay in the stained with anti-CD4 (RM45; BD Biosciences), anti-CD25 (PC61.5; + lo onset and a reduced disease severity relative to controls (Fig. 2A, eBioscience), and anti-CD44 (IM7; BD Biosciences), and CD4 CD44 by guest on September 24, 2021 2 . Table I). Accordingly, flow cytometric analyses at the peak of CD25 naive cells were isolated to purities of 98% on a MoFlo cytometer + (Dako Cytomation). Naive T cells were differentiated into Th17 cells as disease revealed significantly fewer CD45 hematopoietic cells, described (18, 19). including CD4+ and CD8+ T cells, CD19+ Bcells,DCs,CD45+ Mixed glial cultures were prepared from 1- to 2-d-old mice using CD11b+ macrophages, and neutrophils, in the spinal cords of a published protocol (20). In brief, brains were dissected and meninges Map3k82/2 mice compared with WT mice (Fig. 2B). were removed. Brains were mechanically homogenized and passed through + a 70-mm cell strainer (BD Pharmingen). The resulting cell suspension was Intracellular staining showed similar proportions of CD4 T cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated cells expressing IFN-g and GM-CSF in the spinal cord between 2 2 FCS, antibiotics, and 20% L929 cell supernatant, with medium changes WT and Map3k8 / mice (Fig. 2C, upper panel). However, the every 3–4 d. After 10–14 d, the floating and loosely adherent microglial fraction of CD4+ cells producing IL-17A was significantly de- cells were harvested by vigorous shaking of culture flasks (250 rpm, 3 h, ∼ 3 5 creased by 50% in the absence of TPL-2. Consistent with the 37˚C), before being replated at 2.0 10 in 24-well plates and stimulated + the following day. Adherent astrocytes were trypsinized, seeded at densi- overall decrease in CD4 T cell number, the total numbers of ties of 1.0 3 106 per six-well plate, rested overnight in 1% serum, and CD4+ T cells expressing each of these encephalitogenic cytokines stimulated the following day. Microglial cell populations were $98% were significantly reduced in Map3k82/2 mice (Fig. 2C, lower + CD11b , whereas astrocyte cell populations were $85% glial fibrillary panel). The proportions of gd T cells expressing IL-17A and acidic protein+ staining, as determined by flow cytometry. IFN-g in the CNS were unaffected by TPL-2 deficiency, and total Statistical analyses gd T cell numbers were not statistically significantly different from WT (Supplemental Fig. 1A). Immunohistochemistry confirmed re- 6 Data are presented as means SEM. For analysis of clinical scores, a two- duced infiltration of T and B cells and macrophages in the spinal way ANOVAwith Bonferroni correction was applied. An unpaired Student 2/2 t test with two-tailed p values was used for statistical analysis of in vitro cords of Map3k8 mice, accompanied by significantly reduced assays. A nonparametric Mann–Whitney U test was used for statistical degrees of demyelination and neuronal damage (Fig. 2D, 2E). analyses of flow cytometric data. All statistical analyses were calculated A number of proinflammatory cytokines and chemokines are , using GraphPad Prism 5 software. A p value 0.05 was considered sig- produced in the spinal cord by infiltrating reactivated CD4+ T cells nificant. and activated CNS-resident cells (25, 26). Quantitative RT-PCR demonstrated significantly reduced amounts of mRNAs encoding Results most of these proteins in the spinal cords of Map3k82/2 mice at TPL-2 is not essential for in vitro generation of Th17-polarized the peak of disease (Supplemental Fig. 1B). In contrast, steady- cells state levels of Il12a and Il12b mRNAs were increased, consistent TPL-2 is required for efficient polarization of naive CD4+ T cells with earlier in vitro studies (27). to IFN-g–producing Th1 cells in vitro, and in vivo after T. gondii Taken together, these data indicate that TPL-2 deficiency pro- infection (8). To initially determine whether TPL-2 was required tected mice from EAE by limiting CNS inflammation, thereby for polarization to the Th17 cell lineage, CD252CD44loCD4+ reducing demyelination and axonal damage. The Journal of Immunology 3521

FIGURE 1. TPL-2 is not required for in vitro differentiation of Th17 cells. (A–C)CD252CD44hiCD4+ T cells (naive CD4+ T cells) were isolated from WT (Map3k8+/+) and Map3k82/2 mice by FACS sorting and cultured in triplicates for 4 d with anti-CD3/anti-CD28 plus the indicated Th17-polarizing cytokines and the aryl hydrocarbon receptor ligand FICZ. (A and B) IFN-g and IL-17A expression in CD4+ Tcells was determined by intracellular stain- ing and flow cytometry (mean 6 SEM). (C) ELISA of IL-17A pro- Downloaded from ductionbyCD4+ T cells (mean 6 SEM). (D) Expression of the indicated mRNAs in Th17-polarized cell pop- ulations was determined by quantita- tive RT-PCR, presented relative to Hprt1 mRNA (mean 6 SEM). Data are representative of three independent http://www.jimmunol.org/ experiments.

TPL-2 is not required for T cell priming during EAE induce IL-17A production by CD4+ T cells to the same extent as

The disease course of EAE can be considered to occur in two WT BMDCs in an in vitro Th17 differentiation assay (Supplemental by guest on September 24, 2021 stages: a priming phase, in which immunization leads to activa- Fig. 2C, 2D) (19). Our in vitro experiments suggested that TPL-2 expressed in Ag- tion and expansion of peripheral myelin-specific T cells, and an + effector phase, in which infiltrating inflammatory cells cause CNS presenting DCs and in responding CD4 T cells might not be es- damage. The priming phase involves the stimulation and expansion sential for initial generation of Th17 cells in EAE. To investigate this, the in vivo priming and differentiation of Th17 cells was examined of myelin-responsive T cells by activated Ag-presenting DCs in 2/2 LNs. The production of IL-1b, IL-6, and IL-23 by DCs, and other in WT and Map3k8 mice during the early stages of EAE. Anal- innate immune cells, is critical for the initial induction of Th17 ysis of dLNs isolated from mice 9 d after MOG35–55 immunization + cells during an immune response (10), and signaling via each of revealed that both the total cellularity and CD4 T cell numbers were these cytokines is essential for EAE induction (21, 25). Because unaffected by TPL-2 deficiency (Fig. 3A). This suggested that the 2/2 + TPL-2 has an established signaling function in DCs, we initially activation and expansion of MOG35–55-specific Map3k8 CD4 investigated the effect of TPL-2 deficiency on the induction of T cells occurred normally. To quantify the Ag-specific T cell re- Th17-polarizing cytokines by BMDCs in response to heat-inactivated sponse, dLN cells were labeled with CFSE and restimulated in vitro HI M. tuberculosis (Mtb ). with MOG35–55. Consistent with normal priming in EAE, a similar + Immunoblotting of cell lysates revealed that TPL-2 deficiency fraction of MOG35–55-specific CD4 T cells divided within the WT 2/2 blocked the early activation of ERK by MtbHI, whereas a second wave and Map3k8 LN cell populations (Fig. 3B). of ERK-1/2 phosphorylation was TPL-2–independent (Supplemental Although T cell activation and expansion in EAE were unaf- Fig. 2A). p38 activation was also reduced in Map3k82/2 BMDCs, fected by TPL-2 deficiency, it was possible that the ability of similar to the reported effects of TPL-2 deficiency after LPS responding T cells to differentiate into Th17 and/or Th1 cells stimulation (27). Despite these decreases in MAPK activation, following MOG35–55 immunization was impaired. However, re- HI quantitative RT-PCR revealed that Mtb was still able to induce stimulation of dLN cells with either MOG35–55 peptide or PdBu Il6 and Il23p19 mRNAs to similar levels in Map3k82/2 and WT and ionomycin revealed a similar fraction of IFN-g– and IL-17A– BMDCs (Supplemental Fig. 2B). TPL-2 deficiency reduced the producing CD4+ T cells, as well as a similar production of both + induction of Il1b mRNA by ∼40%, whereas the induction of Il12b cytokines by MOG35–55-specific CD4 T cells from immunized 2 2 mRNA was increased. TPL-2 deficiency had similar effects on WT and Map3k8 / mice (Fig. 3C, 3D). MtbHI induction of ERK-1/2 activation and cytokines expression In vivo experiments therefore indicated that CD4+ T cell activa- in BMDMs (Supplemental Fig. 3A, 3B). These data indicated that tion, expansion, and differentiation into Th1 and Th17 effector cells TPL-2 was not essential for the induction of Th17-polarizing occurred independently of TPL-2. These results were consistent 2 2 cytokines by MtbHI in BMDCs and BMDMs, although MtbHI with the ability of Map3k8 / CD4+ T cells to activate ERK-1/2 activation of ERK was largely dependent on TPL-2 expression in (Supplemental Fig. 3C) and proliferate normally in response to both cell types. Consistently, Map3k82/2 BMDCs were able to CD3/CD28 crosslinking (16), and for Map3k82/2 BMDCs to induce 3522 TPL-2 KINASE AND EAE Downloaded from http://www.jimmunol.org/

FIGURE 2. TPL-2 regulates the onset and severity of EAE. (A) Mean clinical scores of WT and Map3k82/2 mice (n = 19/WT; n = 18/Map3k82/2)at various times after immunization with MOG33–55/CFA. Data presented are combined from two independent experiments (n = 9–10/genotype/experiment). (B) Immune cell infiltration in the spinal cords of WT and Map3k82/2 mice (n = 8/genotype) at the peak of disease was determined by flow cytometry using the indicated markers (mean 6 SEM). (C) Intracellular staining for infiltrated IL-17A–, IFN-g–, and GM-CSF–expressing CD4+ T cells in the spinal cords 2/2 2/2 of WT and Map3k8 mice (n = 8/genotype) on day 12 after MOG33–55/CFA immunization. In (D) and (E), EAE was induced in WT and Map3k8 mice, and spinal cords were removed at the peak of disease. (D) Spinal cord sections were stained with Luxol fast blue (LFB) to monitor demyelination. Scale by guest on September 24, 2021 bars, 200 mm in the first panels and 50 mm in the lower panels. Immune cell infiltration was revealed by Ab staining: CD3 for T cells, Mac-3 for macrophages, and B220 for B cells. Axonal damage was visualized by staining for amyloid precursor protein (APP). (E) Quantitation of demyelination, immune infiltration, and axonal damage (mean 6 SEM). Data in (B) and (C) are compiled from two independent experiments (n = 4/genotype/experiment). Data in (D) and (E) are representative of two independent experiments. *p # 0.05, **p # 0.01, ***p # 0.001. differentiation of CD4+ T cells to the Th17 cell lineage in vitro to The potential function of TPL-2 in T cells was also investigated a similar extent as WT BMDCs (Supplemental Fig. 2C). in a passive EAE model, in which encephalitogenic myelin-responsive T cells were transferred into naive recipients (13). dLN cells were TPL-2 does not function within T cells to promote EAE 2/2 isolated from Map3k8 mice 9 d after MOG35–55 immunization Defective T cell priming did not explain the reduced susceptibility and then cultured with MOG35–55 peptide, IL-1b, and IL-23 to 2/2 of Map3k8 mice to EAE. Nevertheless, it remained possible that expand MOG35–55-specific Th17 cells (28). Intracellular cytokine TPL-2 had a T cell–intrinsic function at a later stage in EAE path- staining indicated similar polarization efficiency within WT and ogenesis. This was investigated by mixing BM cells from Tcra2/2 Map3k82/2 CD4+ T cell populations (data not shown). Following mice with WT or Map3k82/2 BM cells (ratio 4:1) before transfer- i.v. injection, the onset and extent of EAE induced by transferred ring them into Rag12/2 hosts. In the resulting chimeras, all of the Map3k82/2 and WT Th17-polarized cells were very similar T cells developed from either WT or TPL-2–deficient donor BM, (Fig. 4B), consistent with the results of active EAE in mixed whereas most (80%) of the other hematopoietic cells were derived BM chimeras. from the TPL-2–sufficient Tcra2/2 BM. Upon induction, the onset CD20 Ab depletion has demonstrated that B cells promote EAE and severity of EAE were essentially identical in both sets of mixed disease progression via production of IL-6 (29, 30), in addition to BM chimeras (Fig. 4A), indicating that there was no T cell–intrinsic their late-acting regulatory role (31, 32). TPL-2 is known to reg- function for TPL-2 in EAE pathogenesis. ulate ERK-1/2 activation in B cells following CD40 and TLR stimulation (3, 33), raising the possibility that the protection of Map3k82/2 mice from EAE might be due to TPL-2 signaling in

Table I. Clinical features of MOG35–55–induced EAE B cells. To investigate this, chimeric mice were generated in which BM from mMT2/2 mice was mixed with Map3k82/2 BM cells 2/2 Mean Mean Day (ratio 4:1) and injected into lethally irradiated Rag1 recipients. Maximum Score of Onset The onset and severity of EAE were similar between mice lacking Incidence (Mean 6 SD) (Mean 6 SD) TPL-2 expression in B cells and WT controls (Supplemental Fig. WT 19/19 (100%) 3.5 6 0.9 10.52 6 2.1 4A). Therefore, the protection from EAE observed in Map3k82/2 2/2 6 6 Map3k8 18/18 (100%) 2.7 0.9 15.07 2.5 mice was not attributable to the absence of TPL-2 in T or B cells. The Journal of Immunology 3523

FIGURE 3. TPL-2 is not required for T cell priming in EAE. WT and Map3k82/2 mice were immunized with MOG33–55/CFA, and dLNs were isolated on day 9. (A) CD4+ T cell numbers were determined by flow cytometry (mean 6 SEM; n = 3). (B) Flow cytometric analysis of the pro- liferation of CFSE-labeled CD4+

T cells cultured for 3 d with MOG33–55 peptide (mean 6 SEM; n = 3). (C)

Frequencies of IL-17A– and IFN-g– Downloaded from expressing CD4+ T cells were deter- mined by flow cytometry after re- stimulation with PdBu/ionomycin or MOG33–55 peptide (mean 6 SEM; n =3).(D)ELISAofIFN-g and IL- 17A production by LN cells restimu- http://www.jimmunol.org/ lated with MOG33–55 peptide (mean 6 SEM; n = 3). Data are representa- tive of at least three independent experiments. by guest on September 24, 2021

TPL-2 signaling in radiation-resistant cells promotes the were observed upon transfer of encephalitogenic Th17-polarized effector phase of EAE cells into Map3k82/2 mice (Fig. 4B). TPL-2–deficient mice were 2 2 Because the priming phase of EAE was normal in Map3k8 / also protected from EAE induced by WT MOG35–55 peptide– mice, this suggested that TPL-2 functioned during the effector primed Th1 cells, suggesting the protection from disease did not phase of the disease. To investigate this, the ability of WT involve a Th cell–specific factor (Supplemental Fig. 4B, 4C). MOG35–55 peptide-primed Th17 cells to induce passive EAE in The effector phase of EAE involves two waves of immune cell 2/2 WT and Map3k8 recipient mice was determined. A delay in infiltration into the CNS (34–36). First, “pioneer” MOG35–55-specific onset of clinical symptoms and a reduction in disease severity CD4+ T cells enter the brain via the choroid plexus. Inflammatory

FIGURE 4. TPL-2 signaling in T cells is not required for EAE development. (A) BM cells from WT and Map3k82/2 mice were mixed in a 1:4 ratio with 2/2 2/2 BM cells from Tcra mice and transferred into Rag1 hosts. After 8 wk, EAE was induced by immunization with MOG33–55/CFA, and clinical scores 2/2 were determined (mean 6 SEM; n = 6). (B) dLN cells from WT and Map3k8 mice were isolated 9 d after immunization with MOG33–55/CFA and restimulated in vitro with MOG33–55 peptide in the presence of recombinant IL-1b and IL-23 for 5 d. These Th17-polarized cells were then transferred into naive WT and Map3k82/2 mice. Graph represents the average clinical score after Th17 cell transfer (mean 6 SEM; n = 5). Data in (A) and (B) are representative of two independent experiments. 3524 TPL-2 KINASE AND EAE cytokines produced by reactivated CD4+ T cells then induce re- duced the secondary infiltration of immune cells into the CNS cruitment of a second wave of infiltrating immune cells. We in- parenchyma. vestigated the recruitment of immune cells to the CNS in passive + TPL-2 signals in both microglia and astrocytes EAE. CD45.1 MOG35–55 peptide–primed Th17 cells generated from WT mice were transferred to CD45.2+ WT or Map3k82/2 CNS-resident microglia and astrocytes have been reported previ- mice. Similar numbers of CD45.1+ CD4+ T cells were detected in ously to play pivotal roles in EAE disease manifestation (20, 37, the spleens and brains of WT and Map3k82/2 mice early after 38). These radiation-resistant cell types produce a number of adoptive transfer (Fig. 5A, left and middle panels). However, at the proinflammatory cytokines and chemokines during EAE, which peak of the disease, significantly more CD45.1+ CD4+ Tcells promote the secondary influx of hematopoietic cells into the CNS accumulated in the brains of WT relative to Map3k82/2 mice (34, 36, 39). Defective activation of one or both of these cell (Fig. 5A, middle panel). Moreover, consistent with a defect in types, therefore, could account for the protection of Map3k82/2 secondary immune cell infiltration into Map3k82/2 brains, fewer mice from EAE development. We explored this possibility by CD45+F4/80+ macrophages were detected 14 d after Th17 cell determining whether TPL-2 deficiency affected cytokine and transfer (Fig. 5A, right panel). The number of CD45.1+CD4+ chemokine induction in primary in vitro–derived microglia and T cells in the spleen at these later time points was similar between astrocytes. the two recipient CD45.2+ genotypes, demonstrating that host Map3k82/2 microglia expressed significantly less Il6, Il1, Cxcl1, TPL-2 expression was not required for CD4+ T cell survival Ccl2,andCcl20 mRNAs in response to LPS stimulation than did (Fig. 5A, left panel). Furthermore, transferred CD4+ T cells in their WT counterparts (Fig. 7A). Additionally, induction of Il1 and 2 2 the CNS of Map3k8 / hosts produced similar fractions of IFN- Ccl2 mRNAs was reduced by TPL-2 deficiency following stimu- Downloaded from g and IL-17 to WT hosts (Fig. 5B). Pioneer Th17 cells, therefore, lation with IFN-g or with TNF plus IFN-g (Fig. 7A, 7B). LPS could infiltrate the CNS of Map3k82/2 mice, but failed to optimally upregulation of mRNAs encoding IL-6, IL-1b, CCL2, CXCL1, trigger a secondary infiltration of immune cells, the major medi- VCAM1, and MMP9 was also significantly reduced in Map3k82/2 ators of myelin damage. astrocytes compared with WT control cells (Fig. 7C). Furthermore, Although transfer of encephalitogenic Th17 cells demonstrated IL-1b induction of Il6, Ccl2,andVcam1 mRNAs, as well as TNF

that the TPL-2–deficient host environment was protective in passive induction of Ccl2 and Mmp9 mRNAs, was reduced by TPL-2 de- http://www.jimmunol.org/ EAE (Fig. 4B), it did not reveal whether TPL-2 promoted EAE by ficiency in astrocytes (Fig. 7C). Defective TNF induction of Ccl2, signaling in hematopoietic cells other than T or B cells. This was Vcam1,andMmp9 mRNAs in Map3k82/2 astrocytes was not res- investigated by generating BM chimeras via transferring WT or cued by costimulation with IL-17A (Fig. 7D). In line with earlier Map3k82/2 BM cells into lethally irradiated Rag12/2 or Rag12/2 studies, stimulation of astrocytes with IL-17A alone had minimal Map3k82/2 recipients. In these chimeras, all lymphocytes, as effect on the expression of mRNAs encoding proinflammatory well as .99% of MHC class II+ cells, were of donor origin cytokines or chemokines in astrocytes (20, 40). (data not shown). Upon EAE induction, deletion of TPL-2 in We next analyzed the activation of MAPK signaling pathways radiation-sensitive cells (Map3k82/2 . Rag12/2 compared with in microglia and astrocytes to gain insight into how TPL-2 defi- Map3k8+/+ . Rag12/2) was not able to protect against disease ciency might impair proinflammatory gene expression in these by guest on September 24, 2021 (Fig. 6A). However, transferring WT BM cells into Rag12/2 cell types. In line with earlier studies using primary macro- hosts that additionally lacked TPL-2 resulted in delayed EAE phagesandDCs(2,3,27),ERK-1/2andp38a phosphorylation onset. Furthermore, lack of TPL-2 expression in the radiation- following LPS or TNF stimulation of microglia was reduced by resistant compartment resulted in a reduction in immune infil- TPL-2 deficiency (Fig. 8A). Analysis of NF-kB1 p105 phos- trates in the spinal cord (Fig. 6B) but did not affect polarization phorylation (data not shown) indicated that this decrease did not of CD4+ T cells to produce IL-17A and IFN-g (Fig. 6C). result from impaired activation of the IkB kinase (IKK) complex Therefore, TPL-2 promoted the effector phase of EAE by sig- (41). Phosphorylation of ERK-1/2 and p38a was not induced by naling in radiation-resistant nonhematopoietic cells, which in- stimulation with either IFN-g or IL-17A (data not shown).

FIGURE 5. TPL-2 regulates the effector phase of

EAE. (A) MOG33–55-specific Th17 cells were gen- erated from CD45.1+ WT mice and then transferred into CD45.2+ WT and Map3k82/2 mice as in Fig. 6C. Numbers of CD45.1+CD4+ T cells in the spleen and brain were determined by flow cytom- etry at the indicated time points after Th17 cell transfer (mean 6 SEM; n = 3). Macrophage num- bers in the brain after transfer were determined by F4/80 staining. (B) Intracellular staining for IL-17A and IFN-g expression in transferred Th17-polarized CD45.1+CD4+ T cells in the spleen and brain on day 14 after transfer (mean 6 SEM, n = 3). Data are representative of at least two independent experi- ments. *p # 0.05. The Journal of Immunology 3525

FIGURE 6. TPL-2 signaling in radiation-resistant cells promotes EAE. (A) BM cells from WT and Map3k82/2 mice were adoptively transferred into le- thally irradiated Rag12/2 or Map3k82/2Rag12/2 hosts, and EAE was induced 8 wk later. Mean clinical scores are shown (mean 6 SEM; n = 4–5). (B and C) EAE was induced in chimeric mice generated as in (A). (B) Total numbers of CD4+ and CD45+ cells in the spinal cords

12 d after MOG33–55 peptide plus CFA immunization were determined by flow cytometry (mean 6 SEM, n = 4). (C) Intracellular staining for IL-17A– and IFN-g– expressing CD4+ T cells in the spinal cord at peak of disease (mean 6 SEM; n = 4). Data are representative of three independent experiments. *p # 0.05, **p # 0.01. Downloaded from http://www.jimmunol.org/ ERK-1 and ERK-2 were basally phosphorylated at high levels in (Supplemental Fig. 4D), which is catalytically inactive (43). In astrocytes, and this was not detectably altered by stimulation with contrast to Map3k82/2 cells, Map3k8D270A/D270A cells expressed LPS or TNF (Fig. 8B). However, LPS and TNF clearly induced similar amounts of ABIN-2 as WT cells (Fig. 7A). Compared to p38a phosphorylation, and this was partially decreased by TPL-2 WT controls, EAE development was delayed and reduced in deficiency following stimulation with either agonist. Addition of severity in Map3k8D270A/D270A mice (Fig. 9C), similar to Map3k82/2 IFN-g or IL-17A did not alter p38a phosphorylation in astrocytes mice. TPL-2 catalytic activity was therefore required to promote (data not shown). EAE, and this was independent of effects on the steady-state In conclusion, our biochemical analyses demonstrated that expression of ABIN-2 protein, or any potential adaptor function of TPL-2 contributed to p38a activation in both microglia and astro- TPL-2. by guest on September 24, 2021 cytes after stimulation with LPS and TNF, and it was also required TPL-2 promotes EAE independently of type I IFN signaling for optimal activation of ERK-1/2 in microglia by these agonists. Together with the qRT-PCR analyses of cytokine and chemokine Development of EAE is exacerbated in mice deficient in type I IFN mRNA expression (Fig. 7), these data were consistent with the receptor (IFNAR), with markedly higher inflammation and de- hypothesis that TPL-2 signaling in both microglia and astrocytes myelination in the CNS compared with WT controls (44). TPL-2/ could contribute to EAE disease development. ERK-1/2 signaling negatively regulates TLR induction of IFN-b (27), raising the possibility that TPL-2 deficiency might reduce TPL-2 kinase activity is required for development of EAE EAE disease severity by augmenting IFN-b production. We in- TPL-2 is associated with the ubiquitin-binding protein ABIN-2, vestigated this possibility genetically by generating Map3k82/2 which is required to maintain TPL-2 stability (42). ABIN-2–de- Ifnar2/2 mice lacking expression of both TPL-2 and the receptor ficient mice have reduced steady-state levels of TPL-2 in multiple for type I IFNs, IFNAR. cell types compared with WT mice (14). In a reciprocal fashion, As reported previously (44), IFNAR deficiency did not affect analysis of cells from Map3k82/2 mice revealed that TPL-2 is EAE disease onset, but increased the severity and duration of the required to maintain steady-state expression of ABIN-2, and both effector phase (Fig. 9D). However, the onset of EAE and maximal splenocytes and BMDMs from Map3k82/2 mice have substan- clinical scores were similar in Map3k82/2 and Map3k82/2 Ifnar2/2 tially reduced amounts of ABIN-2 compared with WT (Fig. 9A). mice. The inhibitory effects of TPL-2 deficiency on EAE devel- This suggested that the resistance of Map3k82/2 mice to EAE opment, therefore, were independent of type I IFN signaling. might result from reduced ABIN-2 expression. However, EAE disease severity was similar between Tnip22/2 (which completely Discussion lack ABIN-2 expression) and WT mice (Fig. 9B). These data We investigated the potential role of the MAP3 kinase TPL-2 in indicated ABIN-2 was not required for EAE development and Th17 cell generation and function. Our results show that TPL-2 was suggested that the low levels of TPL-2 expressed in Tnip22/2 dispensable for the generation of Th17 cells in vitro and during the mice were sufficient to promote maximal disease. priming phase of EAE. Nevertheless, TPL-2 regulated both the Taken together, our analyses of Map3k82/2 and Tnip22/2 mice onset and severity of EAE, functioning in the effector phase of this demonstrated that TPL-2 expression was required for efficient Th17 cell–mediated autoimmune disease model. Importantly, the EAE development. However, these experiments did not establish effects of TPL-2 in EAE required its catalytic activity, suggesting whether this reflected a role for TPL-2 catalytic activity, and it that small molecule inhibitors of TPL-2 might be therapeutically remained possible that TPL-2 promoted disease by functioning as beneficial in MS. IFN-b is widely used for treatment of relapsing- an adaptor protein. To distinguish these possibilities, we generated remitting MS (45). TPL-2 promoted EAE independently of type I a novel knock-in mouse strain expressing mutant TPL-2D270A IFN signaling, raising the possibility that administration of a TPL-2 3526 TPL-2 KINASE AND EAE Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. TPL-2 regulates proinflammatory gene expression in primary microglia and astrocytes. Quantitative RT-PCR of mRNA expression in primary microglia (A, B) and astrocytes (C, D) from WT and Map3k82/2 mice, normalized to Hprt mRNA (mean 6 SEM). Cells were either left untreated (nt) or stimulated with the indicated agonists for 6 h. Data are compiled from three (A, C) or two (B, D) independent experiments. *p # 0.05, **p # 0.01. inhibitor in combination with the established drug IFN-b might be DCs following stimulation with MtbHI (28). MtbHI can potentially more effective for MS therapy. activate multiple pattern recognition receptors on DCs, including Earlier pharmacological studies with the MEK-1/2 inhibitor TLR2, TLR4, TLR9, NOD-2, and C-type lectins (46, 47). TPL-2 U0126 have suggested that ERK-1/2 activation is required for is required for activation of ERK-1/2 in DCs following TLR2, induction of the Th17-polarizing cytokines IL-1b and IL-23 in TLR4, or TLR9 stimulation (4, 27), and it also contributes to the The Journal of Immunology 3527

cytokines, or for MtbHI induction of Th17-polarizing cytokines in BMDCs and BMDMs. Furthermore, mixed BM chimera experi- ments demonstrated that TPL-2 expression in either T or B cells was not required for EAE development. Instead, passive EAE experiments using MOG35–55-specific Th17 cells revealed that TPL-2 signaling in host cells regulated the effector phase of dis- ease in the CNS. A similar requirement for host TPL-2 was found after adoptive transfer of WT MOG35–55-specific Th1 cells. Therefore, TPL-2 did not have a Th17-specific function in the effector phase of EAE. After priming in LNs, Ag-specific Th17 cells traffic through the choroid plexus into the subarachnoid space, where they are reactivated (35). As a consequence of productive T cell/APC in- FIGURE 8. TPL-2 is required for optimal MAPK activation in microglia teractions, Th17 cell cytokines and chemokines are then pro- and astrocytes. Lysates of microglia (A) and astrocytes (B), generated from duced, which activate parenchymal vasculature. This promotes 2/2 WT and Map3k8 mice and stimulated for the indicated time points with a secondary wave of leukocyte infiltration, leading to inflamma- LPS or TNF, were immunoblotted. Phosphorylated protein bands were tory CNS damage and EAE onset (34). Passive EAE experiments quantified by laser densitometry using the Quantity One software package indicated that the initial trafficking of Th17 cells into the brain and are presented as relative density normalized to the respective total Downloaded from protein. Data are representative of three to four independent experiments was not affected by TPL-2 deficiency. Instead, TPL-2 was re- with similar results. quired for the second wave of leukocyte recruitment into the CNS. The expression of several chemokines known to be involved in regulating the migration of leukocytes into the CNS was signifi- activation of ERK in macrophages following NOD-1 and NOD-2 cantly reduced in intact Map3k82/2 mice during EAE, including stimulation, but it is dispensable for activation of ERK-1/2 by CCL2, CCL3, CCL5, and CXCL10, which have each been impli-

the C-type lectin Dectin-1 (4). Consistent with this, stimulation of cated to have positive roles in EAE induction (34). The protective http://www.jimmunol.org/ ERK phosphorylation by MtbHI was largely dependent on TPL-2 effect of TPL-2 deficiency in EAE, therefore, may result from im- expression in BMDCs. However, TPL-2 deficiency only partially paired upregulation of these chemokines in CNS-resident cells. reduced MtbHI induction of IL-1b, and it did not impair induction An important outstanding question is the identity of the of mRNAs encoding IL-6, IL-12p35, or IL-23p19 in these cells, radiation-resistant nonhematopoietic cell type in which TPL-2 sig- which were able to induce normal Th17 cell differentiation in nals to promote EAE. In vitro experiments indicated that TPL-2 vitro. In line with these data, the generation of Th17 cells in the was required for optimal ERK-1/2 and p38a activation in micro- dLNs during the priming phase of EAE was unaffected by TPL-2 glia, resident myeloid-lineage cells in the brain and spinal cord deficiency. It is likely that low levels of TPL-2–independent ERK- (48). Microglia can contribute to EAE disease initiation by pre-

2 2 by guest on September 24, 2021 1/2 activation explain the ability of Map3k8 / BMDCs to induce senting Ags to naive T cells, and they are also a potent source of mRNAs encoding IL-1b and IL-23p19. inflammatory cytokines and chemokines (37, 39). Additionally, Experiments with lethally irradiated BM chimeras demonstrated our in vitro experiments revealed that TPL-2 signaling contributes that TPL-2 functioned in a radiation-resistant cell population to to the activation of p38a in astrocytes, the most abundant cells of promote EAE, implying that TPL-2 signaling was not required in the brain that also produce cytokines and chemokines, and they T cells APCs (DCs, macrophages). These data are consistent with have an important role in regulating the recruitment and function in vitro experiments showing that TPL-2 expression was dis- of T cells in the CNS (39). TPL-2 expression was found to be pensable for Th17 cell differentiation induced with recombinant required for optimal expression of proinflammatory cytokines and

FIGURE 9. TPL-2 kinase activity is required to induce EAE. (A) Lysates were prepared from spleno- cytes (left panel)andBMDMs(right panel) generated from WT, Map3k82/2, Map3k8D270A/D270A,and TNIP22/2 mice and immunoblotted for the indi- cated Ags. (B) Mean clinical scores of WT and Tnip22/2 mice (mean 6 SEM; n = 7–8). Data are compiled from two different experiments (n = 3–4/ genotype/experiment). (C) Mean clinical scores of WT, Map3k8 D270A/D270A, and Map3k82/2 mice at various times after immunization with MOG33–55/ CFA (mean 6 SEM; n = 8–9). Data are compiled from two different experiments (n = 3–4/genotype/ experiment). *p # 0.05, ***p # 0.001. (D) Mean clinical scores of WT, Map3k82/2, Ifnar2/2,and Map3k82/2Ifnar2/2 mice after immunization with

MOG33–55/CFA (mean 6 SEM; n = 9–10). Data are compiled from two different experiments (n = 4–5/ genotype/experiment). 3528 TPL-2 KINASE AND EAE chemokines in both microglia and astrocytes, possibly owing to 8. Watford, W. T., B. D. Hissong, L. R. Durant, H. Yamane, L. M. Muul, Y. Kanno, C. M. Tato, H. L. Ramos, A. E. Berger, L. Mielke, et al. 2008. Tpl2 kinase the contribution of TPL-2 to MAPK activation in these cell types. regulates T cell interferon-g production and host resistance to Toxoplasma Earlier studies with knockout mice have shown the importance of gondii. J. Exp. Med. 205: 2803–2812. the affected cytokines and chemokines for EAE disease develop- 9. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune system: the T helper lymphocyte grows up. Dev. 14: 1693–1711. ment (34, 39). Our data raise the possibility that the reduced sec- 10. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 cells. 2/2 ondary infiltration of inflammatory cells into the CNS in Map3k8 Annu. Rev. Immunol. 27: 485–517. mice compared with WT controls during EAE results from the 11. Becher, B., and B. M. Segal. 2011. TH17 cytokines in autoimmune neuro-in- flammation. Curr. Opin. Immunol. 23: 707–712. decreased expression of cytokines and chemokines by microglia 12. Krishnamoorthy, G., and H. Wekerle. 2009. EAE: an immunologist’s magic eye. and astrocytes. Eur. J. Immunol. 39: 2031–2035. Genetic deletion of IKK2 or NEMO in neuroectodermal-derived 13. Denic, A., A. J. Johnson, A. J. Bieber, A. E. Warrington, M. Rodriguez, and I. Pirko. 2011. The relevance of animal models in multiple sclerosis research. cells ameliorates EAE in mice, which correlates with decreased Pathophysiology 18: 21–29. expression of proinflammatory cytokines and chemokines by CNS- 14. Papoutsopoulou, S., A. Symons, T. Tharmalingham, M. P. Belich, F. Kaiser, D. Kioussis, A. O’Garra, V. Tybulewicz, and S. C. Ley. 2006. ABIN-2 is required resident cells (38). Recently, it has also been shown that condi- for optimal activation of Erk MAP kinase in innate immune responses. Nat. tional deletion of Tak1, which functions upsteam of the IKK Immunol. 7: 606–615. complex, in either astrocytes or microglia also ameliorates EAE 15. Dann, A., H. Poeck, A. L. Croxford, S. Gaupp, K. Kierdorf, M. Knust, D. Pfeifer, C. Maihoefer, S. Endres, U. Kalinke, et al. 2012. Cytosolic RIG-I-like helicases development (49). The IKK complex is a critical positive regulator act as negative regulators of sterile inflammation in the CNS. Nat. Neurosci. 15: of the TPL-2 signaling pathway, inducing proteolysis of its in- 98–106. hibitor NF-kB1 p105 (50, 51) and directly phosphorylating a key 16. Sriskantharajah, S., M. P. Belich, S. Papoutsopoulou, J. Janzen, V. Tybulewicz, B. Seddon, and S. C. Ley. 2009. Proteolysis of NF-kB1 p105 is essential for regulatory serine in the TPL-2 C terminus (52). The protective T cell antigen receptor-induced proliferation. Nat. Immunol. 10: 38–47. Downloaded from effects of IKK2/NEMO deficiency in astrocytes or TAK1 defi- 17. Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, and ciency in astrocytes and microglia, therefore, may be mediated in A. O’Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived den- dritic cells in directing T helper type 1 and 2 cell development: dependency on part by blockade of TPL-2 signaling. The generation of condi- antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197: 101–109. tional knockout mouse strains lacking TPL-2 expression in astro- 18. Veldhoen, M., K. Hirota, A. M. Westendorf, J. Buer, L. Dumoutier, J.-C. Renauld, cytes or microglia will be essential to determine whether TPL-2 and B. Stockinger. 2008. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453: 106–109. signaling in either or both these cell types is important for develop- 19. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. http://www.jimmunol.org/ ment of EAE. 2006. TGFb in the context of an inflammatory cytokine milieu supports de novo 2/2 differentiation of IL-17-producing T cells. Immunity 24: 179–189. Earlier studies of Map3k8 mice identified TPL-2 as a po- 20. Kang, Z., C. Z. Altuntas, M. F. Gulen, C. Liu, N. Giltiay, H. Qin, L. Liu, tential drug target in septic shock and inflammatory bowel disease W. Qian, R. M. Ransohoff, C. Bergmann, et al. 2010. Astrocyte-restricted ab- (2, 5). Interestingly, Mapk1 was recently identified as a disease lation of interleukin-17-induced Act1-mediated signaling ameliorates autoim- mune encephalomyelitis. Immunity 32: 414–425. susceptibility gene locus in MS (53). Mapk1 encodes ERK2, a major 21. Chung, Y., S. H. Chang, G. J. Martinez, X. O. Yang, R. Nurieva, H. S. Kang, downstream target of TPL-2 signaling. These data, together with L. Ma, S. S. Watowich, A. M. Jetten, Q. Tian, and C. Dong. 2009. Critical the present study, suggest that blockade of TPL-2 catalytic activity regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 30: 576–587. might also be therapeutically beneficial in MS. 22. Bettelli, E., T. Korn, M. Oukka, and V. K. Kuchroo. 2008. Induction and effector by guest on September 24, 2021 functions of TH17 cells. Nature. 453: 1051–1057. 23. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, Acknowledgments D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgt directs We thank the National Institute of Medical Research Photographics Depart- the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: ment, National Institute of Medical Research Biological Services, National 1121–1133. Institute of Medical Research flow cytometry service, Damian Carragher 24. Yang, X. O., B. P. Pappu, R. Nurieva, A. Akimzhanov, H. S. Kang, Y. Chung, L. Ma, B. Shah, A. D. Panopoulos, K. S. Schluns, et al. 2008. T helper 17 lineage and Xuemei Wu (Division of Immunoregulation, National Institute of Med- differentiation is programmed by orphan nuclear receptors RORa and RORg. ical Research), Julia Janzen, and other members of the Ley Laboratory for Immunity 28: 29–39. help during the course of this work. 25. McGeachy, M. J., and S. M. Anderton. 2005. Cytokines in the induction and resolution of experimental autoimmune encephalomyelitis. Cytokine 32: 81–84. 26. Wilson, E. H., W. Weninger, and C. A. Hunter. 2010. Trafficking of immune cells Disclosures in the central nervous system. J. Clin. Invest. 120: 1368–1379. The authors have no financial conflicts of interest. 27. Kaiser, F., D. Cook, S. Papoutsopoulou, R. Rajsbaum, X. Wu, H. T. Yang, S. Grant, P. Ricciardi-Castagnoli, P. N. Tsichlis, S. C. Ley, and A. O’Garra. 2009. TPL-2 negatively regulates interferon-beta production in macrophages and myeloid dendritic cells. J. Exp. Med. 206: 1863–1871. References 28. Brereton, C. F., C. E. Sutton, S. J. Lalor, E. C. Lavelle, and K. H. G. Mills. 2009. 1. Gantke, T., S. Sriskantharajah, and S. C. Ley. 2011. Regulation and function of Inhibition of ERK MAPK suppresses IL-23- and IL-1-driven IL-17 production TPL-2, an IkB kinase-regulated MAP kinase kinase kinase. Cell Res. 21: 131–145. and attenuates autoimmune disease. J. Immunol. 183: 1715–1723. 2. Dumitru, C. D., J. D. Ceci, C. Tsatsanis, D. Kontoyiannis, K. Stamatakis, 29. Barr, T. A., P. Shen, S. Brown, V. Lampropoulou, T. Roch, S. Lawrie, B. Fan, J.-H. Lin, C. Patriotis, N. A. Jenkins, N. G. Copeland, G. Kollias, and P. N. Tsichlis. R. A. O’Connor, S. M. Anderton, A. Bar-Or, et al. 2012. B cell depletion therapy 2000. TNF-a induction by LPS is regulated posttranscriptionally via a Tpl2/ ameliorates autoimmune disease through ablation of IL-6-producing B cells. J. ERK-dependent pathway. Cell 103: 1071–1083. Exp. Med. 209: 1001–1010. 3. Eliopoulos, A. G., C.-C. Wang, C. D. Dumitru, and P. N. Tsichlis. 2003. Tpl2 30. Matsushita, T., K. Yanaba, J.-D. Bouaziz, M. Fujimoto, and T. F. Tedder. 2008. transduces CD40 and TNF signals that activate ERK and regulates IgE induction Regulatory B cells inhibit EAE initiation in mice while other B cells promote by CD40. EMBO J. 22: 3855–3864. disease progression. J. Clin. Invest. 118: 3420–3430. 4. Mielke, L. A., K. L. Elkins, L. Wei, R. Starr, P. N. Tsichlis, J. J. O’Shea, and 31. Anderton, S. M., and S. Fillatreau. 2008. Activated B cells in autoimmune W. T. Watford. 2009. Tumor progression locus 2 (Map3k8) is critical for host diseases: the case for a regulatory role. Nat. Clin. Pract. Rheumatol. 4: 657–666. defense against Listeria monocytogenes and IL-1b production. J. Immunol. 183: 32. Fillatreau, S., C. H. Sweenie, M. J. McGeachy, D. Gray, and S. M. Anderton. 7984–7993. 2002. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3: 5. Kontoyiannis, D., G. Boulougouris, M. Manoloukos, M. Armaka, M. Apostolaki, 944–950. T. T. Pizarro, A. Kotlyarov, I. Forster, R. A. Flavell, M. Gaestel, et al. 2002. 33. Banerjee, A., R. Gugasyan, M. McMahon, and S. Gerondakis. 2006. Diverse Genetic dissection of the cellular pathways and signaling mechanisms in mod- Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase eled tumor necrosis factor-induced Crohn’s-like inflammatory bowel disease. J. (ERK) in hemopoietic cells. Proc. Natl. Acad. Sci. USA 103: 3274–3279. Exp. Med. 196: 1563–1574. 34. Prendergast, C. T., and S. M. Anderton. 2009. Immune cell entry to central 6. George, D., and A. Salmeron. 2009. Cot/Tpl-2 protein kinase as a target for the nervous system: current understanding and prospective therapeutic targets. treatment of inflammatory disease. Curr. Top. Med. Chem. 9: 611–622. Endocr. Metab. Immune Disord. Drug Targets 9: 315–327. 7. Watford, W. T., C.-C. Wang, C. Tsatsanis, L. A. Mielke, A. G. Eliopoulos, 35. Reboldi, A., C. Coisne, D. Baumjohann, F. Benvenuto, D. Bottinelli, S. Lira, C. Daskalakis, N. Charles, S. Odom, J. Rivera, J. O’Shea, and P. N. Tsichlis. A. Uccelli, A. Lanzavecchia, B. Engelhardt, and F. Sallusto. 2009. C-C che- 2010. Ablation of tumor progression locus 2 promotes a type 2 Th cell response mokine receptor 6-regulated entry of TH-17 cells into the CNS through the in ovalbumin-immunized mice. J. Immunol. 184: 105–113. choroid plexus is required for the initiation of EAE. Nat. Immunol. 10: 514–523. The Journal of Immunology 3529

36. Zepp, J., L. Wu, and X. Li. 2011. IL-17 receptor signaling and T helper 17- 45. Prinz, M., and K.-P. Knobeloch. 2012. Type I interferons as ambiguous modulators mediated autoimmune demyelinating disease. Trends Immunol. 32: 232–239. of chronic inflammation in the central nervous system. Front. Immunol. 3: 67. 37. Heppner, F. L., M. Greter, D. Marino, J. Falsig, G. Raivich, N. Ho¨velmeyer, 46. Kleinnijenhuis, J., M. Oosting, L. A. B. Joosten, M. G. Netea, and R. Van Crevel. A. Waisman, T. Rulicke,€ M. Prinz, J. Priller, et al. 2005. Experimental autoimmune 2011. Innate immune recognition of Mycobacterium tuberculosis. Clin. Dev. encephalomyelitis repressed by microglial paralysis. Nat. Med. 11: 146–152. Immunol. 2011: 405310. 38. van Loo, G., R. De Lorenzi, H. Schmidt, M. Huth, A. Mildner, M. Schmidt- 47. Marta, M. 2009. Toll-like receptors in multiple sclerosis mouse experimental Supprian, H. Lassmann, M. R. Prinz, and M. Pasparakis. 2006. Inhibition of models. Ann. N. Y. Acad. Sci. 1173: 458–462. transcription factor NF-kB in the central nervous system ameliorates autoim- 48. Prinz, M., J. Priller, S. S. Sisodia, and R. M. Ransohoff. 2011. Heterogeneity of mune encephalomyelitis in mice. Nat. Immunol. 7: 954–961. CNS myeloid cells and their roles in neurodegeneration. Nat. Neurosci. 13: 39. Chastain, E. M. L., D. S. Duncan, J. M. Rodgers, and S. D. Miller. 2011. The role 1227–1235. of antigen presenting cells in multiple sclerosis. Biochim. Biophys. Acta 1812: 49. Goldmann, T., P. Wieghofer, P. F. Muller,€ Y. Wolf, D. Varol, S. Yona, 265–274. S. M. Brendecke, K. Kierdorf, O. Staszewski, M. Datta, et al. 2013. A new type 40. Wang, X., M. Deckert, N. T. Xuan, G. Nishanth, S. Just, A. Waisman, of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune in- M. Naumann, and D. Schluter. 2013. Astrocytic A20 ameliorates experimental flammation. Nat. Neurosci. 16: 1618–1626. autoimmune encephalomyelitis by inhibiting NF-kB and STAT1-dependent. 50. Beinke, S., M. J. Robinson, M. Hugunin, and S. C. Ley. 2004. Lipopolysac- Acta Neuropathol. 126: 711–724. charide activation of the TPL-2/MEK/extracellular signal-regulated kinase 41. Yang, H. T., S. Papoutsopoulou, M. Belich, C. Brender, J. Janzen, T. Gantke, -activated protein kinase cascade is regulated by IkB kinase-induced M. Handley, and S. C. Ley. 2012. Coordinate regulation of TPL-2 and NF-kB proteolysis of NF-kB1 p105. Mol. Cell. Biol. 24: 9658–9667. signaling in macrophages by NF-kB1 p105. Mol. Cell. Biol. 32: 3438–3451. 51. Waterfield, M., W. Jin, W. Reiley, M. Y. Zhang, and S.-C. Sun. 2004. IkBkinaseisan 42. Lang, V., A. Symons, S. J. Watton, J. Janzen, Y. Soneji, S. Beinke, S. Howell, essential component of the Tpl2 signaling pathway. Mol. Cell. Biol. 24: 6040–6048. and S. C. Ley. 2004. ABIN-2 forms a ternary complex with TPL-2 and NF-kB1 52. Roget, K., A. Ben-Addi, A. Mambole-Dema, T. Gantke, J. Janzen, H.-T. Yang, p105 and is essential for TPL-2 protein stability. Mol. Cell. Biol. 24: 5235–5248. N. Shpiro, N. Morrice, D. Abbott, and S. C. Ley. 2012. IkB kinase 2 regulates 43. Salmeron, A., T. B. Ahmad, G. W. Carlile, D. Pappin, R. P. Narsimhan, and TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct S. C. Ley. 1996. Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, phosphorylation of TPL-2 serine 400. Mol. Cell. Biol. 32: 4684–4690. a novel MAP kinase kinase kinase. EMBO J. 15: 817–826. 53. Sawcer, S., G. Hellenthal, M. Pirinen, C. C. Spencer, N. A. Patsopoulos, 44. Prinz, M., H. Schmidt, A. Mildner, K.-P. Knobeloch, U.-K. Hanisch, J. Raasch, L. Moutsianas, A. Dilthey, Z. Su, C. Freeman, S. E. Hunt, et al; International Downloaded from D. Merkler, C. Detje, I. Gutcher, J. Mages, et al. 2008. Distinct and nonredun- Multiple Sclerosis Genetics Consortium; Wellcome Trust Case Control Con- dant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the sortium 2. 2011. Genetic risk and a primary role for cell-mediated immune central nervous system. Immunity 28: 675–686. mechanisms in multiple sclerosis. Nature 476: 214–219. http://www.jimmunol.org/ by guest on September 24, 2021