ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 5, May 2014, pp 1208–1217 DOI 10.1002/art.38327 © 2014, American College of Rheumatology
Alternative p38 MAPKs Are Essential for Collagen-Induced Arthritis Gabriel Criado,1 Ana Risco,2 Dayanira Alsina-Beauchamp,2 Marı´a J. Pe´rez-Lorenzo,1 Alejandra Esco ´s,2 and Ana Cuenda2 node cells from p38␥/␦ⴚ/ⴚ mice. IL-17 and IFN␥ messenger RNA expression in joints was significantly inhibited in p38␥/␦ⴚ/ⴚ mice. Wild-type chimeric mice with p38␥/␦ⴚ/ⴚ bone marrow did not show decreased CIA. Conclusion. Reduced disease severity in p38␥/␦ⴚ/ⴚ mice was associated with lower cytokine production and anticollagen antibody responses than in controls, indicating that p38␥ and p38␦ are crucial regulators of inflammatory joint destruction in CIA. Our findings indicate that p38␥ and p38␦ are potential therapeutic targets in complex diseases, such as rheumatoid arthritis, that involve innate and adaptive immune responses.
Objective. The role of most p38 MAPK isoforms in inflammatory arthritis is not known. This study was undertaken to evaluate p38␥ and p38␦ deficiency in the collagen-induced arthritis (CIA) model. Methods. Wild-type, p38␥ⴚ/ⴚ, p38␦ⴚ/ⴚ, and ⴚ/ⴚ p38␥/␦ mice were immunized with chicken type II collagen, and disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum cytokine levels and in vitro T cell cytokine responses were quantified by flow cytometry and multiplex analysis, and serum anticollagen antibody levels by enzymelinked immunosorbent assay. Cytokine and p38 MAPK isoform expression in joints were determined by quantitative polymerase chain reaction. Results. Compound p38␥ and p38␦ deficiency markedly reduced arthritis severity compared with that in wild-type mice, whereas lack of either p38␥ or p38␦ had an intermediate effect. Joint damage was minimal in arthritic p38␥/␦ⴚ/ⴚ mice compared with wild-type mice. The p38␥/␦ⴚ/ⴚ mice had lower levels of pathogenic anticollagen antibodies and interleukin-1 (IL-1) and tumor necrosis factor ␣ than controls. In vitro T cell assays showed reduced proliferation, interferon-␥ (IFN␥) production, and IL-17 production by lymph
Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and joint destruction that results from continuous release of proinflammatory cytokines, including tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1), as well as other mediators (1,2). MAPK family proteins regulate cellular processes such as synthesis and release of proinflammatory molecules that contribute to RA pathogenesis (3). The p38 MAPK family in particular is a central regulator of proinflammatory cytokine production (4,5). There are 4 p38 MAPKs, p38␣, p38, p38␥, and p38␦, which have very similar protein sequences. They are activated by phosphorylation mediated primarily by MAPK kinase 3 (MKK-3) and MKK-6 in response to a range of cell stresses and in response to inflammatory cytokines (4,6). The p38 ␣ isoform is the most abundant, bestcharacterized isoform and is necessary for a correct immune response. It is activated during both innate and adaptive immune responses, and after activation regulates a variety of genes implicated in inflammation, including TNF␣, IL-1, and IL-6 (5,7). The role of p38␣ in response to and in the regulation of inflammatory pathways has led to efforts to develop small molecular weight inhibitors as therapeutic agents for diseases characterized by chronic inflammation, such as RA (8).
Supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; CP07/250 to Dr. Criado and BFU2010-19734 to Dr. Cuenda) and from the Instituto de Salud Carlos III RETICS Programme (RD08/0075 RIER). Dr. Criado is recipient of a MINECO Miguel Servet Fellowship. Ms AlsinaBeauchamp is recipient of a MINECO FPI Fellowship. 1 Gabriel Criado, PhD, Marı´a J. Pe´rez-Lorenzo, BSc: Instituto de Investigacio ´n Sanitaria and Hospital Universitario 12 de Octubre, Madrid, Spain; 2Ana Risco, PhD, Dayanira Alsina-Beauchamp, MSc, Alejandra Esco ´s, BSc, Ana Cuenda, PhD: Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain. Address correspondence to Ana Cuenda, PhD, Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail: [email protected]. Submitted for publication June 21, 2013; accepted in revised form December 17, 2013. 1208
p38␥ AND p38␦ IN ARTHRITIS
The role of p38␣ in arthritis development has been demonstrated using kinase inhibitors for chemical blockade of its activation in various arthritis models (9–11); however, clinical progress with these compounds has been halted due to lack of effectiveness and to toxicity in clinical trials (3,12). These inhibitors are not completely isoform-specific and can also block the activity of other p38 MAPKs (13,14). In addition, genetic deletion of the upstream p38 activators MKK-3 or MKK-6 confers protection against arthritis (15–18). It is thus particularly important to understand the individual function of each p38 MAPK over the course of arthritis progression. Selective genetic targeting of single isoforms will help to pinpoint this role. Although all p38 MAPK isoforms are expressed in mouse osteoclasts, in mouse fibroblast-like synoviocytes, and in synovial tissue from RA patients (15,19,20), the role of p38␥ and p38␦ in RA development is still largely unknown. Studies in mice deficient in p38␥, p38␦, or both showed that these kinases are essential for the innate immune response (21,22). Combined deletion of p38␥ and p38␦ impairs proinflammatory cytokine production in macrophages and dendritic cells in response to bacterial lipopolysaccharide (LPS), and p38␥/␦-deficient mice are less sensitive to LPS-induced septic shock than wild-type mice, as they show lower TNF␣ and IL-1 levels after challenge (22). Given the essential role of proinflammatory cytokines in RA, these findings suggest that p38␥ and p38␦ are fundamental in arthritis development. In this study, we compared the development of collagen-induced arthritis (CIA) in wild-type mice with that in mice lacking p38␥, p38␦, or both. We found that the combined action of p38␥ and p38␦ is necessary for arthritis progression. TNF␣ and IL-1 levels were lower in serum, and production of IL-17 and IFN␥ was reduced in synovial tissue and lymph nodes (LNs) in p38␥/␦-deficient mice compared to wild-type mice. MATERIALS AND METHODS Mice, antibodies, and reagents. Mice lacking p38␥, p38␦, or p38␥/␦ were backcrossed onto the C57BL/6 background for at least 9 generations (22,23). Mice were housed in specific pathogen–free conditions in accordance with European Union regulations, and the study was approved by local CNB-CSIC ethics review. Antibodies to total and phosphorylated ERK-1/2 forms and phosphorylated p38 MAPK were from Cell Signaling Technology, anti–total p38␣ was from Santa Cruz, and anti-p38␥ and anti-p38␦ antibodies were raised and purified as previously described (24). Induction and assessment of CIA. Mice were immunized intradermally at the tail base with an emulsion of chicken
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type II collagen (CII) in Freund’s complete adjuvant (25). Arthritis was assessed daily by scoring each limb on a scale of 0–3, where 0 ⫽ normal, 1 ⫽ slight swelling and erythema, 2 ⫽ pronounced edematous swelling, and 3 ⫽ joint rigidity, yielding a maximum score of 12 per mouse. At the end of the experiment, paws were removed, fixed in 4% formalin, decalcified with 10% EDTA, and paraffin embedded. Sections (4 M thick) were stained with hematoxylin and eosin, and damage was assessed using a scale of 0–3, where 0 ⫽ normal, 1 ⫽ moderate infiltration with discrete erosion, 2 ⫽ severe infiltration and moderate erosion with preserved joint architecture, and 3 ⫽ severe erosion with loss of joint structure. A mean value for each paw was obtained by assessing distal and proximal interphalangeal joints and the tarsometatarsal joint; addition of scores for 4 paws yielded the total score for each mouse (maximum possible score of 12). Bone marrow transfer. Female wild-type or p38␥/␦⫺/⫺ mice were lethally irradiated (9.25 Gy) and injected intravenously with donor cells (3 ⫻ 106) from male wild-type or p38␥/␦⫺/⫺ mice. The extent of repopulation was determined by polymerase chain reaction (PCR) analysis for the Y chromosome–linked Sry gene of the genomic DNA extracted from blood and by Western blotting to detect p38␥ and p38␦ in splenocytes. Immunoprecipitation and immunoblotting. Mouse paws that had been snap-frozen in liquid nitrogen were thawed, and joint proteins were extracted using buffer A (50 mM Tris HCl [pH 7.5], 1 mM EGTA, 1 mM EDTA, 0.15M NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 50 mM sodium -glycerophosphate, 5 mM pyrophosphate, 0.27M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, and 1% volume/volume Triton X-100) with 0.1% v/v 2mercaptoethanol and complete proteinase inhibitor cocktail (Roche). Lysates were centrifuged (13,000g for 15 minutes at 4°C), and supernatants were removed, snap-frozen, and stored at ⫺80°C. Joint extracts (0.5 mg or 1 mg) were incubated for 2 hours at 4°C with 2 g of anti-p38␥ or anti-p38␦ antibody coupled to protein G–Sepharose. Captured proteins were centrifuged (13,000g), supernatants were discarded, and the beads were washed twice in buffer A containing 0.5M NaCl and then twice in buffer A alone. For immunoblotting, protein samples were resolved in sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked (for 30 minutes) in 50 mM Tris HCl (pH 7.5), 0.15M NaCl, 0.05% v/v Tween (Tris buffered saline–Tween [TBST] buffer) containing 10% weight/ volume dry milk, then incubated in TBST buffer with 10% w/v dry milk and 0.5–1 g/ml antibody (for 2 hours at room temperature or overnight at 4°C). Protein was detected with fluorescence-labeled secondary antibodies (Invitrogen), using an Odyssey infrared imaging system. Cytokine production assay. Cytokine concentrations in T cell supernatants and mouse serum samples were measured using a Luminex-based MilliPlex Mouse cytokine/chemokine immunoassay and the Luminex-based Bio-Plex Mouse Grp I Cytokine 23-Plex Panel (Bio-Rad). IFN␥ and IL-17 were quantified with enzyme-linked immunosorbent assay (ELISA) MAX sets (BioLegend).
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Determination of anticollagen antibodies. Serum levels of anticollagen total IgG, IgG1, and IgG2c were assessed by ELISA (25). A standard curve was generated for each assay by including serial dilutions of a reference sample of pooled sera from arthritic C57BL/6 mice. The lowest dilution of the reference sample was assigned an antibody concentration of 100 arbitrary units (AU), and antibody concentrations for each sample were obtained by reference to the standard curve. Gene expression analysis. RNA was extracted from mouse paws using TRI Reagent (Sigma-Aldrich). Complementary DNA (cDNA) for real-time quantitative PCR (qPCR) was generated from 1 g total RNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) in a 10 l final reaction volume. The quantitative PCRs were performed in triplicate using 3 l/well of 2 serial dilutions (1:50 and 1:500) of each cDNA, 0.3 M of each primer, and 1⫻ FluoCycle SYBR Green Mix for qPCR (Genycell-EuroClone) in 8 l, using MicroAmp Optical 384-well plates (Applied Biosystems). PCRs were carried out in an ABI Prism 7900HT system (Applied Biosystems), and the results were analyzed using the comparative Ct method (⌬⌬Ct) using SDS software version 2.2. The constitutively expressed gene 18S RNA was used as an internal control. The following primer sequences were used: for p38␣, forward 5⬘-AACCAGACAGTGGATATTTGGTC and reverse 5⬘-TGAGCTTCAACTGATCAATATGGT; for p38, forward 5⬘-GTCCTGAAGTTCTGGCAAAGA and reverse 5⬘-CACTGATGAGGTCCTTCTGG; for p38␥, forward 5⬘-ACCT GATGAGTCTCTGGACGA and reverse 5⬘-CCAGATCAGTGCCCATGAAT; for p38␦, forward 5⬘-GGACCCTGAGGAGGAGACA and reverse 5⬘-GTTTGAGATCTCTTTGTAGATGTGTTG; for IL-6, forward 5⬘-GCTACCAAACTGGATATAATCAGG and reverse 5⬘-CAGGTAGCTATGGTACTCCAGAA; for IL-1  , forward 5⬘TTGACGGACCCCAAAAGAT and reverse 5⬘-GAAGCTGGATGCTCTCATCTG; for TNF␣, forward 5⬘-CTGTAGCCCACGTCGTAGC and reverse 5⬘-TTGAGATCCATGCCGTTG; for IFN␥, forward 5⬘-GCAACAGCAAGGCGAAAAAG and reverse 5⬘-TTTCTG GCTGTTACTGCCACG; for IL-17, forward 5⬘-CAGGGAGAGCTTCATCTGTGT and reverse 5⬘-GCTGAGCTTTGAGGGATGAT; and for 18S, forward 5⬘-CGCGGTTCTATTTTGTTGGT and reverse 5⬘-AGTCGGCATCGTTTATGGTC. Analysis of collagen-specific T cell responses. Singlecell suspensions from mouse inguinal LNs were cultured with 50 g/ml of CII or 100 ng/ml of anti-CD3 (clone 145-2C11; BioLegend). Culture supernatants were collected after 48 hours, and cytokine production was quantified by ELISA. The proliferative response to CII was determined by water-soluble tetrazolium 1 reagent (Roche) incorporation in cultured cells after 72 hours. Intracellular cytokine staining and flow cytometry. Single-cell suspensions from mouse LNs were stimulated for 4 hours (at 37°C in 5% CO2) with 20 ng/ml of phorbol myristate acetate (PMA) and 1 g/ml of ionomycin in the presence of 10 g/ml brefeldin A (all from Sigma-Aldrich), and stained with PerCP-conjugated anti-CD4 (clone RM4-5; BD Biosciences). Cells were permeabilized with 0.5% saponin and stained with fluorescein isothiocyanate (FITC)– conjugated anti-IFN␥ (clone XMG1.2; BD Biosciences), phy-
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coerythrin (PE)–conjugated anti–IL-17 (clone TC11-18H10; Miltenyi Biotec), FITC-conjugated IL-10 (clone JES5-16E3), and PE-conjugated IL-4 (clone 11B11; both from BD Biosciences). To detect Treg cells, mouse LN cells were stained with peridinin chlorophyll A protein (PerCP)–conjugated antiCD4, fixed, permeabilized, and stained with PE-conjugated anti-FoxP3 (clone MF-23; BD Biosciences). For flow cytometric analysis, we used a FACSCalibur instrument and CellQuest Pro software (BD Biosciences). Statistical analysis. Data are expressed as the mean ⫾ SD. The area under the curve was used for analysis of the difference in clinical scores. Histology scores, qPCR, and protein quantification results were analyzed by Mann-Whitney U test using GraphPad Prism software. P values less than 0.05 were considered significant.
RESULTS Resistance to CIA in p38␥/␦ⴚ/ⴚ mice. To determine p38␥ and p38␦ involvement in arthritis, we assessed whether lack of p38␥ or p38␦ altered the course of CIA. CIA incidence and onset time in wild-type mice were comparable to those for C57BL/6 mice (⬃50%, 3–5 weeks after immunization) (26), whereas the percentage of animals with arthritis was lower for both p38␥⫺/⫺ mice and p38␦⫺/⫺ mice than for wild-type mice (Figure 1A). Arthritis severity was nonetheless comparable in all 3 groups throughout the course of disease (Figure 1B). Since we previously found that p38␥ and p38␦ have largely redundant functions (22,23), we studied the effect of combined p38␥/p38␦ deletion in the CIA model. The p38␥/␦-null mice showed lower disease incidence and a decrease in clinical score compared to wild-type mice (area under the curve 95.5 ⫾ 27.8 for wild-type versus 46.5 ⫾ 4.0 for p38␥/␦⫺/⫺ mice) (Figures 1A and B). We measured the extent of inflammatory infiltration and bone erosion in wild-type and p38␥/␦⫺/⫺ mice at the end of the experiment (day 42 after immunization) and found that arthritic p38␥/␦⫺/⫺ mice had significantly lower levels of inflammatory infiltrate and bone erosion than wild-type mice (Figure 1C). These data show that the compound p38␥/p38␦ deficiency protected mice against CIA development. Expression and activation of p38 MAPK and ERK-1/2 in CIA. Since it has been shown that all p38 MAPK isoforms are expressed in mouse fibroblast-like synoviocytes and in synovial tissue from RA patients (15,19,20), we then investigated whether or not p38␥ and p38␦ expression and activation vary in the mouse joint after induction of CIA. Levels of protein and messenger RNA (mRNA) for p38␥ and p38␦ were measured in
p38␥ AND p38␦ IN ARTHRITIS
Figure 1. Attenuation of collagen-induced arthritis by deletion of p38␥ and p38␦. A, Arthritis incidence in wild-type (WT) mice, p38␥⫺/⫺ mice, p38␦⫺/⫺ mice, and p38␥/␦⫺/⫺ mice after immunization with type II collagen. B, Clinical scores in wild-type mice, p38␥⫺/⫺ mice, p38␦⫺/⫺ mice, and p38␥/␦⫺/⫺ mice after arthritis onset. The wild-type and p38␥/␦⫺/⫺ groups were compared using the area under the curve method, which resulted in values of 64.24 for wild-type mice and 23.25 for p38␥/␦⫺/⫺ mice. Values are the mean ⫾ SD (n ⫽ 8–12 mice per group). C, Representative hematoxylin and eosin–stained sections of ankle joints from wild-type mice and p38␥/␦⫺/⫺ mice after CIA induction, and quantification of damage in the joints. Values are the mean ⫾ SD (n ⫽ 10 mice per group). ⴱⴱ ⫽ P ⬍ 0.01 by Mann-Whitney test.
joint extracts from wild-type mice and p38␥/␦⫺/⫺ mice that were left untreated, that were immunized with CII and did not develop arthritis, and that were immunized with CII and developed arthritis. The mRNA levels were assessed by qPCR, and the protein levels were assessed by Western blotting, using antibodies specific for each p38 MAPK isoform. The p38␥/␦⫺/⫺ mice did not express p38␥ or p38␦, and we observed no notable differences in p38␥ or p38␦ gene or protein expression in the joints of untreated or treated wild-type mice (Figures 2A and B). Assessment of p38␣ and p38 mRNA showed no differences in p38␣ gene expression between wild-type and p38␥/␦⫺/⫺ mice, whereas p38 mRNA levels decreased
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Figure 2. Expression and activation of p38␥ and p38␦ by collagen immunization. Wild-type (WT) or p38␥/␦⫺/⫺ mice were left untreated or were immunized with type II collagen (CII). At 6 weeks postimmunization, joint extracts were prepared for mRNA and protein analyses. A, Quantitative polymerase chain reaction of p38 MAPK mRNAs in total RNA from the joints of wild-type and p38␥/␦⫺/⫺ mice that were either nonimmunized, immunized and did not develop collageninduced arthritis (CIA⫺), or immunized and did develop CIA (CIA⫹). Values are the mean ⫾ SD from 1 representative experiment of at least 3 (performed in triplicate) with similar results. ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. B, Immunoblotting of joint extracts (50 g) from wild-type and p38␥/␦⫺/⫺ mice with antibodies to active phosphorylated p38 (p-p38␣, p-p38␥), phosphorylated ERK-1/2 (p–ERK-1/2), and total p38␣, p38␥, p38␦, and ERK-1/2. Duplicate lanes are shown; results were similar in 3 independent experiments. C, Quantification of the bands from the immunoblots shown in B, using an Odyssey infrared imaging system. Quantification is represented as p-p38␣ density/p38␣, p-p38␥ density/p38␥, and p–ERK-1/2 density/ERK-1/2 total density. Values are the mean ⫾ SD from 3 experiments performed in duplicate. ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. D, Immunoprecipitation of endogenous p38␥ and p38␦ from wild-type joint extracts (2 mg). Pellets were immunoblotted with anti–p-p38, anti-p38␥, or anti-p38␦ antibodies. Blots are representative of 3 independent experiments. Total extracts were used as a control. NS ⫽ not significant.
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in wild-type mice and increased in p38␥/␦⫺/⫺ mice after CII immunization (Figure 2A). We assessed p38 activation in joint extracts from wild-type and p38␥/␦⫺/⫺ mice. Both p38␣ and p38␥ were activated in wild-type joints, and p38␣ was activated in p38␥/␦⫺/⫺ mice to the same extent as in wild-type joints (Figures 2B and C). We observed no active p38␥ in p38␥/␦⫺/⫺ mouse joint extracts, and we detected no active p38␦ under any experimental conditions (Figure 2D). We also evaluated ERK-1/2 activation in joint extracts. ERK-1/2 was activated after immunization, with no differences between wild-type and p38␥/␦⫺/⫺ mice (Figures 2B and C). These data show that p38␥ and p38␦ do not control either p38␣ or ERK-1/2 activation in CIA, and that p38␥ is activated in the joints of CIItreated wild-type mice. Cytokine expression in arthritic p38␥/␦ⴚ/ⴚ mice. IL-1, TNF␣, and IL-6 are key cytokines in CIA pathogenesis. We therefore evaluated serum levels of these cytokines in p38␥/␦⫺/⫺ and wild-type mice after CIA induction. Serum TNF␣ and IL-1 levels were markedly lower in p38␥/␦⫺/⫺ mice, whereas IL-6 levels were unaffected (Figure 3A). In contrast, TNF␣, IL-1, and IL-6 gene expression in the arthritic joint were similar in p38␥/␦⫺/⫺ and wild-type mice (Figure 3B). IL-17 and IFN␥ gene expression were markedly lower in joints from p38␥/␦⫺/⫺ mice compared with those from wildtype mice (Figure 3B). These results confirm the need for p38␥ and p38␦ for cytokine production in the in vivo inflammatory response, and suggest that p38␥/␦ deficiency limits arthritis progression and severity by reducing systemic production of TNF␣ and IL-1. Anticollagen antibody levels in arthritic p38␥/␦ⴚ/ⴚ mice. CIA pathogenesis is characterized by the generation of anti-CII antibodies, and the IgG2 subclass binds complement effectively and forms anticollagen immune complexes in the synovium (27). To determine whether the reduced severity and incidence of arthritis in p38␥/␦⫺/⫺ mice was due to an impaired antibody response to CII, we measured serum anti-CII–specific total IgG, IgG1, and IgG2c levels by ELISA at 3 and 6 weeks postimmunization, to assess temporal differences between genotypes (Figure 4). Levels of all anticollagen antibodies were similar in wild-type and p38␥/␦-deficient mice at 3 weeks postimmunization. At 6 weeks, serum concentrations of anti-CII total IgG and IgG1 were similar in wild-type and in p38␥/␦⫺/⫺ mice, although disease severity was lower in the knockout mice. In contrast, IgG2c levels were significantly lower in p38␥/␦⫺/⫺ than in wild-type mice. These results
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Figure 3. Altered cytokine production in p38␥/␦-deficient mice in response to type II collagen immunization. A, Serum levels of tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), and IL-6 in wildtype (WT) and p38␥/␦⫺/⫺ mice. Serum from wild-type and p38␥/␦⫺/⫺ mice was collected 42 days after immunization, and cytokine levels were measured by multiplex cytokine assay. Values are the mean ⫾ SD (n ⫽ 6 mice per group). B, Expression of mRNA for TNF␣, IL-1, IL-6, IL-17, and interferon-␥ (IFN␥) in joint extracts from wild-type and p38␥/␦⫺/⫺ arthritic mice. Joint extracts were prepared 42 days postimmunization and quantitative polymerase chain reaction was performed on total RNA. Results were normalized to 18S RNA expression, and x-fold induction was calculated relative to mRNA expression in extracts from untreated wild-type mice. Values are the mean ⫾ SD from 1 representative experiment of 3 with similar results (n ⫽ 5–7 mice per group). ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. CIA ⫽ collagen-induced arthritis; NS ⫽ not significant.
suggest a role for p38␥/␦⫺/⫺ in regulating B cell responses to collagen. T cell response in CIA p38␥/␦ⴚ/ⴚ mice. Induction and propagation of CIA requires functional B and T cells. The lower IL-17 and IFN␥ levels in p38␥/␦⫺/⫺ mouse paws (Figure 3B) suggest that T cell responses to CII are altered in these mice. To evaluate whether p38␥ and p38␦ regulate CII-specific T cell responses in CIA, we collected inguinal LN cells at the end of the experi-
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a significant reduction in Th17 cells in p38␥/␦⫺/⫺ mice (Figure 5C). The frequencies of Th2 cells (defined as
Figure 4. Antibody response to type II collagen (CII) in p38␥/␦deficient mice. Serum levels of total IgG, IgG1, and IgG2c anti-CII antibodies in wild-type (WT) and p38␥/␦⫺/⫺ mice were quantified by enzyme-linked immunosorbent assay at 3 weeks (A) and 6 weeks (B) after collagen immunization. Values are the mean ⫾ SD (n ⫽ 10 mice per group). ⴱ ⫽ P ⱕ 0.05. NS ⫽ not significant.
ments (6 weeks postimmunization) and cultured them in vitro with CII. Compared to LNs from wild-type mice, LNs from p38␥/␦⫺/⫺ mice showed significantly less proliferation in response to CII or anti-CD3 (Figure 5A). Analysis of culture supernatant cytokine production showed that IL-17 and IFN␥ production were significantly reduced in response to CII and anti-CD3 in p38␥/␦⫺/⫺ mice (Figure 5B). These data suggest that p38␥ and p38␦ regulate Th1 and Th17 cell generation in vivo during CIA. We tested this by analyzing Th1 (CD4⫹IFN␥⫹) and Th17 (CD4⫹IL-17⫹) cell frequency in wild-type and p38␥/␦⫺/⫺ mouse LNs at the end of CIA experiments. Flow cytometric analysis showed a trend toward reduced Th1 cell frequency and
Figure 5. Reduced proliferation and cytokine production in response to type II collagen (CII) in lymph nodes from p38␥/␦-deficient mice. Lymph node cells from arthritic wild-type (WT) or p38␥/␦⫺/⫺ mice were isolated and were left unstimulated or were stimulated with 50 g/ml of CII or 100 ng/ml of anti-CD3 antibody. A, Proliferation in cultured cells harvested after 72 hours. B, Levels of tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), IL-17, and interferon-␥ (IFN␥) in culture supernatants collected after 48 hours. Cytokine production was quantified by enzyme-linked immunosorbent assay and multiplex cytokine assays. In A and B, values are the mean ⫾ SEM (n ⫽ 9–10 mice per group). ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. C, Reduced frequency of IL-17–producing cells in lymph nodes from p38␥/␦deficient mice. Single-cell suspensions of lymph node cells were assessed for cytokine-producing and Treg cells (see Materials and Methods). Representative dot plots of CD4⫹-gated cells, quantification of cytokine-producing cells, and quantification of FoxP3 and FoxP3:IL-17 ratio are shown. Values are the mean ⫾ SEM (n ⫽ 10 mice per group). ⴱⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant.
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Figure 6. No decrease in the incidence or severity of collagen-induced arthritis in wild-type (WT) chimeric mice with p38␥/␦⫺/⫺ bone marrow. A, Arthritis incidence in the indicated groups 42 days after immunization with type II collagen. B, Clinical score 8 days after arthritis onset. C, Serum levels of total IgG1 and IgG2c anti-CII antibodies 6 weeks after collagen immunization, as quantified by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD from 3 independent experiments (n ⫽ 24–30 mice per group). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant.
CD4⫹IL4⫹/IL10⫹) and Treg cells (CD4⫹FoxP3⫹) were nonetheless similar in wild-type mice and p38␥/␦⫺/⫺ mice, which led to an increased Treg:Th17 ratio in LNs from p38␥/␦⫺/⫺ mice (Figure 5C) and prevented the development of CIA. CIA in p38␥/␦ bone marrow chimeras. Although the above data indicate that B and T cell responses are reduced in p38␥/␦⫺/⫺ mice during CIA, nonhematopoietic cells that can play a role in arthritis development also express p38␥ and p38␦. To assess the contribution of p38␥ and p38␦ in nonhematopoietic cells to arthritis progression in CIA, we generated wild-type and p38␥/␦ chimeric mice with adoptive bone marrow transplantation. After a 6-week reconstitution phase to achieve chimerism, mice were immunized with chicken CII (Figure 6). Bone marrow chimerism did not affect the phenotype outcome, and p38␥/␦⫺/⫺ mice that received either p38␥/␦⫺/⫺ (p38␥/␦⫺/⫺3 p38␥/␦⫺/⫺) or wild-type (WT3p38␥/␦⫺/⫺) bone marrow grafts showed absence of clinical arthritis, while wild-type mice that received wild-type (WT3 WT) or p38␥/␦⫺/⫺ (p38␥/␦⫺/⫺3 WT)
bone marrow grafts had comparable incidence and clinical scores (Figures 6A and B). Likewise, IgG2c levels were reduced in p38␥/␦⫺/⫺ mice, regardless of the origin of bone marrow cells (Figure 6C). This suggests that p38␥/␦ expression in the nonhematopoietic compartment drives arthritis development and controls the activation of B and T cells in CIA. DISCUSSION We used mice lacking p38␥ and/or p38␦ to analyze the effect of these isoforms on the development of CIA. Studies using p38 MAPK–specific inhibitors and MKK-3–deficient or MKK-6–deficient mice suggested that p38␣ and/or p38 are involved in this process. However, results from knockout mice showed p38 to be completely dispensable for arthritis development (28). Full p38␣ knockout is fatal to the embryo (29–32). Nonetheless, in an arthritis model induced by TNF␣ overexpression, p38␣ deletion using inducible Mx-Cre causes a decrease in paw swelling as well as in joint
p38␥ AND p38␦ IN ARTHRITIS
inflammation and bone damage (19). In contrast, in a passive K/BxN arthritis model, chronic arthritis severity is increased in mice with specific p38␣ deletion in macrophages (33). In this study, we demonstrated the role of p38␥ and p38␦ as essential components in arthritis pathogenesis. Combined deletion of p38␥ and p38␦ led to a marked reduction in disease incidence and severity in the CIA model. The results were confirmed by histologic evaluation. Lack of p38␥ or p38␦ alone had a partial effect on CIA incidence, indicating that p38␥ and p38␦ have largely redundant functions. The protective effect of p38␥/p38␦ deficiency in arthritis was similar to that observed in mice lacking MKK-3 and MKK-6, which lie upstream in this pathway (15–18). In addition, serum TNF␣ and IL-1 levels, but not IL-6 levels, were considerably reduced in p38␥/␦⫺/⫺ mice compared with CII-immunized wild-type mice. Similar reductions in serum cytokine levels are also observed in LPS/Dgalactosamine treated p38␥/␦⫺/⫺ mice compared with wild-type mice (22). In situ analysis showed increased cytokine mRNA levels in arthritic wild-type and p38␥/ ␦⫺/⫺ mouse paw extracts compared to healthy mice. In the case of IL-17 and IFN␥, we observed a significant decrease in mRNA levels in arthritic p38␥/␦⫺/⫺mice compared to arthritic wild-type mice. Our data suggest that the function of p38␥ and p38␦ in CIA progression is effected mainly via proinflammatory cytokines such as TNF␣, IL-1, IL-17, and IFN␥. We also found differences in the anticollagen antibody response between wild-type and p38␥/␦⫺/⫺ mice, suggesting that the reduced disease incidence and severity in p38␥/␦⫺/⫺ mice compared with wild-type mice are partially attributable to an impaired immune response. We found that activation and proliferation defects in p38␥/␦⫺/⫺ mouse B cells affect in vivo antibody responses. The p38␥/␦⫺/⫺ mice had significantly lower IgG2 levels than wild-type mice and showed defective class-switching in response to immunization with a T cell–dependent antigen (Risco A and Cuenda A: unpublished observations). Lack of both p38␥ and p38␦ nonetheless did not affect B cell ability to class-switch to IgG in vitro, indicating that the reduced in vivo antibody response to T cell–dependent antigen is not B cell autonomous and is probably due to intrinsic non–B cell factors such as cell context (Risco A and Cuenda A: unpublished observations). Impaired cytokine production might be implicated in the defective T cell– dependent antibody response. Our in vitro assays of T cell activity showed a decreased proliferative response to CII in p38␥/␦⫺/⫺ cells compared with those from wild-
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type mice. Collagen-induced TNF␣, IFN␥, and IL-17 production in LNs were significantly lower in the culture supernatants of p38␥/␦⫺/⫺ mouse T cells, consistent with our findings in serum (for TNF␣) and arthritic joints (for IFN␥ and IL-17). Moreover, p38␥/␦⫺/⫺ mice showed a lower Th17 cell frequency and a greater Treg:Th17 ratio than wild-type mice, both of which are linked to successful therapy in RA (34). The effects of p38␥ and p38␦ deficiency on IL-17 production are especially interesting, in light of clinical trials that target this cytokine in RA and other inflammatory conditions (35). IL-17 is a critical regulator of joint inflammation and bone erosion (36). Deficiency in this cytokine or its receptor suppresses inflammation and joint damage in CIA, and IL-17 injection into normal mouse ankles induces synovitis and cartilage destruction (35,37). Decreased serum production of anticollagen IgG2c in p38␥/␦⫺/⫺ mice might also be IL-17 dependent, since this cytokine mediates antibody class-switching to IgG2a/c (38). The p38 isoform is also highly active in the joints of patients with RA and in mouse models of inflammatory arthritis (39,40). We examined p38 MAPK in CIA and found that both p38␣ and p38␥ are activated in wild-type mice, whereas no p38␦ phosphorylation was detected under our experimental conditions. This phosphorylation pattern coincides with that reported for synovial tissue from RA patients, in which p38␣ and p38␥, but not p38␦, are phosphorylated (20). These results suggest that p38␥ is the p38 MAPK isoform with a central role in arthritis development. Nonetheless, deletion of both p38␥ and p38␦ isoforms is necessary to see a significant difference in arthritis score, which indicates the importance of p38␦ in this model. Our data imply that p38␦ compensates for the lack of p38␥. Using cells that lack several family members in combination with specific kinase inhibitors, we previously demonstrated functional compensation by these closely related protein kinases when one member is not expressed. Thus, p38␥ substrates can be phosphorylated by p38␦ in p38␥⫺/⫺ cells (22,23). Alternatively, p38␦ might have a role in CIA at a location (tissue or cells) other than joints. We cannot rule out the possibility that our experimental tools are insufficiently sensitive to detect its phosphorylation under these experimental conditions, since p38␦ expression in joints is very low compared to other mouse tissues (results not shown). The p38␥ and p38␦ isoforms are expressed by several cell types involved in CIA progression, yet the contribution of p38␥ and p38␦ expressed by specific hematopoietic and nonhematopoietic cell types remains
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to be determined. Bone marrow transfer experiments indicated that p38␥ and p38␦ expression by nonhematopoietic stromal cells drives CIA progression, but is not necessary in the hematopoietic compartment. This observation is unexpected, given that p38␥/␦⫺/⫺ mice have reduced IL-17– and IFN␥-producing T cells. It is nonetheless supported by a study showing that synovial stromal cells induce increased Th17 cell expansion in the human system (41), and is also consistent with the view that local synovial inflammatory cells are responsible for the perpetuation of autoimmune inflammation (42,43). Our results indicate that p38␥ and p38␦ isoforms likely play roles in both hematopoietic and nonhematopoietic compartments. Further studies are needed to determine whether these phenomena are dependent on p38␥ and p38␦ expression by synovial cells. In conclusion, our data show that p38␥ and p38␦ regulate the immune response in CIA and suppress clinical disease and bone destruction. The crucial role of p38␥/␦ in synovial inflammation, bone erosion, and TNF␣, IL-1, IFN␥, and IL-17 production suggests that it could serve as a target of therapy in RA as an alternative to traditional p38␣ inhibitors, which have proven minimally effective in human disease (44). ACKNOWLEDGMENTS We thank the antibody purification teams (Division of Signal Transduction Therapy, University of Dundee, Dundee, UK) coordinated by H. McLauchlan and J. Hastie for generation and purification of antibodies, A. C. Gonzalo for technical support, and C. Mark for editorial assistance. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cuenda had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Criado, Cuenda. Acquisition of data. Criado, Risco, Alsina-Beauchamp, Pe´rez-Lorenzo, Esco ´s, Cuenda. Analysis and interpretation of data. Criado, Risco, Alsina-Beauchamp, Cuenda.
REFERENCES 1. Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–61. 2. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008;118:3537–45. 3. Cohen P. Targeting protein kinases for the development of anti-inflammatory drugs. Curr Opin Cell Biol 2009;21:317–24. 4. Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, function and role in human diseases. Biochim Biophys Acta 2007;1773:1358–75.
5. Gaestel M, Kotlyarov A, Kracht M. Targeting innate immunity protein kinase signalling in inflammation. Nat Rev Drug Discov 2009;8:480–99. 6. Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Teran B, Sabio G, Davis RJ, et al. Differential activation of p38MAPK isoforms by MKK6 and MKK3. Cell Signal 2010;22:660–7. 7. Rincon M, Davis RJ. Regulation of the immune response by stress-activated protein kinases. Immunol Rev 2009;228:212–24. 8. Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA. p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med 2009;15:369–79. 9. Nishikawa M, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogenactivated protein kinase inhibitor FR167653. Arthritis Rheum 2003;48:2670–81. 10. Wada Y, Nakajima-Yamada T, Yamada K, Tsuchida J, Yasumoto T, Shimozato T, et al. R-130823, a novel inhibitor of p38 MAPK, ameliorates hyperalgesia and swelling in arthritis models. Eur J Pharmacol 2005;506:285–95. 11. Hill RJ, Dabbagh K, Phippard D, Li C, Suttmann RT, Welch M, et al. Pamapimod, a novel p38 mitogen-activated protein kinase inhibitor: preclinical analysis of efficacy and selectivity. J Pharmacol Exp Ther 2008;327:610–9. 12. Genovese MC. Inhibition of p38: has the fat lady sung? [editorial]. Arthritis Rheum 2009;60:317–20. 13. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408:297–315. 14. Kuma Y, Sabio G, Bain J, Shpiro N, Marquez R, Cuenda A. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo. J Biol Chem 2005;280:19472–9. 15. Yoshizawa T, Hammaker D, Boyle DL, Corr M, Flavell R, Davis R, et al. Role of MAPK kinase 6 in arthritis: distinct mechanism of action in inflammation and cytokine expression. J Immunol 2009; 183:1360–7. 16. Hammaker D, Topolewski K, Edgar M, Yoshizawa T, Fukushima A, Boyle DL, et al. Decreased collagen-induced arthritis severity and adaptive immunity in MKK-6–deficient mice. Arthritis Rheum 2012;64:678–87. 17. Inoue T, Boyle DL, Corr M, Hammaker D, Davis RJ, Flavell RA, et al. Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis. Proc Natl Acad Sci U S A 2006;103:5484–9. 18. Hegen M, Gaestel M, Nickerson-Nutter CL, Lin LL, Telliez JB. MAPKAP kinase 2-deficient mice are resistant to collageninduced arthritis. J Immunol 2006;177:1913–7. 19. Bohm C, Hayer S, Kilian A, Zaiss MM, Finger S, Hess A, et al. The ␣-isoform of p38 MAPK specifically regulates arthritic bone loss. J Immunol 2009;183:5938–47. 20. Korb A, Tohidast-Akrad M, Cetin E, Axmann R, Smolen J, Schett G. Differential tissue expression and activation of p38 MAPK ␣, , ␥, and ␦ isoforms in rheumatoid arthritis. Arthritis Rheum 2006; 54:2745–56. 21. Ittner A, Block H, Reichel CA, Varjosalo M, Gehart H, Sumara G, et al. Regulation of PTEN activity by p38␦-PKD1 signaling in neutrophils confers inflammatory responses in the lung. J Exp Med 2012;209:2229–46. 22. Risco A, del Fresno C, Mambol A, Alsina-Beauchamp D, MacKenzie KF, Yang HT, et al. p38␥ and p38␦ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation. Proc Natl Acad Sci U S A 2012;109:11200–5. 23. Sabio G, Arthur JS, Kuma Y, Peggie M, Carr J, Murray-Tait V, et al. p38␥ regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP. EMBO J 2005;24:1134–45. 24. Cuenda A, Cohen P, Buee-Scherrer V, Goedert M. Activation of
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25.
26. 27.
28. 29. 30.
31. 32.
33.
34.
stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6): comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J 1997;16: 295–305. Inglis JJ, Criado G, Medghalchi M, Andrews M, Sandison A, Feldmann M, et al. Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen. Arthritis Res Ther 2007;9:R113. Campbell IK, Hamilton JA, Wicks IP. Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur J Immunol 2000;30:1568–75. Wooley PH, Luthra HS, Stuart JM, David CS. Type II collageninduced arthritis in mice. I. Major histocompatibility complex (I region) linkage and antibody correlates. J Exp Med 1981;154: 688–700. Beardmore VA, Hinton HJ, Eftychi C, Apostolaki M, Armaka M, Darragh J, et al. Generation and characterization of p38 (MAPK11) gene-targeted mice. Mol Cell Biol 2005;25:10454–64. Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, et al. Essential role of p38␣ MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 2000;6:109–16. Mudgett JS, Ding J, Guh-Siesel L, Chartrain NA, Yang L, Gopal S, et al. Essential role for p38␣ mitogen-activated protein kinase in placental angiogenesis. Proc Natl Acad Sci U S A 2000;97: 10454–9. Tamura K, Sudo T, Senftleben U, Dadak AM, Johnson R, Karin M. Requirement for p38␣ in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 2000;102:221–31. Allen M, Svensson L, Roach M, Hambor J, McNeish J, Gabel CA. Deficiency of the stress kinase p38␣ results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells. J Exp Med 2000;191: 859–70. Guma M, Hammaker D, Topolewski K, Corr M, Boyle DL, Karin M, et al. Antiinflammatory functions of p38 in mouse models of rheumatoid arthritis: advantages of targeting upstream kinases MKK-3 or MKK-6. Arthritis Rheum 2012;64:2887–95. Samson M, Audia S, Janikashvili N, Ciudad M, Trad M, Fraszczak J, et al. Inhibition of interleukin-6 function corrects Th17/Treg cell
1217
35.
36. 37.
38.
39.
40.
41.
42.
43. 44.
imbalance in patients with rheumatoid arthritis. Arthritis Rheum 2012;64:2499–503. Chabaud M, Lubberts E, Joosten L, van Den Berg W, Miossec P. IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis. Arthritis Res 2001;3: 168–77. Peck A, Mellins ED. Breaking old paradigms: Th17 cells in autoimmune arthritis. Clin Immunol 2009;132:295–304. Lubberts E, Joosten LA, van de Loo FA, Schwarzenberger P, Kolls J, van den Berg WB. Overexpression of IL-17 in the knee joint of collagen type II immunized mice promotes collagen arthritis and aggravates joint destruction. Inflamm Res 2002;51:102–4. Mitsdoerffer M, Lee Y, Jager A, Kim HJ, Korn T, Kolls JK, et al. Proinflammatory T helper type 17 cells are effective B-cell helpers. Proc Natl Acad Sci U S A 2010;107:14292–7. Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal– regulated kinase, c-JUN N-terminal kinase, and p38 mitogenactivated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12. Zwerina J, Hayer S, Redlich K, Bobacz K, Kollias G, Smolen JS, et al. Activation of p38 MAPK is a key step in tumor necrosis factor–mediated inflammatory bone destruction. Arthritis Rheum 2006;54:463–72. Eljaafari A, Tartelin ML, Aissaoui H, Chevrel G, Osta B, Lavocat F, et al. Bone marrow–derived and synovium-derived mesenchymal cells promote Th17 cell expansion and activation through caspase 1 activation: contribution to the chronicity of rheumatoid arthritis. Arthritis Rheum 2012;64:2147–57. Armaka M, Apostolaki M, Jacques P, Kontoyiannis DL, Elewaut D, Kollias G. Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J Exp Med 2008;205:331–7. Pablos JL, Canete JD. Immunopathology of rheumatoid arthritis. Curr Top Med Chem 2013;13:705–11. Clark AR, Dean JL. The p38 MAPK pathway in rheumatoid arthritis: a sideways look. Open Rheumatol J 2012;6:209–19.
Alternative p38 MAPKs Are Essential for Collagen-Induced Arthritis Gabriel Criado,1 Ana Risco,2 Dayanira Alsina-Beauchamp,2 Marı´a J. Pe´rez-Lorenzo,1 Alejandra Esco ´s,2 and Ana Cuenda2 node cells from p38␥/␦ⴚ/ⴚ mice. IL-17 and IFN␥ messenger RNA expression in joints was significantly inhibited in p38␥/␦ⴚ/ⴚ mice. Wild-type chimeric mice with p38␥/␦ⴚ/ⴚ bone marrow did not show decreased CIA. Conclusion. Reduced disease severity in p38␥/␦ⴚ/ⴚ mice was associated with lower cytokine production and anticollagen antibody responses than in controls, indicating that p38␥ and p38␦ are crucial regulators of inflammatory joint destruction in CIA. Our findings indicate that p38␥ and p38␦ are potential therapeutic targets in complex diseases, such as rheumatoid arthritis, that involve innate and adaptive immune responses.
Objective. The role of most p38 MAPK isoforms in inflammatory arthritis is not known. This study was undertaken to evaluate p38␥ and p38␦ deficiency in the collagen-induced arthritis (CIA) model. Methods. Wild-type, p38␥ⴚ/ⴚ, p38␦ⴚ/ⴚ, and ⴚ/ⴚ p38␥/␦ mice were immunized with chicken type II collagen, and disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum cytokine levels and in vitro T cell cytokine responses were quantified by flow cytometry and multiplex analysis, and serum anticollagen antibody levels by enzymelinked immunosorbent assay. Cytokine and p38 MAPK isoform expression in joints were determined by quantitative polymerase chain reaction. Results. Compound p38␥ and p38␦ deficiency markedly reduced arthritis severity compared with that in wild-type mice, whereas lack of either p38␥ or p38␦ had an intermediate effect. Joint damage was minimal in arthritic p38␥/␦ⴚ/ⴚ mice compared with wild-type mice. The p38␥/␦ⴚ/ⴚ mice had lower levels of pathogenic anticollagen antibodies and interleukin-1 (IL-1) and tumor necrosis factor ␣ than controls. In vitro T cell assays showed reduced proliferation, interferon-␥ (IFN␥) production, and IL-17 production by lymph
Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and joint destruction that results from continuous release of proinflammatory cytokines, including tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1), as well as other mediators (1,2). MAPK family proteins regulate cellular processes such as synthesis and release of proinflammatory molecules that contribute to RA pathogenesis (3). The p38 MAPK family in particular is a central regulator of proinflammatory cytokine production (4,5). There are 4 p38 MAPKs, p38␣, p38, p38␥, and p38␦, which have very similar protein sequences. They are activated by phosphorylation mediated primarily by MAPK kinase 3 (MKK-3) and MKK-6 in response to a range of cell stresses and in response to inflammatory cytokines (4,6). The p38 ␣ isoform is the most abundant, bestcharacterized isoform and is necessary for a correct immune response. It is activated during both innate and adaptive immune responses, and after activation regulates a variety of genes implicated in inflammation, including TNF␣, IL-1, and IL-6 (5,7). The role of p38␣ in response to and in the regulation of inflammatory pathways has led to efforts to develop small molecular weight inhibitors as therapeutic agents for diseases characterized by chronic inflammation, such as RA (8).
Supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; CP07/250 to Dr. Criado and BFU2010-19734 to Dr. Cuenda) and from the Instituto de Salud Carlos III RETICS Programme (RD08/0075 RIER). Dr. Criado is recipient of a MINECO Miguel Servet Fellowship. Ms AlsinaBeauchamp is recipient of a MINECO FPI Fellowship. 1 Gabriel Criado, PhD, Marı´a J. Pe´rez-Lorenzo, BSc: Instituto de Investigacio ´n Sanitaria and Hospital Universitario 12 de Octubre, Madrid, Spain; 2Ana Risco, PhD, Dayanira Alsina-Beauchamp, MSc, Alejandra Esco ´s, BSc, Ana Cuenda, PhD: Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain. Address correspondence to Ana Cuenda, PhD, Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail: [email protected]. Submitted for publication June 21, 2013; accepted in revised form December 17, 2013. 1208
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The role of p38␣ in arthritis development has been demonstrated using kinase inhibitors for chemical blockade of its activation in various arthritis models (9–11); however, clinical progress with these compounds has been halted due to lack of effectiveness and to toxicity in clinical trials (3,12). These inhibitors are not completely isoform-specific and can also block the activity of other p38 MAPKs (13,14). In addition, genetic deletion of the upstream p38 activators MKK-3 or MKK-6 confers protection against arthritis (15–18). It is thus particularly important to understand the individual function of each p38 MAPK over the course of arthritis progression. Selective genetic targeting of single isoforms will help to pinpoint this role. Although all p38 MAPK isoforms are expressed in mouse osteoclasts, in mouse fibroblast-like synoviocytes, and in synovial tissue from RA patients (15,19,20), the role of p38␥ and p38␦ in RA development is still largely unknown. Studies in mice deficient in p38␥, p38␦, or both showed that these kinases are essential for the innate immune response (21,22). Combined deletion of p38␥ and p38␦ impairs proinflammatory cytokine production in macrophages and dendritic cells in response to bacterial lipopolysaccharide (LPS), and p38␥/␦-deficient mice are less sensitive to LPS-induced septic shock than wild-type mice, as they show lower TNF␣ and IL-1 levels after challenge (22). Given the essential role of proinflammatory cytokines in RA, these findings suggest that p38␥ and p38␦ are fundamental in arthritis development. In this study, we compared the development of collagen-induced arthritis (CIA) in wild-type mice with that in mice lacking p38␥, p38␦, or both. We found that the combined action of p38␥ and p38␦ is necessary for arthritis progression. TNF␣ and IL-1 levels were lower in serum, and production of IL-17 and IFN␥ was reduced in synovial tissue and lymph nodes (LNs) in p38␥/␦-deficient mice compared to wild-type mice. MATERIALS AND METHODS Mice, antibodies, and reagents. Mice lacking p38␥, p38␦, or p38␥/␦ were backcrossed onto the C57BL/6 background for at least 9 generations (22,23). Mice were housed in specific pathogen–free conditions in accordance with European Union regulations, and the study was approved by local CNB-CSIC ethics review. Antibodies to total and phosphorylated ERK-1/2 forms and phosphorylated p38 MAPK were from Cell Signaling Technology, anti–total p38␣ was from Santa Cruz, and anti-p38␥ and anti-p38␦ antibodies were raised and purified as previously described (24). Induction and assessment of CIA. Mice were immunized intradermally at the tail base with an emulsion of chicken
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type II collagen (CII) in Freund’s complete adjuvant (25). Arthritis was assessed daily by scoring each limb on a scale of 0–3, where 0 ⫽ normal, 1 ⫽ slight swelling and erythema, 2 ⫽ pronounced edematous swelling, and 3 ⫽ joint rigidity, yielding a maximum score of 12 per mouse. At the end of the experiment, paws were removed, fixed in 4% formalin, decalcified with 10% EDTA, and paraffin embedded. Sections (4 M thick) were stained with hematoxylin and eosin, and damage was assessed using a scale of 0–3, where 0 ⫽ normal, 1 ⫽ moderate infiltration with discrete erosion, 2 ⫽ severe infiltration and moderate erosion with preserved joint architecture, and 3 ⫽ severe erosion with loss of joint structure. A mean value for each paw was obtained by assessing distal and proximal interphalangeal joints and the tarsometatarsal joint; addition of scores for 4 paws yielded the total score for each mouse (maximum possible score of 12). Bone marrow transfer. Female wild-type or p38␥/␦⫺/⫺ mice were lethally irradiated (9.25 Gy) and injected intravenously with donor cells (3 ⫻ 106) from male wild-type or p38␥/␦⫺/⫺ mice. The extent of repopulation was determined by polymerase chain reaction (PCR) analysis for the Y chromosome–linked Sry gene of the genomic DNA extracted from blood and by Western blotting to detect p38␥ and p38␦ in splenocytes. Immunoprecipitation and immunoblotting. Mouse paws that had been snap-frozen in liquid nitrogen were thawed, and joint proteins were extracted using buffer A (50 mM Tris HCl [pH 7.5], 1 mM EGTA, 1 mM EDTA, 0.15M NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 50 mM sodium -glycerophosphate, 5 mM pyrophosphate, 0.27M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, and 1% volume/volume Triton X-100) with 0.1% v/v 2mercaptoethanol and complete proteinase inhibitor cocktail (Roche). Lysates were centrifuged (13,000g for 15 minutes at 4°C), and supernatants were removed, snap-frozen, and stored at ⫺80°C. Joint extracts (0.5 mg or 1 mg) were incubated for 2 hours at 4°C with 2 g of anti-p38␥ or anti-p38␦ antibody coupled to protein G–Sepharose. Captured proteins were centrifuged (13,000g), supernatants were discarded, and the beads were washed twice in buffer A containing 0.5M NaCl and then twice in buffer A alone. For immunoblotting, protein samples were resolved in sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked (for 30 minutes) in 50 mM Tris HCl (pH 7.5), 0.15M NaCl, 0.05% v/v Tween (Tris buffered saline–Tween [TBST] buffer) containing 10% weight/ volume dry milk, then incubated in TBST buffer with 10% w/v dry milk and 0.5–1 g/ml antibody (for 2 hours at room temperature or overnight at 4°C). Protein was detected with fluorescence-labeled secondary antibodies (Invitrogen), using an Odyssey infrared imaging system. Cytokine production assay. Cytokine concentrations in T cell supernatants and mouse serum samples were measured using a Luminex-based MilliPlex Mouse cytokine/chemokine immunoassay and the Luminex-based Bio-Plex Mouse Grp I Cytokine 23-Plex Panel (Bio-Rad). IFN␥ and IL-17 were quantified with enzyme-linked immunosorbent assay (ELISA) MAX sets (BioLegend).
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Determination of anticollagen antibodies. Serum levels of anticollagen total IgG, IgG1, and IgG2c were assessed by ELISA (25). A standard curve was generated for each assay by including serial dilutions of a reference sample of pooled sera from arthritic C57BL/6 mice. The lowest dilution of the reference sample was assigned an antibody concentration of 100 arbitrary units (AU), and antibody concentrations for each sample were obtained by reference to the standard curve. Gene expression analysis. RNA was extracted from mouse paws using TRI Reagent (Sigma-Aldrich). Complementary DNA (cDNA) for real-time quantitative PCR (qPCR) was generated from 1 g total RNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) in a 10 l final reaction volume. The quantitative PCRs were performed in triplicate using 3 l/well of 2 serial dilutions (1:50 and 1:500) of each cDNA, 0.3 M of each primer, and 1⫻ FluoCycle SYBR Green Mix for qPCR (Genycell-EuroClone) in 8 l, using MicroAmp Optical 384-well plates (Applied Biosystems). PCRs were carried out in an ABI Prism 7900HT system (Applied Biosystems), and the results were analyzed using the comparative Ct method (⌬⌬Ct) using SDS software version 2.2. The constitutively expressed gene 18S RNA was used as an internal control. The following primer sequences were used: for p38␣, forward 5⬘-AACCAGACAGTGGATATTTGGTC and reverse 5⬘-TGAGCTTCAACTGATCAATATGGT; for p38, forward 5⬘-GTCCTGAAGTTCTGGCAAAGA and reverse 5⬘-CACTGATGAGGTCCTTCTGG; for p38␥, forward 5⬘-ACCT GATGAGTCTCTGGACGA and reverse 5⬘-CCAGATCAGTGCCCATGAAT; for p38␦, forward 5⬘-GGACCCTGAGGAGGAGACA and reverse 5⬘-GTTTGAGATCTCTTTGTAGATGTGTTG; for IL-6, forward 5⬘-GCTACCAAACTGGATATAATCAGG and reverse 5⬘-CAGGTAGCTATGGTACTCCAGAA; for IL-1  , forward 5⬘TTGACGGACCCCAAAAGAT and reverse 5⬘-GAAGCTGGATGCTCTCATCTG; for TNF␣, forward 5⬘-CTGTAGCCCACGTCGTAGC and reverse 5⬘-TTGAGATCCATGCCGTTG; for IFN␥, forward 5⬘-GCAACAGCAAGGCGAAAAAG and reverse 5⬘-TTTCTG GCTGTTACTGCCACG; for IL-17, forward 5⬘-CAGGGAGAGCTTCATCTGTGT and reverse 5⬘-GCTGAGCTTTGAGGGATGAT; and for 18S, forward 5⬘-CGCGGTTCTATTTTGTTGGT and reverse 5⬘-AGTCGGCATCGTTTATGGTC. Analysis of collagen-specific T cell responses. Singlecell suspensions from mouse inguinal LNs were cultured with 50 g/ml of CII or 100 ng/ml of anti-CD3 (clone 145-2C11; BioLegend). Culture supernatants were collected after 48 hours, and cytokine production was quantified by ELISA. The proliferative response to CII was determined by water-soluble tetrazolium 1 reagent (Roche) incorporation in cultured cells after 72 hours. Intracellular cytokine staining and flow cytometry. Single-cell suspensions from mouse LNs were stimulated for 4 hours (at 37°C in 5% CO2) with 20 ng/ml of phorbol myristate acetate (PMA) and 1 g/ml of ionomycin in the presence of 10 g/ml brefeldin A (all from Sigma-Aldrich), and stained with PerCP-conjugated anti-CD4 (clone RM4-5; BD Biosciences). Cells were permeabilized with 0.5% saponin and stained with fluorescein isothiocyanate (FITC)– conjugated anti-IFN␥ (clone XMG1.2; BD Biosciences), phy-
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coerythrin (PE)–conjugated anti–IL-17 (clone TC11-18H10; Miltenyi Biotec), FITC-conjugated IL-10 (clone JES5-16E3), and PE-conjugated IL-4 (clone 11B11; both from BD Biosciences). To detect Treg cells, mouse LN cells were stained with peridinin chlorophyll A protein (PerCP)–conjugated antiCD4, fixed, permeabilized, and stained with PE-conjugated anti-FoxP3 (clone MF-23; BD Biosciences). For flow cytometric analysis, we used a FACSCalibur instrument and CellQuest Pro software (BD Biosciences). Statistical analysis. Data are expressed as the mean ⫾ SD. The area under the curve was used for analysis of the difference in clinical scores. Histology scores, qPCR, and protein quantification results were analyzed by Mann-Whitney U test using GraphPad Prism software. P values less than 0.05 were considered significant.
RESULTS Resistance to CIA in p38␥/␦ⴚ/ⴚ mice. To determine p38␥ and p38␦ involvement in arthritis, we assessed whether lack of p38␥ or p38␦ altered the course of CIA. CIA incidence and onset time in wild-type mice were comparable to those for C57BL/6 mice (⬃50%, 3–5 weeks after immunization) (26), whereas the percentage of animals with arthritis was lower for both p38␥⫺/⫺ mice and p38␦⫺/⫺ mice than for wild-type mice (Figure 1A). Arthritis severity was nonetheless comparable in all 3 groups throughout the course of disease (Figure 1B). Since we previously found that p38␥ and p38␦ have largely redundant functions (22,23), we studied the effect of combined p38␥/p38␦ deletion in the CIA model. The p38␥/␦-null mice showed lower disease incidence and a decrease in clinical score compared to wild-type mice (area under the curve 95.5 ⫾ 27.8 for wild-type versus 46.5 ⫾ 4.0 for p38␥/␦⫺/⫺ mice) (Figures 1A and B). We measured the extent of inflammatory infiltration and bone erosion in wild-type and p38␥/␦⫺/⫺ mice at the end of the experiment (day 42 after immunization) and found that arthritic p38␥/␦⫺/⫺ mice had significantly lower levels of inflammatory infiltrate and bone erosion than wild-type mice (Figure 1C). These data show that the compound p38␥/p38␦ deficiency protected mice against CIA development. Expression and activation of p38 MAPK and ERK-1/2 in CIA. Since it has been shown that all p38 MAPK isoforms are expressed in mouse fibroblast-like synoviocytes and in synovial tissue from RA patients (15,19,20), we then investigated whether or not p38␥ and p38␦ expression and activation vary in the mouse joint after induction of CIA. Levels of protein and messenger RNA (mRNA) for p38␥ and p38␦ were measured in
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Figure 1. Attenuation of collagen-induced arthritis by deletion of p38␥ and p38␦. A, Arthritis incidence in wild-type (WT) mice, p38␥⫺/⫺ mice, p38␦⫺/⫺ mice, and p38␥/␦⫺/⫺ mice after immunization with type II collagen. B, Clinical scores in wild-type mice, p38␥⫺/⫺ mice, p38␦⫺/⫺ mice, and p38␥/␦⫺/⫺ mice after arthritis onset. The wild-type and p38␥/␦⫺/⫺ groups were compared using the area under the curve method, which resulted in values of 64.24 for wild-type mice and 23.25 for p38␥/␦⫺/⫺ mice. Values are the mean ⫾ SD (n ⫽ 8–12 mice per group). C, Representative hematoxylin and eosin–stained sections of ankle joints from wild-type mice and p38␥/␦⫺/⫺ mice after CIA induction, and quantification of damage in the joints. Values are the mean ⫾ SD (n ⫽ 10 mice per group). ⴱⴱ ⫽ P ⬍ 0.01 by Mann-Whitney test.
joint extracts from wild-type mice and p38␥/␦⫺/⫺ mice that were left untreated, that were immunized with CII and did not develop arthritis, and that were immunized with CII and developed arthritis. The mRNA levels were assessed by qPCR, and the protein levels were assessed by Western blotting, using antibodies specific for each p38 MAPK isoform. The p38␥/␦⫺/⫺ mice did not express p38␥ or p38␦, and we observed no notable differences in p38␥ or p38␦ gene or protein expression in the joints of untreated or treated wild-type mice (Figures 2A and B). Assessment of p38␣ and p38 mRNA showed no differences in p38␣ gene expression between wild-type and p38␥/␦⫺/⫺ mice, whereas p38 mRNA levels decreased
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Figure 2. Expression and activation of p38␥ and p38␦ by collagen immunization. Wild-type (WT) or p38␥/␦⫺/⫺ mice were left untreated or were immunized with type II collagen (CII). At 6 weeks postimmunization, joint extracts were prepared for mRNA and protein analyses. A, Quantitative polymerase chain reaction of p38 MAPK mRNAs in total RNA from the joints of wild-type and p38␥/␦⫺/⫺ mice that were either nonimmunized, immunized and did not develop collageninduced arthritis (CIA⫺), or immunized and did develop CIA (CIA⫹). Values are the mean ⫾ SD from 1 representative experiment of at least 3 (performed in triplicate) with similar results. ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. B, Immunoblotting of joint extracts (50 g) from wild-type and p38␥/␦⫺/⫺ mice with antibodies to active phosphorylated p38 (p-p38␣, p-p38␥), phosphorylated ERK-1/2 (p–ERK-1/2), and total p38␣, p38␥, p38␦, and ERK-1/2. Duplicate lanes are shown; results were similar in 3 independent experiments. C, Quantification of the bands from the immunoblots shown in B, using an Odyssey infrared imaging system. Quantification is represented as p-p38␣ density/p38␣, p-p38␥ density/p38␥, and p–ERK-1/2 density/ERK-1/2 total density. Values are the mean ⫾ SD from 3 experiments performed in duplicate. ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. D, Immunoprecipitation of endogenous p38␥ and p38␦ from wild-type joint extracts (2 mg). Pellets were immunoblotted with anti–p-p38, anti-p38␥, or anti-p38␦ antibodies. Blots are representative of 3 independent experiments. Total extracts were used as a control. NS ⫽ not significant.
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in wild-type mice and increased in p38␥/␦⫺/⫺ mice after CII immunization (Figure 2A). We assessed p38 activation in joint extracts from wild-type and p38␥/␦⫺/⫺ mice. Both p38␣ and p38␥ were activated in wild-type joints, and p38␣ was activated in p38␥/␦⫺/⫺ mice to the same extent as in wild-type joints (Figures 2B and C). We observed no active p38␥ in p38␥/␦⫺/⫺ mouse joint extracts, and we detected no active p38␦ under any experimental conditions (Figure 2D). We also evaluated ERK-1/2 activation in joint extracts. ERK-1/2 was activated after immunization, with no differences between wild-type and p38␥/␦⫺/⫺ mice (Figures 2B and C). These data show that p38␥ and p38␦ do not control either p38␣ or ERK-1/2 activation in CIA, and that p38␥ is activated in the joints of CIItreated wild-type mice. Cytokine expression in arthritic p38␥/␦ⴚ/ⴚ mice. IL-1, TNF␣, and IL-6 are key cytokines in CIA pathogenesis. We therefore evaluated serum levels of these cytokines in p38␥/␦⫺/⫺ and wild-type mice after CIA induction. Serum TNF␣ and IL-1 levels were markedly lower in p38␥/␦⫺/⫺ mice, whereas IL-6 levels were unaffected (Figure 3A). In contrast, TNF␣, IL-1, and IL-6 gene expression in the arthritic joint were similar in p38␥/␦⫺/⫺ and wild-type mice (Figure 3B). IL-17 and IFN␥ gene expression were markedly lower in joints from p38␥/␦⫺/⫺ mice compared with those from wildtype mice (Figure 3B). These results confirm the need for p38␥ and p38␦ for cytokine production in the in vivo inflammatory response, and suggest that p38␥/␦ deficiency limits arthritis progression and severity by reducing systemic production of TNF␣ and IL-1. Anticollagen antibody levels in arthritic p38␥/␦ⴚ/ⴚ mice. CIA pathogenesis is characterized by the generation of anti-CII antibodies, and the IgG2 subclass binds complement effectively and forms anticollagen immune complexes in the synovium (27). To determine whether the reduced severity and incidence of arthritis in p38␥/␦⫺/⫺ mice was due to an impaired antibody response to CII, we measured serum anti-CII–specific total IgG, IgG1, and IgG2c levels by ELISA at 3 and 6 weeks postimmunization, to assess temporal differences between genotypes (Figure 4). Levels of all anticollagen antibodies were similar in wild-type and p38␥/␦-deficient mice at 3 weeks postimmunization. At 6 weeks, serum concentrations of anti-CII total IgG and IgG1 were similar in wild-type and in p38␥/␦⫺/⫺ mice, although disease severity was lower in the knockout mice. In contrast, IgG2c levels were significantly lower in p38␥/␦⫺/⫺ than in wild-type mice. These results
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Figure 3. Altered cytokine production in p38␥/␦-deficient mice in response to type II collagen immunization. A, Serum levels of tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), and IL-6 in wildtype (WT) and p38␥/␦⫺/⫺ mice. Serum from wild-type and p38␥/␦⫺/⫺ mice was collected 42 days after immunization, and cytokine levels were measured by multiplex cytokine assay. Values are the mean ⫾ SD (n ⫽ 6 mice per group). B, Expression of mRNA for TNF␣, IL-1, IL-6, IL-17, and interferon-␥ (IFN␥) in joint extracts from wild-type and p38␥/␦⫺/⫺ arthritic mice. Joint extracts were prepared 42 days postimmunization and quantitative polymerase chain reaction was performed on total RNA. Results were normalized to 18S RNA expression, and x-fold induction was calculated relative to mRNA expression in extracts from untreated wild-type mice. Values are the mean ⫾ SD from 1 representative experiment of 3 with similar results (n ⫽ 5–7 mice per group). ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. CIA ⫽ collagen-induced arthritis; NS ⫽ not significant.
suggest a role for p38␥/␦⫺/⫺ in regulating B cell responses to collagen. T cell response in CIA p38␥/␦ⴚ/ⴚ mice. Induction and propagation of CIA requires functional B and T cells. The lower IL-17 and IFN␥ levels in p38␥/␦⫺/⫺ mouse paws (Figure 3B) suggest that T cell responses to CII are altered in these mice. To evaluate whether p38␥ and p38␦ regulate CII-specific T cell responses in CIA, we collected inguinal LN cells at the end of the experi-
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a significant reduction in Th17 cells in p38␥/␦⫺/⫺ mice (Figure 5C). The frequencies of Th2 cells (defined as
Figure 4. Antibody response to type II collagen (CII) in p38␥/␦deficient mice. Serum levels of total IgG, IgG1, and IgG2c anti-CII antibodies in wild-type (WT) and p38␥/␦⫺/⫺ mice were quantified by enzyme-linked immunosorbent assay at 3 weeks (A) and 6 weeks (B) after collagen immunization. Values are the mean ⫾ SD (n ⫽ 10 mice per group). ⴱ ⫽ P ⱕ 0.05. NS ⫽ not significant.
ments (6 weeks postimmunization) and cultured them in vitro with CII. Compared to LNs from wild-type mice, LNs from p38␥/␦⫺/⫺ mice showed significantly less proliferation in response to CII or anti-CD3 (Figure 5A). Analysis of culture supernatant cytokine production showed that IL-17 and IFN␥ production were significantly reduced in response to CII and anti-CD3 in p38␥/␦⫺/⫺ mice (Figure 5B). These data suggest that p38␥ and p38␦ regulate Th1 and Th17 cell generation in vivo during CIA. We tested this by analyzing Th1 (CD4⫹IFN␥⫹) and Th17 (CD4⫹IL-17⫹) cell frequency in wild-type and p38␥/␦⫺/⫺ mouse LNs at the end of CIA experiments. Flow cytometric analysis showed a trend toward reduced Th1 cell frequency and
Figure 5. Reduced proliferation and cytokine production in response to type II collagen (CII) in lymph nodes from p38␥/␦-deficient mice. Lymph node cells from arthritic wild-type (WT) or p38␥/␦⫺/⫺ mice were isolated and were left unstimulated or were stimulated with 50 g/ml of CII or 100 ng/ml of anti-CD3 antibody. A, Proliferation in cultured cells harvested after 72 hours. B, Levels of tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), IL-17, and interferon-␥ (IFN␥) in culture supernatants collected after 48 hours. Cytokine production was quantified by enzyme-linked immunosorbent assay and multiplex cytokine assays. In A and B, values are the mean ⫾ SEM (n ⫽ 9–10 mice per group). ⴱ ⫽ P ⱕ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. C, Reduced frequency of IL-17–producing cells in lymph nodes from p38␥/␦deficient mice. Single-cell suspensions of lymph node cells were assessed for cytokine-producing and Treg cells (see Materials and Methods). Representative dot plots of CD4⫹-gated cells, quantification of cytokine-producing cells, and quantification of FoxP3 and FoxP3:IL-17 ratio are shown. Values are the mean ⫾ SEM (n ⫽ 10 mice per group). ⴱⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant.
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Figure 6. No decrease in the incidence or severity of collagen-induced arthritis in wild-type (WT) chimeric mice with p38␥/␦⫺/⫺ bone marrow. A, Arthritis incidence in the indicated groups 42 days after immunization with type II collagen. B, Clinical score 8 days after arthritis onset. C, Serum levels of total IgG1 and IgG2c anti-CII antibodies 6 weeks after collagen immunization, as quantified by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD from 3 independent experiments (n ⫽ 24–30 mice per group). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant.
CD4⫹IL4⫹/IL10⫹) and Treg cells (CD4⫹FoxP3⫹) were nonetheless similar in wild-type mice and p38␥/␦⫺/⫺ mice, which led to an increased Treg:Th17 ratio in LNs from p38␥/␦⫺/⫺ mice (Figure 5C) and prevented the development of CIA. CIA in p38␥/␦ bone marrow chimeras. Although the above data indicate that B and T cell responses are reduced in p38␥/␦⫺/⫺ mice during CIA, nonhematopoietic cells that can play a role in arthritis development also express p38␥ and p38␦. To assess the contribution of p38␥ and p38␦ in nonhematopoietic cells to arthritis progression in CIA, we generated wild-type and p38␥/␦ chimeric mice with adoptive bone marrow transplantation. After a 6-week reconstitution phase to achieve chimerism, mice were immunized with chicken CII (Figure 6). Bone marrow chimerism did not affect the phenotype outcome, and p38␥/␦⫺/⫺ mice that received either p38␥/␦⫺/⫺ (p38␥/␦⫺/⫺3 p38␥/␦⫺/⫺) or wild-type (WT3p38␥/␦⫺/⫺) bone marrow grafts showed absence of clinical arthritis, while wild-type mice that received wild-type (WT3 WT) or p38␥/␦⫺/⫺ (p38␥/␦⫺/⫺3 WT)
bone marrow grafts had comparable incidence and clinical scores (Figures 6A and B). Likewise, IgG2c levels were reduced in p38␥/␦⫺/⫺ mice, regardless of the origin of bone marrow cells (Figure 6C). This suggests that p38␥/␦ expression in the nonhematopoietic compartment drives arthritis development and controls the activation of B and T cells in CIA. DISCUSSION We used mice lacking p38␥ and/or p38␦ to analyze the effect of these isoforms on the development of CIA. Studies using p38 MAPK–specific inhibitors and MKK-3–deficient or MKK-6–deficient mice suggested that p38␣ and/or p38 are involved in this process. However, results from knockout mice showed p38 to be completely dispensable for arthritis development (28). Full p38␣ knockout is fatal to the embryo (29–32). Nonetheless, in an arthritis model induced by TNF␣ overexpression, p38␣ deletion using inducible Mx-Cre causes a decrease in paw swelling as well as in joint
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inflammation and bone damage (19). In contrast, in a passive K/BxN arthritis model, chronic arthritis severity is increased in mice with specific p38␣ deletion in macrophages (33). In this study, we demonstrated the role of p38␥ and p38␦ as essential components in arthritis pathogenesis. Combined deletion of p38␥ and p38␦ led to a marked reduction in disease incidence and severity in the CIA model. The results were confirmed by histologic evaluation. Lack of p38␥ or p38␦ alone had a partial effect on CIA incidence, indicating that p38␥ and p38␦ have largely redundant functions. The protective effect of p38␥/p38␦ deficiency in arthritis was similar to that observed in mice lacking MKK-3 and MKK-6, which lie upstream in this pathway (15–18). In addition, serum TNF␣ and IL-1 levels, but not IL-6 levels, were considerably reduced in p38␥/␦⫺/⫺ mice compared with CII-immunized wild-type mice. Similar reductions in serum cytokine levels are also observed in LPS/Dgalactosamine treated p38␥/␦⫺/⫺ mice compared with wild-type mice (22). In situ analysis showed increased cytokine mRNA levels in arthritic wild-type and p38␥/ ␦⫺/⫺ mouse paw extracts compared to healthy mice. In the case of IL-17 and IFN␥, we observed a significant decrease in mRNA levels in arthritic p38␥/␦⫺/⫺mice compared to arthritic wild-type mice. Our data suggest that the function of p38␥ and p38␦ in CIA progression is effected mainly via proinflammatory cytokines such as TNF␣, IL-1, IL-17, and IFN␥. We also found differences in the anticollagen antibody response between wild-type and p38␥/␦⫺/⫺ mice, suggesting that the reduced disease incidence and severity in p38␥/␦⫺/⫺ mice compared with wild-type mice are partially attributable to an impaired immune response. We found that activation and proliferation defects in p38␥/␦⫺/⫺ mouse B cells affect in vivo antibody responses. The p38␥/␦⫺/⫺ mice had significantly lower IgG2 levels than wild-type mice and showed defective class-switching in response to immunization with a T cell–dependent antigen (Risco A and Cuenda A: unpublished observations). Lack of both p38␥ and p38␦ nonetheless did not affect B cell ability to class-switch to IgG in vitro, indicating that the reduced in vivo antibody response to T cell–dependent antigen is not B cell autonomous and is probably due to intrinsic non–B cell factors such as cell context (Risco A and Cuenda A: unpublished observations). Impaired cytokine production might be implicated in the defective T cell– dependent antibody response. Our in vitro assays of T cell activity showed a decreased proliferative response to CII in p38␥/␦⫺/⫺ cells compared with those from wild-
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type mice. Collagen-induced TNF␣, IFN␥, and IL-17 production in LNs were significantly lower in the culture supernatants of p38␥/␦⫺/⫺ mouse T cells, consistent with our findings in serum (for TNF␣) and arthritic joints (for IFN␥ and IL-17). Moreover, p38␥/␦⫺/⫺ mice showed a lower Th17 cell frequency and a greater Treg:Th17 ratio than wild-type mice, both of which are linked to successful therapy in RA (34). The effects of p38␥ and p38␦ deficiency on IL-17 production are especially interesting, in light of clinical trials that target this cytokine in RA and other inflammatory conditions (35). IL-17 is a critical regulator of joint inflammation and bone erosion (36). Deficiency in this cytokine or its receptor suppresses inflammation and joint damage in CIA, and IL-17 injection into normal mouse ankles induces synovitis and cartilage destruction (35,37). Decreased serum production of anticollagen IgG2c in p38␥/␦⫺/⫺ mice might also be IL-17 dependent, since this cytokine mediates antibody class-switching to IgG2a/c (38). The p38 isoform is also highly active in the joints of patients with RA and in mouse models of inflammatory arthritis (39,40). We examined p38 MAPK in CIA and found that both p38␣ and p38␥ are activated in wild-type mice, whereas no p38␦ phosphorylation was detected under our experimental conditions. This phosphorylation pattern coincides with that reported for synovial tissue from RA patients, in which p38␣ and p38␥, but not p38␦, are phosphorylated (20). These results suggest that p38␥ is the p38 MAPK isoform with a central role in arthritis development. Nonetheless, deletion of both p38␥ and p38␦ isoforms is necessary to see a significant difference in arthritis score, which indicates the importance of p38␦ in this model. Our data imply that p38␦ compensates for the lack of p38␥. Using cells that lack several family members in combination with specific kinase inhibitors, we previously demonstrated functional compensation by these closely related protein kinases when one member is not expressed. Thus, p38␥ substrates can be phosphorylated by p38␦ in p38␥⫺/⫺ cells (22,23). Alternatively, p38␦ might have a role in CIA at a location (tissue or cells) other than joints. We cannot rule out the possibility that our experimental tools are insufficiently sensitive to detect its phosphorylation under these experimental conditions, since p38␦ expression in joints is very low compared to other mouse tissues (results not shown). The p38␥ and p38␦ isoforms are expressed by several cell types involved in CIA progression, yet the contribution of p38␥ and p38␦ expressed by specific hematopoietic and nonhematopoietic cell types remains
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to be determined. Bone marrow transfer experiments indicated that p38␥ and p38␦ expression by nonhematopoietic stromal cells drives CIA progression, but is not necessary in the hematopoietic compartment. This observation is unexpected, given that p38␥/␦⫺/⫺ mice have reduced IL-17– and IFN␥-producing T cells. It is nonetheless supported by a study showing that synovial stromal cells induce increased Th17 cell expansion in the human system (41), and is also consistent with the view that local synovial inflammatory cells are responsible for the perpetuation of autoimmune inflammation (42,43). Our results indicate that p38␥ and p38␦ isoforms likely play roles in both hematopoietic and nonhematopoietic compartments. Further studies are needed to determine whether these phenomena are dependent on p38␥ and p38␦ expression by synovial cells. In conclusion, our data show that p38␥ and p38␦ regulate the immune response in CIA and suppress clinical disease and bone destruction. The crucial role of p38␥/␦ in synovial inflammation, bone erosion, and TNF␣, IL-1, IFN␥, and IL-17 production suggests that it could serve as a target of therapy in RA as an alternative to traditional p38␣ inhibitors, which have proven minimally effective in human disease (44). ACKNOWLEDGMENTS We thank the antibody purification teams (Division of Signal Transduction Therapy, University of Dundee, Dundee, UK) coordinated by H. McLauchlan and J. Hastie for generation and purification of antibodies, A. C. Gonzalo for technical support, and C. Mark for editorial assistance. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cuenda had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Criado, Cuenda. Acquisition of data. Criado, Risco, Alsina-Beauchamp, Pe´rez-Lorenzo, Esco ´s, Cuenda. Analysis and interpretation of data. Criado, Risco, Alsina-Beauchamp, Cuenda.
REFERENCES 1. Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–61. 2. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008;118:3537–45. 3. Cohen P. Targeting protein kinases for the development of anti-inflammatory drugs. Curr Opin Cell Biol 2009;21:317–24. 4. Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, function and role in human diseases. Biochim Biophys Acta 2007;1773:1358–75.
5. Gaestel M, Kotlyarov A, Kracht M. Targeting innate immunity protein kinase signalling in inflammation. Nat Rev Drug Discov 2009;8:480–99. 6. Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Teran B, Sabio G, Davis RJ, et al. Differential activation of p38MAPK isoforms by MKK6 and MKK3. Cell Signal 2010;22:660–7. 7. Rincon M, Davis RJ. Regulation of the immune response by stress-activated protein kinases. Immunol Rev 2009;228:212–24. 8. Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA. p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med 2009;15:369–79. 9. Nishikawa M, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogenactivated protein kinase inhibitor FR167653. Arthritis Rheum 2003;48:2670–81. 10. Wada Y, Nakajima-Yamada T, Yamada K, Tsuchida J, Yasumoto T, Shimozato T, et al. R-130823, a novel inhibitor of p38 MAPK, ameliorates hyperalgesia and swelling in arthritis models. Eur J Pharmacol 2005;506:285–95. 11. Hill RJ, Dabbagh K, Phippard D, Li C, Suttmann RT, Welch M, et al. Pamapimod, a novel p38 mitogen-activated protein kinase inhibitor: preclinical analysis of efficacy and selectivity. J Pharmacol Exp Ther 2008;327:610–9. 12. Genovese MC. Inhibition of p38: has the fat lady sung? [editorial]. Arthritis Rheum 2009;60:317–20. 13. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408:297–315. 14. Kuma Y, Sabio G, Bain J, Shpiro N, Marquez R, Cuenda A. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo. J Biol Chem 2005;280:19472–9. 15. Yoshizawa T, Hammaker D, Boyle DL, Corr M, Flavell R, Davis R, et al. Role of MAPK kinase 6 in arthritis: distinct mechanism of action in inflammation and cytokine expression. J Immunol 2009; 183:1360–7. 16. Hammaker D, Topolewski K, Edgar M, Yoshizawa T, Fukushima A, Boyle DL, et al. Decreased collagen-induced arthritis severity and adaptive immunity in MKK-6–deficient mice. Arthritis Rheum 2012;64:678–87. 17. Inoue T, Boyle DL, Corr M, Hammaker D, Davis RJ, Flavell RA, et al. Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis. Proc Natl Acad Sci U S A 2006;103:5484–9. 18. Hegen M, Gaestel M, Nickerson-Nutter CL, Lin LL, Telliez JB. MAPKAP kinase 2-deficient mice are resistant to collageninduced arthritis. J Immunol 2006;177:1913–7. 19. Bohm C, Hayer S, Kilian A, Zaiss MM, Finger S, Hess A, et al. The ␣-isoform of p38 MAPK specifically regulates arthritic bone loss. J Immunol 2009;183:5938–47. 20. Korb A, Tohidast-Akrad M, Cetin E, Axmann R, Smolen J, Schett G. Differential tissue expression and activation of p38 MAPK ␣, , ␥, and ␦ isoforms in rheumatoid arthritis. Arthritis Rheum 2006; 54:2745–56. 21. Ittner A, Block H, Reichel CA, Varjosalo M, Gehart H, Sumara G, et al. Regulation of PTEN activity by p38␦-PKD1 signaling in neutrophils confers inflammatory responses in the lung. J Exp Med 2012;209:2229–46. 22. Risco A, del Fresno C, Mambol A, Alsina-Beauchamp D, MacKenzie KF, Yang HT, et al. p38␥ and p38␦ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation. Proc Natl Acad Sci U S A 2012;109:11200–5. 23. Sabio G, Arthur JS, Kuma Y, Peggie M, Carr J, Murray-Tait V, et al. p38␥ regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP. EMBO J 2005;24:1134–45. 24. Cuenda A, Cohen P, Buee-Scherrer V, Goedert M. Activation of
p38␥ AND p38␦ IN ARTHRITIS
25.
26. 27.
28. 29. 30.
31. 32.
33.
34.
stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6): comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J 1997;16: 295–305. Inglis JJ, Criado G, Medghalchi M, Andrews M, Sandison A, Feldmann M, et al. Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen. Arthritis Res Ther 2007;9:R113. Campbell IK, Hamilton JA, Wicks IP. Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur J Immunol 2000;30:1568–75. Wooley PH, Luthra HS, Stuart JM, David CS. Type II collageninduced arthritis in mice. I. Major histocompatibility complex (I region) linkage and antibody correlates. J Exp Med 1981;154: 688–700. Beardmore VA, Hinton HJ, Eftychi C, Apostolaki M, Armaka M, Darragh J, et al. Generation and characterization of p38 (MAPK11) gene-targeted mice. Mol Cell Biol 2005;25:10454–64. Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, et al. Essential role of p38␣ MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 2000;6:109–16. Mudgett JS, Ding J, Guh-Siesel L, Chartrain NA, Yang L, Gopal S, et al. Essential role for p38␣ mitogen-activated protein kinase in placental angiogenesis. Proc Natl Acad Sci U S A 2000;97: 10454–9. Tamura K, Sudo T, Senftleben U, Dadak AM, Johnson R, Karin M. Requirement for p38␣ in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 2000;102:221–31. Allen M, Svensson L, Roach M, Hambor J, McNeish J, Gabel CA. Deficiency of the stress kinase p38␣ results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells. J Exp Med 2000;191: 859–70. Guma M, Hammaker D, Topolewski K, Corr M, Boyle DL, Karin M, et al. Antiinflammatory functions of p38 in mouse models of rheumatoid arthritis: advantages of targeting upstream kinases MKK-3 or MKK-6. Arthritis Rheum 2012;64:2887–95. Samson M, Audia S, Janikashvili N, Ciudad M, Trad M, Fraszczak J, et al. Inhibition of interleukin-6 function corrects Th17/Treg cell
1217
35.
36. 37.
38.
39.
40.
41.
42.
43. 44.
imbalance in patients with rheumatoid arthritis. Arthritis Rheum 2012;64:2499–503. Chabaud M, Lubberts E, Joosten L, van Den Berg W, Miossec P. IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis. Arthritis Res 2001;3: 168–77. Peck A, Mellins ED. Breaking old paradigms: Th17 cells in autoimmune arthritis. Clin Immunol 2009;132:295–304. Lubberts E, Joosten LA, van de Loo FA, Schwarzenberger P, Kolls J, van den Berg WB. Overexpression of IL-17 in the knee joint of collagen type II immunized mice promotes collagen arthritis and aggravates joint destruction. Inflamm Res 2002;51:102–4. Mitsdoerffer M, Lee Y, Jager A, Kim HJ, Korn T, Kolls JK, et al. Proinflammatory T helper type 17 cells are effective B-cell helpers. Proc Natl Acad Sci U S A 2010;107:14292–7. Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal– regulated kinase, c-JUN N-terminal kinase, and p38 mitogenactivated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12. Zwerina J, Hayer S, Redlich K, Bobacz K, Kollias G, Smolen JS, et al. Activation of p38 MAPK is a key step in tumor necrosis factor–mediated inflammatory bone destruction. Arthritis Rheum 2006;54:463–72. Eljaafari A, Tartelin ML, Aissaoui H, Chevrel G, Osta B, Lavocat F, et al. Bone marrow–derived and synovium-derived mesenchymal cells promote Th17 cell expansion and activation through caspase 1 activation: contribution to the chronicity of rheumatoid arthritis. Arthritis Rheum 2012;64:2147–57. Armaka M, Apostolaki M, Jacques P, Kontoyiannis DL, Elewaut D, Kollias G. Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J Exp Med 2008;205:331–7. Pablos JL, Canete JD. Immunopathology of rheumatoid arthritis. Curr Top Med Chem 2013;13:705–11. Clark AR, Dean JL. The p38 MAPK pathway in rheumatoid arthritis: a sideways look. Open Rheumatol J 2012;6:209–19.
