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IL-37 Alleviates Rheumatoid Arthritis by Suppressing IL-17 and IL-17−Triggering Cytokine Production and Limiting Th17 Cell Proliferation Liang Ye, Bo Jiang, Jun Deng, Jing Du, Wen Xiong, Youfei Guan, Zhongyang Wen, Kunzhao Huang and Zhong Huang J Immunol 2015; 194:5110-5119; Prepublished online 27 April 2015; doi: 10.4049/jimmunol.1401810 http://www.jimmunol.org/content/194/11/5110

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http://www.jimmunol.org/content/suppl/2015/04/25/jimmunol.140181 0.DCSupplemental.html This article cites 30 articles, 4 of which you can access for free at: http://www.jimmunol.org/content/194/11/5110.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Supplementary Material

The Journal of Immunology

IL-37 Alleviates Rheumatoid Arthritis by Suppressing IL-17 and IL-17–Triggering Cytokine Production and Limiting Th17 Cell Proliferation Liang Ye,*,†,‡ Bo Jiang,x Jun Deng,{ Jing Du,‖ Wen Xiong,# Youfei Guan,** Zhongyang Wen,*,†,‡ Kunzhao Huang,*,†,‡ and Zhong Huang*,†,‡

R

heumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by the infiltration of activated immune cells and the production of inflammatory factors that lead to the formation of synovial hyperplasia and pannus, as well as the destruction of cartilage and joints (1, 2). Although the etiology and pathogenesis of RA are not fully understood, recent in vivo animal models and in vitro human studies demonstrated that IL-17 can be considered a decisive mediator in the pathogenesis of RA (3). IL-17 (also known as IL-17A) is the signature cytokine of the newly discovered CD4+ Th17 subsets that are known to have

*Institute of Biological Therapy, Shenzhen University, Shenzhen 518060, China; † Department of Pathogen Biology and Immunology, Shenzhen University School of Medicine, Shenzhen 518060, China; ‡Shenzhen City Shenzhen University Immunodiagnostic Technology Platform, Shenzhen 518060, China; xMaoming City People’s Hospital, Maoming 525000, Guangdong, China; {Department of Pathology, University of Hong Kong, Hong Kong 999077, China; ‖Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen 518036, China; #Shenzhen Blood Center, Shenzhen 518035, Guangdong, China; and **Shenzhen University Diabetes Center, Shenzhen University Health Science Center, Shenzhen 518060, China Received for publication July 16, 2014. Accepted for publication March 31, 2015. This work was supported by Grant 81273305 from the National Natural Science Foundation of China and by Grant JCYJ20140711144858545 from the Special Fund for Shenzhen Strategic Emerging Industry Development. Address correspondence and reprint requests to Dr. Zhong Huang, Institute of Biological Therapy, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060, Guangdong, China. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: Ad-EV, empty adenovirus; Ad–IL-37, adenovirus encoding human IL-37; anti-CCP, anti-cyclic citrullinated peptide Ab; CIA, collageninduced arthritis; CII, bovine type II collagen; CRP, C-reactive protein; DAS28, 28joint disease activity score; DC, dendritic cell; ESR, erythrocyte sedimentation rate; HC, healthy control; Ion, ionomycin; RA, rheumatoid arthritis; RF, rheumatoid factor; rh, recombinant human. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401810

potent proinflammatory functions (3, 4). Based on data from animal models of autoimmune arthritis, IL-17 is recognized as one of the important factors in the pathogenesis of RA (3). Recent studies revealed that the expression of IL-17 is increased in serum, synovial fluid, and synovium of patients with RA compared with healthy controls (HCs) (4). Moreover, IL-17–transgenic mice are prone to develop collagen-induced arthritis (CIA), whereas blocking of endogenous IL-17 with its receptor in CIA results in the suppression of arthritis and reduced joint destruction (4, 5). IL-37, a new member of the IL-1 family of cytokines, is produced by various types of cells, including PBMCs, epithelial cells, and macrophages (6). IL-37 has five splice variants: IL-37a to IL-37e (6). Only IL-37b and IL-37c contain exons 1 and 2 and express an N-terminal prodomain that includes a potential caspase-1 cleavage site (7). IL-37b is the largest isoform, has significant sequence similarity with IL-18, and is the most studied. The IL-37b precursor is cleaved by caspase-1 into mature IL-37b (6, 7). IL-37b has been identified as a natural suppressor of innate immunity (8). Recently, IL-37 was shown to be highly expressed in the serum and PBMCs of patients with systemic lupus erythematosus (9) and in the synovial tissues and plasma of patients with RA (8, 10). We recently revealed that recombinant human (rh)IL-37 suppresses the expression of proinflammatory cytokines IL-1b and IL-6 in PBMCs from patients with systemic lupus erythematosus in vitro (9). In vivo, mice transgenic for IL-37 exhibited markedly reduced production of IL-17 and other proinflammatory cytokines, including IL-1b and IL-6, following LPS stimulation (11). Other studies showed that IL-37 is strongly expressed by CD4+ T cells and macrophages in human psoriatic plaques, which may inhibit the excessive inflammation in the pathogenesis of psoriasis (12). Based on the data described above, it is postulated that IL-37, through the production of activated immune cells, may play a prominent role in the pathogenesis of RA by curbing IL-17 or other proinflammatory cytokines.

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IL-37, a new member of the IL-1 cytokine family, is a natural inhibitor of innate immunity associated with autoimmune diseases. This study was undertaken to evaluate whether IL-37 has antiarthritic effects in patients with rheumatoid arthritis (RA) and in mice with collagen-induced arthritis (CIA). In this study, we analyzed the expression of IL-37 in PBMCs, serum, and lymphocytes from RA patients as well as CD4+ T cells polarized under Th1/Th2/Th17 conditions. The role of IL-37 was assessed by investigating the effects of recombinant human (rh)IL-37 and an adenovirus encoding human IL-37 (Ad–IL-37) on Th17 cells and Th17related cytokines in RA patients and CIA mice. We found that active RA patients showed higher IL-37 levels compared with patients with inactive RA and healthy controls. Upregulated IL-37 expression also was found in CD3+ T cells and CD4+ T cells from RA patients and in Th1/Th17-differentiation conditions. rhIL-37 markedly decreased IL-17 expression and Th17 cell frequency in PBMCs and CD4+ T cells from RA patients. Furthermore, IL-37 exerted a more suppressive effect on Th17 cell proliferation, whereas it had little or no effect on Th17 cell differentiation. IL-17 and IL-17–driving cytokine production were significantly reduced in synovium and joint cells from CIA mice receiving injections of Ad–IL-37. Our findings indicate that IL-37 plays a potent immunosuppressive role in the pathogenesis of human RA and CIA models via the downregulation of IL-17 and IL-17–triggering cytokine production and the curbing of Th17 cell proliferation. The Journal of Immunology, 2015, 194: 5110–5119.

The Journal of Immunology In this study, we investigated the expression of IL-37 in patients with active and inactive RA. We also analyzed the expression pattern of IL-37 in cell populations isolated from the PBMCs of patients with RA, as well as CD4+ T cells under Th1/Th2/Th17differentiation conditions. The effect of IL-37 on the modulation of IL-17 and IL-17–driving cytokines (IL-1b and IL-6) in vitro and in vivo was examined in this study. We further analyzed the effect of IL-37 on Th17 cell differentiation and proliferation. To our knowledge, this is the first study to show the antiinflammatory properties of IL-37 in both human RA and the CIA model through the inhibition of IL-17 and IL-17–triggering cytokine production and the suppression of Th17 cell proliferation, which may contribute to its antiarthritic effects.

Materials and Methods Patients and HCs

Preparation of human PBMCs Peripheral blood samples were obtained from patients with RA and HCs. Serum samples were stored at 280˚C until cytokines were determined. PBMCs from patients and HCs were isolated using lymphocyte separation medium (TBD Science), according to the manufacturer’s instructions. The collected cells were used for cell cultures or stored at 280˚C until RNA extraction.

Cell sorting Human PBMCs were stained using human-specific Abs, including FITCconjugated anti-CD3, PE-conjugated anti-CD19, FITC-conjugated antiCD4, and PE-conjugated anti-CD8 (all from BD Biosciences), and CD3+ T cells, CD19+ T cells, CD4+ T cells, and CD8+ T cells, respectively, were sorted using a FACSAria II cell sorter (BD Biosciences).

T cell differentiation and proliferation in vitro For human T cell differentiation, CD4+ T cells purified by flow cytometry were cultured in RPMI 1640 medium (HyClone, Thermo Fisher Scientific), supplemented with 10% FCS (Hangzhou Sijiqing Biological Engineering Materials) and 100 IU/ml penicillin and 100 mg/ml streptomycin (Beyotime), in the presence of anti-CD3 (1 mg/ml) and anti-CD28 (1 mg/ml). The conditions for the different Th cell subsets are as follows: Th1 conditions: IL-12 (10 ng/ml), IL-2 (10 U/ml), and anti–IL-4 (10 mg/ml); Th2 conditions: IL-4 (20 ng/ml) and anti–IL-12 and anti–IFN-g (10 mg/ml); and Th17 conditions: TGF-b (1 ng/ml), IL-6 (20 ng/ml), IL-1b (10 ng/ml), and anti– IFN-g, anti–IL-4, and anti–IL-12 (10 mg/ml); (all from eBioscience). Cells were cultured for 5 d and then restimulated for 4 or 24 h with PMA (500 ng/ml) and ionomycin (Ion; 500 ng/ml), or cells were treated for 4 or 24 h with LPS (1 mg/ml). Cells and culture supernatants were analyzed by real-time PCR and ELISA, respectively, for cytokines IL-37, IFN-g, IL-17, and IL-4. For murine Th17 cell differentiation, naive CD4+CD62L+ T cells from spleens of DBA mice were purified using a CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec), and purity was routinely 95%. Purified naive T cells were cultured with anti-CD3– and anti-CD28–coated 24-well plates with TGF-b (1 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml), with or without rhIL-37 (0, 50, and 100 ng/ml), for 72 h. For the final 5 h of incubation, PMA (50 ng/ml), Ion (1 mg/ml), and monensin (10003) were added to each well and mixed thoroughly. IL-17 and RORgt were stained with anti-mouse IL-17-PE and anti-mouse RORgt-PE Abs, respectively (BD Biosciences), and cells were measured with a flow cytometer (BD Biosciences) and analyzed using FlowJo software. For Th17 cell proliferation, purified naive CD4+ T cells were labeled with CFSE and cultured for 3 d in the presence of rhIL-37 (0, 50, and 100

ng/ml) under Th17-polarizing conditions, as described above. Th17 proliferation was measured with a flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Cell culture and IL-37 treatment RPMI 1640 (HyClone, Thermo Fisher Scientific), supplemented with 10% FCS (Hangzhou Sijiqing Biological Engineering Materials) and 100 IU/ml penicillin and 100 mg/ml streptomycin (Beyotime), was used as culture medium. PBMCs and purified CD4+ T cells were cultured in RPMI 1640 medium in a humidified atmosphere of 5% CO2 at 37˚C. PBMCs from patients with RA were cultured at a concentration of 1.5 3 106 cells/ml in 24-well plates in the absence or presence of rhIL-37 (9) (100 ng/ml) for 24 h and then stimulated with LPS (1 mg/ml) for 6 h; culture supernatants were harvested and frozen at 280˚C for later cytokine analysis by ELISA.

Flow cytometric analysis Anti-human CD4-FITC and anti-human IL-17–PE were purchased from eBioscience. Anti-mouse CD4-allophycocyanin, anti-mouse IL-17–PE, and anti-mouse RORgt-PE were purchased from BD Biosciences. Human samples for intracellular staining of IL-17, PMA and Ion (500 ng/ml each), and brefeldin A (1 mg/ml) were added to cultures for the last for 12 h before flow cytometric analysis. Mouse samples for staining of IL-17 and RORgt, PMA (50 ng/ml), Ion (1 mg/ml), and monensin (10003) were added to cultures for the last 5 h before flow cytometric analysis, as previously described (16).

Construction of a recombinant adenovirus encoding human IL-37 Briefly, the human IL-37 gene (GenBank: AF167368) was amplified by PCR, using human PBMC cDNA as the template, and ligated to the pMd18T vector (Takara) for the construction of pMd–18T–IL-37 vector plasmid. Subsequently, double digestion with EcoRV and XhoI (both from Takara) was conducted on plasmid pMd–18T–IL-37 and shuttle vector pShuttle-IRES-hrGFP2 (Stratagene), respectively. Recovered products were connected with T4 DNA ligase (Takara), and the ligation mixture was transformed into DH5a bacteria (TransGen Biotech). Kana-resistant transformants were selected by plating the transformation mixture on Luria-Bertani agar plates supplemented with kanamycin (Sangon Biotech). After incubation overnight, the recombinant plasmid DNA was isolated and further confirmed via double digestion; the confirmed recombinant adenoviral vector was named pShuttle–IRES–hrGFP2–IL-37. Thereafter, pShuttle–IRES–hrGFP2–IL-37 was linearized by restriction endonuclease PmeI (Thermo Fisher Scientific) and subsequently cotransformed with adenoviral backbone vector pAdEasy-1 (Stratagene) in Escherichia coli BJ5183 cells (Stratagene) for homologous recombination. Then it was digested by PacI (Thermo Fisher Scientific) and transfected into HEK293 cells (Stratagene) with Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen), to generate an adenovirus encoding human IL-37 (Ad–IL-37). The empty adenovirus (Ad-EV) control used in this study was an empty pShuttle-IRES-hrGFP2 shuttle vector with no insert. Seven days later, green fluorescence could be seen under a fluorescence microscope. The concentration of the Ad–IL-37 was 3.5 3 109 PFU, as determined by plaque assay.

Adenovirus-mediated IL-37 gene expression HEK293 cells were infected with Ad–IL-37 (0, 105, or 107 PFU) for 72 h, and cells were collected. Subsequently, Ad–IL-37 was detected in cell lysates by probing for Flag, and equal loading was determined by b-actin. The infection efficiency of Ad-EV and Ad–IL-37 in HEK293 cells was observed through GFP reporter fluorescence. The hind joints of DBA/1 mice were injected intra-articularly with 1 3 107 PFU Ad–IL-37 or Ad-EV containing GFP reporter fluorescence. Seven days later, fluorescence was measured in vivo using the IVIS imaging system (Caliper Life Sciences), and IL-37 expression was examined in different tissues by real-time PCR.

CIA induction and Ad–IL-37 treatment DBA/1J mice were purchased from The Jackson Laboratory. Briefly, mice (age 8–12 wk) were immunized intradermally at the base of tail with 200 ml bovine type II collagen (CII; Chondrex) emulsified in CFA containing Mycobacterium tuberculosis (Chondrex). On day 21, these mice were given a boost immunization of 80 ml CII dissolved in CFA. Ad–IL-37 (107 PFU) or Ad-EV (107 PFU) was injected intra-articularly into the knee joints on days 17 and 23 after the first CII immunization. Clinical arthritis was evaluated as previously described (17).

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A total of 49 patients with RA, from Peking University Shenzhen Hospital, was included in the current study. The classification of RA fulfilled the 1987 and 2010 American College of Rheumatology criteria (13, 14). RA patients were divided into active (n = 26) and inactive (n = 23) groups, according to their 28-joint disease activity score (DAS28); active disease is defined as DAS28 $ 3.2, based on the European League Against Rheumatism diagnostic criteria (15). At the time of sample acquisition, 38 patients were being treated with prednisone, and 11 patients were not. HC peripheral blood samples were obtained from 33 healthy volunteers. RA patients and HCs did not exhibit significant differences in terms of mean age or male/ female distribution (Supplemental Table I). The study was approved by the Review Board for the Peking University Shenzhen Hospital. Informed consent was obtained from the recruited subjects.

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5112 Preparation of synovial tissues and joint cells and collection of synovial fluids Synovial tissues, joint cells, and synovial fluids were collected according to a previously described protocol (18). Synovial fluid samples were stored at 280˚C prior to assay. Joint cells were cultured onto 24-well plates in RPMI 1640 medium (Hyclone, Thermo Fisher Scientific) containing 10% FCS (Hangzhou Sijiqing Biological Engineering Materials) and incubated for 12 h in the presence of rhIL-37 (100 ng/ml).

RNA extraction and real-time PCR RNA samples were extracted by TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. cDNA was prepared using the iScript cDNA Synthesis Kit (Bio-Rad). The primer sequences are summarized in Supplemental Table II. Real-time PCR amplification reactions were prepared with the SYBR Green PCR Kit (Bio-Rad) and performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems). PCR products were verified by melting curve analysis. Relative expression levels of target genes were calculated by normalization to b-actin values using the 22ΔΔct method.

Human and mouse cytokine levels were measured by ELISA, according to the manufacturer’s instructions (eBioscience). Concentrations of CIIspecific IgG1 and IgG2a Abs (eBioscience) in the serum and synovial fluid of mice were measured by ELISA, as described previously (18).

Histopathologic analysis Mice were anesthetized and killed on day 32, and knee joints were removed for histopathologic examination. Tissue sections were prepared and stained with H&E. Histopathologic scoring of joint damage was performed under blinded conditions, according to a widely used scoring system for evaluating synovitis, cartilage degradation, and bone erosion (18).

Statistical analysis Data are expressed as mean (6 SEM) or median (range) and were analyzed using GraphPad Prism v5.00 software (GraphPad). A two-tailed Student t test was used for the statistical comparison of two groups. Where appropriate, two-way ANOVA, followed by the Bonferroni post test for multiple comparisons, and a Mann–Whitney U test for nonparametric data were used. The Spearman correlation test was used to assess the association between serum IL-37 levels and different variables. The p values # 0.05 were considered significant.

Results Increased IL-37 mRNA and serum protein levels in patients with active RA and their correlation with the inflammation response To investigate the potential role of IL-37 in patients with RA, 49 RA patients and 33 age- and gender-matched HCs were recruited (Supplemental Table I). Distinct differences in IL-37 mRNA and serum protein levels were present between active RA patients and inactive RA patients (Fig. 1A, 1B). Patients with active RA showed higher IL-37 mRNA (Fig. 1A) and serum IL-37 (Fig. 1B) protein levels than did HCs. However, we did not observe differences in IL-37 mRNA and protein levels between inactive RA patients and HCs (Fig. 1A, 1B). To observe the effect of prednisolone on IL-37 expression, RA patients were divided into treated and untreated groups. We found that prednisolone-treated RA patients showed lower IL-37 levels than did RA patients before treatment (Fig. 1C). However, there was no difference in the levels of IL-37 in RA patients not treated with prednisolone compared with RA patients before treatment (Fig. 1C). At the time of the highest inflammatory activity during the observation period, as determined by maximal C-reactive protein (CRP) and/or erythrocyte sedimentation rate (ESR) values, serum levels of IL37 correlated with CRP (Supplemental Fig. 1A, R = 0.4141, p = 0.0031) and ESR (Supplemental Fig. 1B, R = 0.3973, p = 0.0047). Similarly, there was a positive correlation between serum IL-37 levels and DAS28 (Supplemental Fig. 1C, R = 0.4172, p = 0.0029). No significant correlation was observed between serum

levels of IL-37 and rheumatoid factor (RF) or anti-cyclic citrullinated peptide Abs (anti-CCPs) (Supplemental Fig. 1D, 1E). IL-17 and IL-17–driving cytokines (IL-1b and IL-6) play an important role in the initiation and progression of CIA. We next assessed whether serum IL-37 levels correlated with these cytokines. As seen in Supplemental Fig. 1F–H, serum IL-37 concentrations correlated positively with the levels of serum IL-17 (R = 0.3388, p = 0.0173), IL-1b (R = 0.3554, p = 0.0122), and IL-6 (R = 0.3215, p = 0.0243) in RA patients. Upregulated IL-37 mRNA expression in peripheral CD3+ T cells and CD4+ T cells from patients with RA Considering the involvement of T and B cells in the pathogenesis of RA, we investigated the expression levels of IL-37 in T and B cells from PBMCs of RA patients and HCs. We found that IL-37 mRNA expression was significantly higher in CD3+ and CD4+ T cells from RA patients compared with HCs (Fig. 1D). In contrast, no change in IL-37 mRNA expression in CD8+ T cells or CD19+ B cells was observed between RA patients and HCs (Fig. 1D). Together, these results demonstrate that IL-37 expression in peripheral immune cells may be involved in the pathogenesis of RA. Increased IL-37 production in CD4+ T cells under Th1- and Th17-polarizing conditions To further investigate whether CD4+ Th cell (Th1, Th2, and Th17) differentiation regulates the expression of IL-37, CD4+ T cells from PBMCs of HCs were primed in vitro for 5 d under Th1-, Th2-, or Th17-polarizing conditions. Cells were then restimulated with PMA/Ion or LPS and examined for IL-37 expression using real-time PCR and ELISA. The results showed that both PMA/Ion and LPS clearly induced IL-37 and IFN-g mRNA expression in CD4+ T cells under Th1-polarizing conditions (Fig. 2A), consistent with elevated IL-37 and IL-17 mRNA levels in CD4+ T cells under Th17-polarizing conditions (Fig. 2B). Although IL-4 mRNA expression was highly increased in CD4+ T cells polarized under Th2 conditions, the expression of IL-37 mRNA in CD4+ T cells polarized under Th2 conditions was unaffected by treatment with PMA/Ion or LPS (Fig. 2C). Notably, IL-37 protein also was stably expressed in CD4+ T cells under Th1- and Th17-polarizing conditions via stimulation of PMA/Ion or LPS (Fig. 2D). Taken together, these results indicate that IL-37 is consistently elevated during CD4+ T cell activation, especially activated Th1 and Th17 cells. IL-37 suppresses proinflammatory cytokine expression in PBMCs from patients with RA To determine whether IL-37 inhibits proinflammatory cytokine production in RA, we expressed and purified rhIL-37 protein (9). Our results revealed that rhIL-37 downregulated proinflammatory cytokine expression in PBMCs from HCs in a dose-dependent manner (optimum concentration of rhIL-37 was 100 and 200 ng/ml) (data not shown). To further address the effects of IL-37 on proinflammatory cytokines in PBMCs from RA patients, PBMCs from 49 RA patients and 33 HCs were either left untreated or treated with rhIL-37 (100 ng/ml) for 6 or 24 h, and cells and culture supernatants were harvested for real-time PCR and ELISA analysis, respectively. We found that the transcriptional levels of proinflammatory cytokines IL-17 (Fig. 3A), IL-1b (Fig. 3B), and IL-6 (Fig. 3C) in PBMCs of RA patients were significantly suppressed by rhIL-37. rhIL-37 also markedly reduced the secretion of IL-17 (Fig. 3D), IL-1b (Fig. 3E), and IL-6 (Fig. 3F) in PBMCs of RA patients. However, the cytokine mRNA (Fig. 3A–C) and protein (data not shown) levels in PBMCs of HCs were not noticeably changed by treatment with rhIL-37.

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ELISA

IL-37 SUPPRESSES Th17 RESPONSE IN ARTHRITIS

The Journal of Immunology

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IL-37 inhibits IL-17 expression in CD4+ T cells from patients with RA and decreases RA Th17 cells To further investigate the effects of IL-37 on IL-17 expression from CD4+ T cells in patients with RA, CD4+ T cells from PBMCs of RA patients and HCs were cultured or not with rhIL-37. We demonstrated that the expression of IL-17 mRNA in CD4+ T cells from patients with RA was markedly higher than that in cells from HCs (Fig. 4A). More importantly, rhIL-37 significantly reduced the expression of IL-17 mRNA in CD4+ T cells from patients with RA, as determined by real-time PCR, but IL-17 mRNA expression in HCs was not inhibited by treatment with rhIL-37 (Fig. 4A). To further analyze the levels of IL-17–driving cytokines IL-1b and IL-6, we next examined the levels of these cytokines in CD4+ T cells of RA patients. Consistent with the above findings, IL-1b (Fig. 4B) and IL-6 (Fig. 4C) mRNA levels were significantly higher in CD4+ T cells of RA patients. Notably, rhIL-37 also significantly downregulated the expression of IL-1b (Fig. 4B) and IL-6 (Fig. 4C) mRNA in CD4+ T cells from patients with RA. We further observed that the mean percentage of Th17 cells was significantly reduced by treatment with rhIL-37 (4.2% without rhIL-37 versus 2.0% with rhIL-37) (Fig. 4D). However, IL-1b is not required for IL-37–mediated suppression of IL-17 expression in CD4+ T cells from RA patients (Supplemental Fig. 2). Collectively, these data indicated that IL-37 suppresses IL-17 and IL17–triggering cytokine expression and reduces the proportion of Th17 cells; the suppressive effect of IL-37 was independent of IL-1b. IL-37 limits Th17 cell proliferation from naive CD4+ T cells in culture but not Th17 cell differentiation To evaluate whether IL-37 exerts any direct effects on Th17 cell differentiation and proliferation in vitro, naive CD4+ T cells purified from the spleens of normal mice were cultured with IL-37 under Th17-polarizing conditions for 3 d, and the frequency of IL17+CD4+ T cells was analyzed by flow cytometry. As shown in Fig. 5A, IL-37 did not significantly change the percentage of IL17+CD4+ T cells, it only slightly reduced Th17 cell differentiation. Interestingly, upon treatment with IL-37 (100 ng/ml), the number of Th17 cells in the total CD4+ T cell population was markedly reduced (Fig. 5B). Because the differentiation of Th17 cells is

governed by the transcript factor RORgt (5), we investigated whether IL-37 affects the expression of RORgt in naive T cells at the stage of Th17 cell differentiation. As shown in Fig. 5C, the expression of RORgt showed no obvious changes upon IL-37 treatment, indicating the minor effect of IL-37 on Th17 cell differentiation. To further investigate whether IL-37 can suppress Th17 cell proliferation, CFSE-labeled naive CD4+ T cells were

FIGURE 2. IL-37 expression in CD4+ T cells under Th1/Th2/Th17-polarizing conditions. CD4+ T cells from PBMCs of HCs were cultured under Th1-, Th2-, and Th17-polarizing conditions. The cells were cultured for 5 d and then restimulated for 4 h with PMA (500 ng/ml) and Ion (500 ng/ml), or cells were treated for 4 h with LPS (1 mg/ml). The mRNA levels of IL-37 and IFN-g (Th1 polarization) (A), IL-37 and IL-17 (Th17 polarization) (B), and IL-37 and IL-4 (Th2 polarization) (C) were assessed by real-time PCR. (D) CD4+ T cells from PBMCs of HCs were prepared in Th1- and Th17polarizing conditions. The cells were stimulated with PMA (500 ng/ml) and Ion (500 ng/ml) for 24 h, or the cells were treated for 24 h with LPS (1 mg/ml), and the supernatants were analyzed for IL-37 by ELISA. Data are mean 6 SEM (n = 3). *p , 0.05, **p , 0.01.

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FIGURE 1. Comparison of IL-37 levels between RA patients and HCs. (A) Expression of IL-37 mRNA in PBMCs from RA patients (n = 49), distributed according to disease activity (active, n = 26; inactive, n = 23), and HCs (n = 33) was determined using real-time PCR. (B) Serum IL-37 levels in RA patients (n = 49), distributed according to disease activity (active, n = 26; inactive, n = 23), and HCs (n = 33) were determined by ELISA. (C) IL-37 protein levels in serum from prednisolone-treated (n = 38) or untreated (n = 11) RA patients were assessed by ELISA. Each symbol represents an individual RA patient or HC. Horizontal lines indicate median values. (D) IL-37 mRNA expression in CD3+ T cells, CD4+ T cells, CD8+ T cells, and CD19+ T cells from PBMCs of RA patients (n = 9) and HCs (n = 9) was assessed by real-time PCR. Data are mean 6 SEM. *p , 0.05, **p , 0.01. ns, not significant.

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cultured with IL-37 (50 and 100 ng/ml) under Th17-differentiation conditions. Flow cytometric analysis revealed that Th17 cell proliferation was significantly suppressed by 100 ng/ml IL-37 (Fig. 5D). Taken together, these data suggest that although IL37 efficaciously inhibits the proliferation of Th17 cells, it does not readily suppresses Th17 cell differentiation. Overexpression of Ad–IL-37 in HEK293 cells and mice joints Because the mouse IL-37 sequence has not been reported, we constructed recombinant Ad–IL-37 to further investigate the antiinflammatory properties of IL-37. To verify that Ad–IL-37 was capable of expressing IL-37, HEK293 cells were infected with 0, 105, or 107 PFU of Ad–IL-37 for 72 h, and IL-37 protein expression was determined by Western blot. We observed adenovirus-mediated IL-37 expression (105 or 107 PFU) from HEK293 cell lysates (Fig. 6A). Fluorescence microscopy further

confirmed that the infection efficiency of 107 PFU Ad-EV or Ad– IL-37 was near 100% in HEK293 cells (Fig. 6B). In vivo measurement of GFP reporter fluorescence did not differ between Ad– IL-37–expressing mice and Ad-EV control mice showing high expression in the joints (Fig. 6C). Subsequently, we used real-time PCR to assess IL-37 expression in the joint, spleen, liver, and kidney of mice with intra-articular injection of 107 PFU of Ad-EV and Ad–IL-37. We found that Ad–IL-37 strongly expressed IL-37 only in the joints of mice, with no change in spleen, liver, or kidney (Fig. 6D). Thus, we used 107 PFU of Ad–IL-37 and Ad-EV for subsequent experiments. Local intra-articular injection of Ad–IL-37 attenuates the development of CIA To confirm whether locally elevated IL-37 concentrations can alleviate CIA progression, we injected Ad–IL-37 and Ad-EV

FIGURE 4. IL-37 downregulated Th17-related cytokine expression in CD4+ T cells from patients with RA and reduced Th17 cells. CD4+ T cells from PBMCs of RA patients and HCs were cultured in the absence or presence of rhIL-37 (100 ng/ml) for 24 h and then stimulated with LPS (1 mg/ml) for 6 h. The mRNA levels of IL-17 (A), IL-1b (B), and IL-6 (C) were measured by real-time PCR. (D) Frequencies of IL-17+CD4+ cells (Th17 cells) were quantified in RA peripheral blood CD4+ T cells treated with PMA, Ion, and brefeldin A in the absence or presence of rhIL-37 (100 ng/ml). Data are integrated from three independent experiments. All data are mean 6 SEM. *p , 0.05, **p , 0.01.

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FIGURE 3. IL-37 inhibited the expression of IL-17, IL-1b, and IL-6 in PBMCs from patients with RA. PBMCs from RA patients (n = 49) and HCs (n = 33) were stimulated or not with rhIL-37 (100 ng/ml) for 6 h, and the mRNA levels of IL-17 (A), IL-1b (B), and IL-6 (C) were detected by real-time PCR. PBMCs from RA patients (n = 49) were stimulated or not with rhIL-37 (100 ng/ml) for 24 h and then incubated with LPS (1 mg/ml) for 6 h, and IL-17 (D), IL-1b (E), and IL-6 (F) levels in supernatant were assessed by ELISA. *p , 0.05, **p , 0.01.

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(107 PFU each) intra-articularly into the knee joint of DBA/1J mice on days 17 and 23 after the first immunization (Fig. 7A). The results showed that the incidence and symptoms of arthritis in Ad–IL-37– treated mice were significantly reduced compared with Ad-EV– treated mice (Fig. 7B, 7C). Histopathologic examination showed that, compared with Ad-EV–treated mice with CIA, the knee joints of Ad–IL-37–treated mice with CIA exhibited a significant reduction in synovial hyperplasia, pannus formation, cartilage damage, and bone erosion (Fig. 7D, 7E). To ascertain whether the CII-specific humoral immune response was modulated by Ad–IL-37

treatment, serum and synovial fluid from Ad–IL-37–treated and AdEV–treated mice were analyzed for the presence of CII-specific IgG1 and IgG2a Abs. Interestingly, no obvious changes in the levels of serum and synovial fluid CII-specific IgG1 Abs were observed in either group (Fig. 7F). In contrast, synovial fluid levels of CII-specific IgG2a Abs were dramatically lower in Ad-IL-37–treated mice compared with Ad-EV–treated mice, whereas serum CII–specific IgG2a Ab levels were not affected (Fig. 7G). Taken together, these results indicate that local expression of IL-37 contributes directly to the suppression of joint inflammation and pathologic changes in CIA.

FIGURE 6. Adenovirus-mediated overexpression of IL-37 in HEK293 cells and in mice joints. (A) HEK293 cells were infected with 0, 105, or 107 PFU of Ad–IL37 for 72 h, and IL-37 protein expression was determined by Western blot. (B) Infection efficiency of AdEV or Ad–IL-37 was near 100% in HEK293 cells, as indicated by GFP reporter fluorescence. Original magnification 3200. (C) The joints of mice were injected with 107 PFU of Ad-EV and Ad–IL-37, and GFP reporter fluorescence was measured in vivo using the IVIS imaging system. (D) IL-37 mRNA expression was determined by real-time PCR in the indicated tissues of mice with intra-articular injection of 107 PFU of Ad-EV or Ad–IL-37. Data are integrated from three independent experiments (mean 6 SEM). **p , 0.01.

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FIGURE 5. IL-37 inhibited murine Th17 cell proliferation but not Th17 cell differentiation. (A) Naive CD4+ T cells from normal mice were cultured under Th17-polarizing conditions in the presence of IL-37 (0, 50, and 100 ng/ml) for 72 h, and then the percentage of CD4+IL-17+ cells was determined by flow cytometry. The flow cytometric profiles are representative of four independent experiments with similar results. (B) Total numbers of IL-17+ CD4 cells in each treatment described in (A) were enumerated by cell counting. Data are mean 6 SEM (n = 4). (C) The expression of RORgt in naive CD4+ T cells under Th17-polarizing conditions in the presence of IL-37 (0, 50, and 100 ng/ml) was determined by flow cytometry. The shaded line graph represents isotype staining. Results represent four independent experiments. (D) CFSE-labeled purified naive splenic CD4+ T cells were cultured with IL-37 (0, 50, and 100 ng/ml) in Th17-polarizing medium for 3 d, and cells were stimulated with PMA, Ion, and monensin for 5 h. Th17 cells were analyzed by flow cytometry; each peak of Th17 cells represents their division in different culture conditions. Results are representative of four independent experiments. *p , 0.05.

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The anti-inflammatory effects of Ad–IL-37 are associated with downregulated IL-17 and IL-17–triggering cytokines in the joints of mice with CIA Proinflammatory cytokines (IL-17, IL-6, and IL-1b) are implicated in the pathogenesis of RA (2). Therefore, we asked whether IL-37 inhibits the expression of these proinflammatory factors in vivo. Compared with Ad-EV–treated mice with CIA, IL-17 levels were significantly decreased in the synovial fluid of Ad–IL-37–treated mice with CIA (Fig. 8A). IL-17–triggering cytokines (IL-6 and IL-1b) also were significantly reduced in the synovial fluid of Ad– IL-37–treated mice with CIA (Fig. 8A). Consistent with the above findings, the expression of IL-17 (Fig. 8B), IL-1b (Fig. 8C), and IL-6 (Fig. 8D) was dramatically downregulated in synovium and joint cells from Ad–IL-37–treated mice with CIA. Importantly, our results also revealed that rhIL-37 could significantly decrease IL-17 and IL-17–triggering cytokine (IL-1b and IL-6) mRNA expression in joint cells from CIA mice in vitro (Fig. 8E). These results suggest that local overexpression of IL-37 could curb IL-17 and IL-17–triggering cytokine (IL-1b and IL-6) production in joints of mice with CIA.

Discussion IL-37 was shown to be involved in autoimmune disease as an antiinflammatory cytokine and an inhibitor of both innate and acquired immune responses (6, 8). However, the source of IL-37–expressing cells in RA is unclear, and the effect of IL-37 in the pathogenesis of RA remains unknown. In this study, we provide a detailed analysis of IL-37 expression in various immune cells from patients with active/inactive RA and HCs. More importantly, we show for the first time, to our knowledge, that IL-37 markedly

suppressed the production of IL-17 and IL-17–triggering cytokines and the frequency of Th17 cells in CD4+ T cells and PBMCs in RA patients; these immunosuppressive effects of IL-37 appear to be independent of IL-1b. Additionally, injection of Ad–IL-37 effectively reduced the clinical and histologic scores in CIA mice by downregulating IL-17 and IL-17–triggering cytokine production. The antiarthritic effects of IL-37 on the production of IL-17 can be attributed, in part, to its inhibitory effects on Th17 cell proliferation but not Th17 cell differentiation. Recent data demonstrated that IL-37 expression was significantly elevated in the plasma and synovium of patients with RA (8, 10). Consistent with these findings, our results also revealed higher levels of IL-37 in PBMCs and serum from RA patients compared with HCs. Additionally, we clearly observed that IL-37 mRNA and protein levels were significantly higher in 26 patients with active disease than in 23 patients with inactive disease and 33 HCs. To the best of our knowledge, the monitoring of RA disease activity is traditionally based on clinical observations and laboratory values, such as DAS28, CRP, ESR, RF, and anti-CCP. Although CRP concentrations and ESR are reliable biochemical indicators of the acute-phase response in RA, neither is sufficient for distinguishing among RA patients with active disease. Consistent with previous results (10), serum IL-37 correlated closely with DAS28 and CRP. It is important to mention that serum IL-37 level also correlated with ESR, but it lacked an association with other laboratory values (RF and anti-CCP). Given that IL-17 and IL-17–triggering cytokines (IL-1b and IL-6) are involved in RA inflammation responses (2), a significantly positive correlation was observed between serum IL-37 levels and these three cytokines. It is worth noting that IL-1b and IL-6 are the main stim-

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FIGURE 7. Local overexpression of IL37 ameliorates the pathology of CIA. (A) Protocol for CIA induction and IL-37 administration in CII-immunized DBA/1J mice. Mice (age 8–12 wk) were immunized intradermally at the base of tail with 200 ml CII emulsified in CFA. On day 21, mice were given a boost immunization of 80 ml CII dissolved in CFA. Ad–IL-37 (107 PFU) or Ad-EV (107 PFU) was injected intraarticularly into the knee joint on days 17 and 23 after the first immunization. Mice were killed on day 32 or later for experimental analysis. Incidence (B) and mean clinical scores (C) of CIA in mice treated with Ad–IL-37 or Ad-EV. Data are from five independent experiments (n = 3/group in each experiment). (D) Representative H&E staining of knee joints of Ad–IL37– or Ad-EV–treated mice with CIA. The images on the right are enlargements of the boxed areas in the images on the left. (E) Evaluation of synovitis, pannus, and erosion of bone and cartilage in the knee joint sections of Ad–IL-37– or Ad-EV–treated mice with CIA. Levels of CII-specific IgG1 (F) and IgG2a (G) Abs in serum and synovial fluid from CIA mice treated with Ad-EV or Ad–IL-37 were assessed by ELISA (n = 5). Data are mean 6 SEM. *p , 0.05, **p , 0.01.

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ulators of the-acute phase response in RA (19), and IL-17 had a significant correlation with the inflammation marker CRP and ESR (20). Hence, these data suggest that IL-17 and IL-17–driving cytokines enhance the acute-phase response, as well as stimulate anti-inflammatory cytokine IL-37 expression to downregulate excessive inflammation during the pathogenic process of RA. Furthermore, our findings found that there was no association between IL-37 and RF/anti-CCP, indicating the preponderant role of T cells in the pathogenesis of RA, because RF and antiCCP Ab production were mainly derived from plasma cells and B lymphocytes (21). The cumulative evidence suggested that the inflammatory response induced the upregulation of IL-37, which exerts an immunosuppressive role in autoimmune diseases (6, 8). An interesting question that emerges from these findings is “What is the source of IL-37 expression in the process of RA inflammatory response?” Considering the crucial role of T and B cells in the pathogenesis of RA (1), we attempted to investigate whether T and B cells are critical for IL-37 expression in RA patients. Our results indicate that IL-37 is predominantly detected in CD3+ and CD4+ T cells, but not in CD8+ T cells or CD19+ B cells, from patients with RA. Thus, increased IL-37 expression in CD3+ and CD4+ T cells might be due to the activation of T cells in RA patients. Meanwhile, the expression of IL-37 in activated CD3+ and CD4+ T cells could be involved in the adaptive immune response in RA. Of note, numerous CD4+ T cells, but not CD8+ T cells, accumulate mainly in RA periphery and joint and may be involved in the pathogenesis of RA (1, 22). These data probably reflect the fact that, in RA periphery–activated immune cells, CD3+ and

CD4+ T cells are the mostly like source of IL-37. Therefore, IL37–mediated anti-inflammatory effects could be mediated by CD3+ and CD4+ T cells. Despite all of this, the presence of immune cells and the expression of IL-37 may be affected by RA disease activity. Further research is necessary to determine the expression of IL-37 immune cells during the different stages of RA disease activity. Many studies demonstrated that the balance of CD4+ T cell subsets (Th1, Th2, and Th17) is involved in the pathogenesis of RA via secretion of IFN-g, IL-4, and IL-17 (2, 5). In this study, we showed that Th1- and Th17-polarizing conditions induced stable IL-37 expression, but the expression of IL-37 in CD4+ T cells polarized under Th2 conditions was unaffected. Furthermore, Th1-secreted cytokine IFN-g and Th17-secreted cytokine IL-17 were increased in CD4+ T cells polarized under Th1 and Th17 conditions, respectively. It was shown that Th1-secreted cytokine IFN-g was highly effective in inducing IL-37, whereas Th2secreted cytokine IL-4 plus GM-CSF inhibited constitutive IL37 expression (8). Actually, as mentioned above (Supplemental Fig. 1F), there was an obvious correlation between IL-37 and Th17-secreted cytokine IL-17. These observations suggest that Th1/Th17 cytokines (IFN-g and IL-17) may play a part or a key role in inducing IL-37 production in CD4+ T cells. In contrast, Th2 cytokine IL-4 may provide an inhibitor signal for IL-37 production in CD4+ T cells. The observations accumulated thus far indicate that Th17 cells and their signature cytokine IL-17 are involved in the development, as well as the progression, of RA (3, 4). Increased IL-17 expression in serum and synovial tissue from patients with RA was demon-

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FIGURE 8. IL-37 suppressed the expression of inflammatory factors in joints of CIA mice. (A) Concentrations of synovial fluid cytokines (IL-17, IL-1b, and IL-6) from CIA mice treated with AdEV or Ad–IL-37 were measured by ELISA. Expression of IL-17 (B), IL-1b (C), and IL-6 (D) in synovial tissues and joint cells of CIA mice treated with Ad-EV or Ad–IL-37 was determined by realtime PCR. (E) Joint cells from mice with CIA were cultured or not with rhIL-37 (100 ng/ml) for 12 h. The expression of IL-17, IL-6, and IL-1b mRNA was determined by real-time PCR. All data are mean 6 SEM (n = 3). *p , 0.05, **p , 0.01.

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ducing proinflammatory cytokine IL-1b and IL-6 secretion in autoimmune diseases (4, 5). Therefore, it is plausible to suggest that overproduction of IL-37 curbed RA disease activity and might be involved in suppressing the IL-17–mediated inflammatory response. Notably, the importance of IL-37 in modulating the inflammatory response is also supported by recent data showing that decreased IL-37 expression in patients with Behc¸et’s disease triggers the Th17-inflammation response (29). Although the molecular mechanism of IL-37 in autoimmune disease remains unknown, recent studies showed that IL-37 acts as an extracellular cytokine by binding IL-18R and IL-1R8 for its anti-inflammatory properties (30). In addition, IL-37 fails to inhibit IL-17–triggering cytokine (IL-1b and IL-6) production and the MAPK signaling pathway in DCs from IL-1R8–deficient mice (30). A recent study showed that IL-17 expression was dramatically enhanced in IL1R8–deficient mice (31). These results suggest that IL-37 may suppress IL-17 and IL-17–triggering cytokine production in the development of RA via binding IL-1R8 and curbing the MAPK signaling pathway in DCs. Nevertheless, further investigations and manipulations of IL-37 signaling may improve therapeutic options for RA patients. In summary, we demonstrate for the first time, to our knowledge, that IL-37 levels were elevated in active RA and were associated particularly with Th17 cytokines and with Th17/Th1 differentiation. More importantly, the results reveal an anti-inflammatory effect of IL-37 in human RA and the CIA model. IL-37 may mediate its antiarthritic effects through inhibition of IL-17 and IL-17–triggering cytokines and via suppression of Th17 cell proliferation. These data suggest a possible therapeutic significance for IL-37 in RA.

Acknowledgments We thank the patients for donating samples to the study. We also thank the staff of the Clinical Medicine Laboratory of Peking University Shenzhen Hospital in Shenzhen for help collecting and processing clinical samples.

Disclosures The authors have no financial conflicts of interest.

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strated in some studies (4). In accordance with previous data, we found higher levels of IL-17 in serum and PBMCs from RA patients compared with HCs. One intriguing finding of our study is that IL-17 production in CD4+ T cells and PBMCs from RA patients was significantly suppressed by rIL-37 treatment. Strikingly, IL-37 also markedly inhibited the percentage of IL-17+ CD4+ T cells from RA patients in vitro. It was shown recently that cytokines IL-1b and IL-6 are very potent at inducing IL-17 secretion from CD4+ T cells and driving Th17 cell differentiation (4, 5). In our study, although IL-37 also was able to inhibit IL-17– triggering cytokine IL-1b and IL-6 expression in CD4+ T cells and PBMCs from RA patients, IL-37 did not appear to exert its antiinflammatory role via IL-1b, because inhibition of anti–IL-1b– neutralizing Ab did not affect the frequency of IL-17+CD4+ T cells in patients with RA. These results indicate that the antiinflammatory role of IL-37 in IL-17 production in RA seems to be independent of IL-1b. Although IL-1b is considered the critical cytokine for IL-17 production in CD4+ T cells and Th17 cell differentiation (23, 24), consistent with our results, IL-1 is selectively required for IL-17 secretion and Th17 cell induction (24), because IL-23 or IL-1 and IL-23, but not IL-1 alone, induced potent IL-17 gene transcription. Moreover, recent evidence suggests that, in addition to IL-1, Th17 cell differentiation is driven by the synergy of other cytokines, such as IL-6, TGF-b, IL-21, and IL-23 (4, 23, 25, 26). Based on this information, we speculate that IL-37 exerts directly suppressive effects on IL-17 production involved in regulating Th17 cell differentiation or proliferation. In stark contrast, our results suggest that IL-37 failed to significantly limit the differentiation of Th17 cells, because there was no obvious alteration in the frequency of IL-17+CD4+ T cells in naive CD4+ T cells treated with IL-37 under Th17 cell–differentiation conditions. Likewise, the transcription factor RORgt, which is critical for Th17 cell generation (5), also was found to be largely resistant to direct suppression by IL-37. Of great interest, IL-37 dramatically limited Th17 cell proliferation in culture. Our present study identified that in vitro administration of IL-37 inhibited IL17 production that was associated with restriction of Th17 cell proliferation. Despite these results indicating that IL-37 has little or no effect on Th17 cell differentiation directly, it is likely that the immunosuppressive effect of IL-37 on Th17 cell and IL-17 production is through regulating dendritic cell (DC) function or phenotype. Actually, recent evidence suggests that IL-37 significantly suppressed Ag-specific adaptive immunity through the induction of tolerogenic DCs and the promotion of IL-10 expression on DCs (27). Similarly, the presence of IL-37 markedly inhibited DC function and Th17 cell differentiation–related cytokine production (e.g. IL-6 and TGF-b) (6, 8). Notably, DCs are the main APCs mediating Th17 responses by secreting anti-inflammatory or proinflammatory cytokines, such as IL-6, TGF-b, and IL-10 (28). These results indicate that IL-37 is capable of regulating DC function and phenotype directly or indirectly and, thereby, inhibiting Th17- and IL-17–mediated inflammatory responses in RA. In vivo, we attempted to confirm our findings of IL-37 as an antiinflammatory cytokine in mice with CIA by local intra-articular injection of Ad–IL-37. As expected, overexpression of IL-37 in the CIA model resulted in a delayed onset of disease and alleviated the severity of clinical symptoms. Furthermore, the antiarthritic effect of IL-37 in CIA was associated with a reduction in IL-17– and IL-17–triggering cytokines (IL-1b and IL-6). Pathogenic Th17 cells secreting their signature proinflammatory cytokine, IL-17, were shown to promote inflammatory responses and to contribute to the pathogenesis of RA (3, 4). More importantly, it is generally regarded that IL-17 is the critical cytokine for in-

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IL-37, a new member of the IL-1 cytokine family, is a natural inhibitor of innate immunity associated with autoimmune diseases. This study was underta...
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