of April 3, 2018.This information is current as

ProliferationCytokine Production and Limiting Th17 Cell

Triggering−Suppressing IL-17 and IL-17 IL-37 Alleviates Rheumatoid Arthritis by

Guan, Zhongyang Wen, Kunzhao Huang and Zhong HuangLiang Ye, Bo Jiang, Jun Deng, Jing Du, Wen Xiong, Youfei

ol.1401810http://www.jimmunol.org/content/early/2015/04/25/jimmun

published online 27 April 2015J Immunol 

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The Journal of Immunology

IL-37 Alleviates Rheumatoid Arthritis by Suppressing IL-17and IL-17–Triggering Cytokine Production and LimitingTh17 Cell Proliferation

Liang Ye,*,†,‡ Bo Jiang,x Jun Deng,{ Jing Du,‖ Wen Xiong,# Youfei Guan,**

Zhongyang Wen,*,†,‡ Kunzhao Huang,*,†,‡ and Zhong Huang*,†,‡

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 investi-

gating the effects of recombinant human (rh)IL-37 and an adenovirus encoding human IL-37 (Ad–IL-37) on Th17 cells and Th17-

related 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 fre-

quency in PBMCs and CD4+ T cells from RA patients. Furthermore, IL-37 exerted a more suppressive effect on Th17 cell prolif-

eration, 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: 000–000.

Rheumatoid arthritis (RA) is a chronic inflammatory au-toimmune disease characterized by the infiltration ofactivated immune cells and the production of inflam-

matory factors that lead to the formation of synovial hyperplasiaand pannus, as well as the destruction of cartilage and joints (1, 2).Although the etiology and pathogenesis of RA are not fully un-derstood, recent in vivo animal models and in vitro human studiesdemonstrated that IL-17 can be considered a decisive mediator inthe 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

potent proinflammatory functions (3, 4). Based on data from an-imal models of autoimmune arthritis, IL-17 is recognized as oneof the important factors in the pathogenesis of RA (3). Recentstudies revealed that the expression of IL-17 is increased in serum,synovial fluid, and synovium of patients with RA comparedwith healthy controls (HCs) (4). Moreover, IL-17–transgenic miceare prone to develop collagen-induced arthritis (CIA), whereasblocking of endogenous IL-17 with its receptor in CIA results inthe 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, andmacrophages (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 anN-terminal prodomain that includes a potential caspase-1 cleavagesite (7). IL-37b is the largest isoform, has significant sequencesimilarity with IL-18, and is the most studied. The IL-37b precursoris cleaved by caspase-1 into mature IL-37b (6, 7). IL-37b has beenidentified 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 im-

mune cells, may play a prominent role in the pathogenesis of RA by

curbing IL-17 or other proinflammatory cytokines.

*Institute of Biological Therapy, Shenzhen University, Shenzhen 518060, China;†Department of Pathogen Biology and Immunology, Shenzhen University Schoolof Medicine, Shenzhen 518060, China; ‡Shenzhen City Shenzhen University Immu-nodiagnostic Technology Platform, Shenzhen 518060, China; xMaoming City Peo-ple’s Hospital, Maoming 525000, Guangdong, China; {Department of Pathology,University of Hong Kong, Hong Kong 999077, China; ‖Department of LaboratoryMedicine, Peking University Shenzhen Hospital, Shenzhen 518036, China; #ShenzhenBlood Center, Shenzhen 518035, Guangdong, China; and **Shenzhen UniversityDiabetes 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 ScienceFoundation of China and by Grant JCYJ20140711144858545 from the Special Fundfor Shenzhen Strategic Emerging Industry Development.

Address correspondence and reprint requests to Dr. Zhong Huang, Institute of Bio-logical Therapy, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060,Guangdong, China. E-mail address: zhuang809@126.com

The online version of this article contains supplemental material.

Abbreviations used in this article: Ad-EV, empty adenovirus; Ad–IL-37, adenovirusencoding human IL-37; anti-CCP, anti-cyclic citrullinated peptide Ab; CIA, collagen-induced arthritis; CII, bovine type II collagen; CRP, C-reactive protein; DAS28, 28-joint disease activity score; DC, dendritic cell; ESR, erythrocyte sedimentation rate;HC, healthy control; Ion, ionomycin; RA, rheumatoid arthritis; RF, rheumatoid fac-tor; rh, recombinant human.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

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In this study, we investigated the expression of IL-37 in patientswith active and inactive RA. We also analyzed the expressionpattern of IL-37 in cell populations isolated from the PBMCs ofpatients with RA, as well as CD4+ T cells under Th1/Th2/Th17-differentiation conditions. The effect of IL-37 on the modulationof IL-17 and IL-17–driving cytokines (IL-1b and IL-6) in vitroand in vivo was examined in this study. We further analyzed theeffect of IL-37 on Th17 cell differentiation and proliferation.To our knowledge, this is the first study to show the anti-inflammatory properties of IL-37 in both human RA and theCIA model through the inhibition of IL-17 and IL-17–triggeringcytokine production and the suppression of Th17 cell prolifera-tion, which may contribute to its antiarthritic effects.

Materials and MethodsPatients and HCs

A total of 49 patients with RA, from Peking University Shenzhen Hospital,was included in the current study. The classification of RA fulfilled the 1987and 2010 American College of Rheumatology criteria (13, 14). RA patientswere divided into active (n = 26) and inactive (n = 23) groups, according totheir 28-joint disease activity score (DAS28); active disease is defined asDAS28 $ 3.2, based on the European League Against Rheumatism di-agnostic criteria (15). At the time of sample acquisition, 38 patients werebeing treated with prednisone, and 11 patients were not. HC peripheralblood samples were obtained from 33 healthy volunteers. RA patients andHCs did not exhibit significant differences in terms of mean age or male/female distribution (Supplemental Table I). The study was approved by theReview Board for the Peking University Shenzhen Hospital. Informedconsent was obtained from the recruited subjects.

Preparation of human PBMCs

Peripheral blood samples were obtained from patients with RA and HCs.Serum samples were stored at280˚C until cytokines were determined. PBMCsfrom patients and HCs were isolated using lymphocyte separation medium(TBD Science), according to the manufacturer’s instructions. The collectedcells were used for cell cultures or stored at 280˚C until RNA extraction.

Cell sorting

Human PBMCs were stained using human-specific Abs, including FITC-conjugated anti-CD3, PE-conjugated anti-CD19, FITC-conjugated anti-CD4, and PE-conjugated anti-CD8 (all from BD Biosciences), and CD3+

T cells, CD19+ T cells, CD4+ T cells, and CD8+ T cells, respectively, weresorted 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 cytometrywere cultured in RPMI 1640 medium (HyClone, Thermo Fisher Scientific),supplemented with 10% FCS (Hangzhou Sijiqing Biological EngineeringMaterials) and 100 IU/ml penicillin and 100 mg/ml streptomycin (Beyo-time), in the presence of anti-CD3 (1 mg/ml) and anti-CD28 (1 mg/ml). Theconditions 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 Th17conditions: 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). Cellswere 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 andELISA, respectively, for cytokines IL-37, IFN-g, IL-17, and IL-4.

For murine Th17 cell differentiation, naive CD4+CD62L+ T cells fromspleens of DBA mice were purified using a CD4+CD62L+ T Cell IsolationKit II (Miltenyi Biotec), and purity was routinely 95%. Purified naiveT cells were cultured with anti-CD3– and anti-CD28–coated 24-well plateswith TGF-b (1 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml), with orwithout rhIL-37 (0, 50, and 100 ng/ml), for 72 h. For the final 5 h of in-cubation, PMA (50 ng/ml), Ion (1 mg/ml), and monensin (10003) wereadded to each well and mixed thoroughly. IL-17 and RORgt were stainedwith anti-mouse IL-17-PE and anti-mouse RORgt-PE Abs, respectively(BD Biosciences), and cells were measured with a flow cytometer (BDBiosciences) and analyzed using FlowJo software.

For Th17 cell proliferation, purified naive CD4+ T cells were labeledwith 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 pro-liferation was measured with a flow cytometer (BD Biosciences) and an-alyzed using FlowJo software.

Cell culture and IL-37 treatment

RPMI 1640 (HyClone, Thermo Fisher Scientific), supplemented with10% FCS (Hangzhou Sijiqing Biological Engineering Materials) and 100IU/ml penicillin and 100 mg/ml streptomycin (Beyotime), was used asculture medium. PBMCs and purified CD4+ T cells were cultured inRPMI 1640 medium in a humidified atmosphere of 5% CO2 at 37˚C.PBMCs from patients with RAwere cultured at a concentration of 1.53 106

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 super-natants were harvested and frozen at 280˚C for later cytokine analysis byELISA.

Flow cytometric analysis

Anti-human CD4-FITC and anti-human IL-17–PE were purchased fromeBioscience. Anti-mouse CD4-allophycocyanin, anti-mouse IL-17–PE,and anti-mouse RORgt-PE were purchased from BD Biosciences. Humansamples 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 hbefore flow cytometric analysis. Mouse samples for staining of IL-17 andRORgt, PMA (50 ng/ml), Ion (1 mg/ml), and monensin (10003) wereadded to cultures for the last 5 h before flow cytometric analysis, as pre-viously described (16).

Construction of a recombinant adenovirus encoding humanIL-37

Briefly, the human IL-37 gene (GenBank: AF167368) was amplified byPCR, using human PBMC cDNA as the template, and ligated to the pMd-18T vector (Takara) for the construction of pMd–18T–IL-37 vector plas-mid. Subsequently, double digestion with EcoRV and XhoI (both fromTakara) was conducted on plasmid pMd–18T–IL-37 and shuttle vectorpShuttle-IRES-hrGFP2 (Stratagene), respectively. Recovered productswere connected with T4 DNA ligase (Takara), and the ligation mixturewas transformed into DH5a bacteria (TransGen Biotech). Kana-resistanttransformants were selected by plating the transformation mixture onLuria-Bertani agar plates supplemented with kanamycin (Sangon Biotech).After incubation overnight, the recombinant plasmid DNA was isolatedand further confirmed via double digestion; the confirmed recombinantadenoviral vector was named pShuttle–IRES–hrGFP2–IL-37. Thereafter,pShuttle–IRES–hrGFP2–IL-37 was linearized by restriction endonucleasePmeI (Thermo Fisher Scientific) and subsequently cotransformed withadenoviral backbone vector pAdEasy-1 (Stratagene) in Escherichia coliBJ5183 cells (Stratagene) for homologous recombination. Then it wasdigested by PacI (Thermo Fisher Scientific) and transfected into HEK293cells (Stratagene) with Lipofectamine 2000, according to the manu-facturer’s instructions (Invitrogen), to generate an adenovirus encodinghuman IL-37 (Ad–IL-37). The empty adenovirus (Ad-EV) control used inthis study was an empty pShuttle-IRES-hrGFP2 shuttle vector with noinsert. Seven days later, green fluorescence could be seen under a fluores-cence 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 celllysates 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 wasobserved through GFP reporter fluorescence. The hind joints of DBA/1mice were injected intra-articularly with 13 107 PFU Ad–IL-37 or Ad-EVcontaining GFP reporter fluorescence. Seven days later, fluorescence wasmeasured 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 mlbovine type II collagen (CII; Chondrex) emulsified in CFA containingMycobacterium tuberculosis (Chondrex). On day 21, these mice weregiven 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 kneejoints on days 17 and 23 after the first CII immunization. Clinical arthritiswas evaluated as previously described (17).

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Preparation of synovial tissues and joint cells and collection ofsynovial fluids

Synovial tissues, joint cells, and synovial fluids were collected according toa previously described protocol (18). Synovial fluid samples were stored at280˚C prior to assay. Joint cells were cultured onto 24-well plates inRPMI 1640 medium (Hyclone, Thermo Fisher Scientific) containing 10%FCS (Hangzhou Sijiqing Biological Engineering Materials) and incubatedfor 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 themanufacturer’s instructions. cDNA was prepared using the iScript cDNASynthesis Kit (Bio-Rad). The primer sequences are summarized in Sup-plemental Table II. Real-time PCR amplification reactions were preparedwith the SYBR Green PCR Kit (Bio-Rad) and performed using the ABI 7500Fast Real-Time PCR System (Applied Biosystems). PCR products wereverified by melting curve analysis. Relative expression levels of target geneswere calculated by normalization to b-actin values using the 22ΔΔct method.

ELISA

Human and mouse cytokine levels were measured by ELISA, accordingto the manufacturer’s instructions (eBioscience). Concentrations of CII-specific IgG1 and IgG2a Abs (eBioscience) in the serum and synovial fluidof mice were measured by ELISA, as described previously (18).

Histopathologic analysis

Mice were anesthetized and killed on day 32, and knee joints were removedfor histopathologic examination. Tissue sections were prepared and stainedwith H&E. Histopathologic scoring of joint damage was performed underblinded conditions, according to a widely used scoring system for evalu-ating synovitis, cartilage degradation, and bone erosion (18).

Statistical analysis

Data are expressed as mean (6 SEM) or median (range) and were analyzedusing GraphPad Prism v5.00 software (GraphPad). A two-tailed Studentt test was used for the statistical comparison of two groups. Where ap-propriate, two-way ANOVA, followed by the Bonferroni post test formultiple comparisons, and a Mann–Whitney U test for nonparametric datawere used. The Spearman correlation test was used to assess the associ-ation between serum IL-37 levels and different variables. The p values #0.05 were considered significant.

ResultsIncreased IL-37 mRNA and serum protein levels in patientswith active RA and their correlation with the inflammationresponse

To investigate the potential role of IL-37 in patients with RA, 49RA patients and 33 age- and gender-matched HCs were recruited(Supplemental Table I). Distinct differences in IL-37 mRNA andserum protein levels were present between active RA patients andinactive RA patients (Fig. 1A, 1B). Patients with active RAshowed higher IL-37 mRNA (Fig. 1A) and serum IL-37 (Fig. 1B)protein levels than did HCs. However, we did not observe dif-ferences in IL-37 mRNA and protein levels between inactive RApatients and HCs (Fig. 1A, 1B). To observe the effect of pred-nisolone on IL-37 expression, RA patients were divided intotreated and untreated groups. We found that prednisolone-treatedRA patients showed lower IL-37 levels than did RA patients be-fore treatment (Fig. 1C). However, there was no difference in thelevels of IL-37 in RA patients not treated with prednisolonecompared with RA patients before treatment (Fig. 1C). At the timeof the highest inflammatory activity during the observation period,as determined by maximal C-reactive protein (CRP) and/orerythrocyte sedimentation rate (ESR) values, serum levels of IL-37 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-37levels 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 cit-rullinated peptide Abs (anti-CCPs) (Supplemental Fig. 1D, 1E).IL-17 and IL-17–driving cytokines (IL-1b and IL-6) play an im-portant role in the initiation and progression of CIA. We nextassessed whether serum IL-37 levels correlated with these cyto-kines. As seen in Supplemental Fig. 1F–H, serum IL-37 concen-trations 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 Tand B cells in the pathogenesis ofRA, we investigated the expression levels of IL-37 in T and B cellsfrom PBMCs of RA patients and HCs. We found that IL-37 mRNAexpression was significantly higher in CD3+ and CD4+ T cells fromRA patients compared with HCs (Fig. 1D). In contrast, no changein IL-37 mRNA expression in CD8+ T cells or CD19+ B cells wasobserved between RA patients and HCs (Fig. 1D). Together, theseresults demonstrate that IL-37 expression in peripheral immunecells may be involved in the pathogenesis of RA.

Increased IL-37 production in CD4+ T cells under Th1- andTh17-polarizing conditions

To further investigate whether CD4+ Th cell (Th1, Th2, and Th17)differentiation regulates the expression of IL-37, CD4+ T cellsfrom PBMCs of HCs were primed in vitro for 5 d under Th1-,Th2-, or Th17-polarizing conditions. Cells were then restimulatedwith PMA/Ion or LPS and examined for IL-37 expression usingreal-time PCR and ELISA. The results showed that both PMA/Ionand LPS clearly induced IL-37 and IFN-g mRNA expression inCD4+ T cells under Th1-polarizing conditions (Fig. 2A), consis-tent with elevated IL-37 and IL-17 mRNA levels in CD4+ T cellsunder Th17-polarizing conditions (Fig. 2B). Although IL-4mRNA expression was highly increased in CD4+ T cells polar-ized under Th2 conditions, the expression of IL-37 mRNA inCD4+ T cells polarized under Th2 conditions was unaffected bytreatment with PMA/Ion or LPS (Fig. 2C). Notably, IL-37 pro-tein also was stably expressed in CD4+ T cells under Th1- andTh17-polarizing conditions via stimulation of PMA/Ion or LPS(Fig. 2D). Taken together, these results indicate that IL-37 isconsistently elevated during CD4+ T cell activation, especiallyactivated Th1 and Th17 cells.

IL-37 suppresses proinflammatory cytokine expression inPBMCs from patients with RA

To determine whether IL-37 inhibits proinflammatory cytokineproduction in RA, we expressed and purified rhIL-37 protein (9).Our results revealed that rhIL-37 downregulated proinflammatorycytokine expression in PBMCs from HCs in a dose-dependentmanner (optimum concentration of rhIL-37 was 100 and 200 ng/ml)(data not shown). To further address the effects of IL-37 onproinflammatory cytokines in PBMCs from RA patients, PBMCsfrom 49 RA patients and 33 HCs were either left untreated ortreated with rhIL-37 (100 ng/ml) for 6 or 24 h, and cells andculture supernatants were harvested for real-time PCR and ELISAanalysis, respectively. We found that the transcriptional levels ofproinflammatory cytokines IL-17 (Fig. 3A), IL-1b (Fig. 3B), andIL-6 (Fig. 3C) in PBMCs of RA patients were significantly sup-pressed by rhIL-37. rhIL-37 also markedly reduced the secretionof IL-17 (Fig. 3D), IL-1b (Fig. 3E), and IL-6 (Fig. 3F) in PBMCsof RA patients. However, the cytokine mRNA (Fig. 3A–C) andprotein (data not shown) levels in PBMCs of HCs were not no-ticeably changed by treatment with rhIL-37.

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IL-37 inhibits IL-17 expression in CD4+ T cells from patientswith RA and decreases RA Th17 cells

To further investigate the effects of IL-37 on IL-17 expression fromCD4+ T cells in patients with RA, CD4+ T cells from PBMCs ofRA patients and HCs were cultured or not with rhIL-37. Wedemonstrated that the expression of IL-17 mRNA in CD4+ T cellsfrom patients with RAwas markedly higher than that in cells fromHCs (Fig. 4A). More importantly, rhIL-37 significantly reducedthe expression of IL-17 mRNA in CD4+ T cells from patients withRA, as determined by real-time PCR, but IL-17 mRNA expressionin HCs was not inhibited by treatment with rhIL-37 (Fig. 4A). Tofurther analyze the levels of IL-17–driving cytokines IL-1b andIL-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 significantlyhigher in CD4+ T cells of RA patients. Notably, rhIL-37 alsosignificantly downregulated the expression of IL-1b (Fig. 4B) andIL-6 (Fig. 4C) mRNA in CD4+ T cells from patients with RA. Wefurther observed that the mean percentage of Th17 cells wassignificantly reduced by treatment with rhIL-37 (4.2% withoutrhIL-37 versus 2.0% with rhIL-37) (Fig. 4D). However, IL-1b isnot required for IL-37–mediated suppression of IL-17 expressionin CD4+ T cells from RA patients (Supplemental Fig. 2). Col-lectively, these data indicated that IL-37 suppresses IL-17 and IL-17–triggering cytokine expression and reduces the proportion ofTh17 cells; the suppressive effect of IL-37 was independent of IL-1b.

IL-37 limits Th17 cell proliferation from naive CD4+ T cells inculture but not Th17 cell differentiation

To evaluate whether IL-37 exerts any direct effects on Th17 celldifferentiation and proliferation in vitro, naive CD4+ T cells pu-rified from the spleens of normal mice were cultured with IL-37under Th17-polarizing conditions for 3 d, and the frequency of IL-17+CD4+ T cells was analyzed by flow cytometry. As shown inFig. 5A, IL-37 did not significantly change the percentage of IL-17+CD4+ T cells, it only slightly reduced Th17 cell differentiation.Interestingly, upon treatment with IL-37 (100 ng/ml), the numberof Th17 cells in the total CD4+ T cell population was markedlyreduced (Fig. 5B). Because the differentiation of Th17 cells is

governed by the transcript factor RORgt (5), we investigatedwhether IL-37 affects the expression of RORgt in naive T cells atthe stage of Th17 cell differentiation. As shown in Fig. 5C, theexpression of RORgt showed no obvious changes upon IL-37treatment, indicating the minor effect of IL-37 on Th17 cell dif-ferentiation. To further investigate whether IL-37 can suppressTh17 cell proliferation, CFSE-labeled naive CD4+ T cells were

FIGURE 1. Comparison of IL-37 levels be-

tween 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 indi-

vidual 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 6SEM. *p , 0.05, **p , 0.01. ns, not signifi-

cant.

FIGURE 2. IL-37 expression in CD4+ T cells under Th1/Th2/Th17-po-

larizing 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 Th17-

polarizing 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 6SEM (n = 3). *p , 0.05, **p , 0.01.

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cultured with IL-37 (50 and 100 ng/ml) under Th17-differentiationconditions. Flow cytometric analysis revealed that Th17 cellproliferation was significantly suppressed by 100 ng/ml IL-37(Fig. 5D). Taken together, these data suggest that although IL-37 efficaciously inhibits the proliferation of Th17 cells, it doesnot 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, weconstructed recombinant Ad–IL-37 to further investigate the anti-inflammatory properties of IL-37. To verify that Ad–IL-37 wascapable of expressing IL-37, HEK293 cells were infected with 0,105, or 107 PFU of Ad–IL-37 for 72 h, and IL-37 proteinexpression was determined by Western blot. We observedadenovirus-mediated IL-37 expression (105 or 107 PFU) fromHEK293 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 mea-surement of GFP reporter fluorescence did not differ between Ad–IL-37–expressing mice and Ad-EV control mice showing highexpression in the joints (Fig. 6C). Subsequently, we used real-timePCR to assess IL-37 expression in the joint, spleen, liver, andkidney of mice with intra-articular injection of 107 PFU of Ad-EVand Ad–IL-37. We found that Ad–IL-37 strongly expressed IL-37only in the joints of mice, with no change in spleen, liver, orkidney (Fig. 6D). Thus, we used 107 PFU of Ad–IL-37 and Ad-EVfor subsequent experiments.

Local intra-articular injection of Ad–IL-37 attenuates thedevelopment of CIA

To confirm whether locally elevated IL-37 concentrations canalleviate CIA progression, we injected Ad–IL-37 and Ad-EV

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.

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 inde-

pendent experiments. All data are mean 6 SEM.

*p , 0.05, **p , 0.01.

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(107 PFU each) intra-articularly into the knee joint of DBA/1J miceon days 17 and 23 after the first immunization (Fig. 7A). The resultsshowed 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 showedthat, compared with Ad-EV–treated mice with CIA, the kneejoints of Ad–IL-37–treated mice with CIA exhibited a significantreduction in synovial hyperplasia, pannus formation, cartilagedamage, and bone erosion (Fig. 7D, 7E). To ascertain whether theCII-specific humoral immune response was modulated by Ad–IL-37

treatment, serum and synovial fluid from Ad–IL-37–treated and Ad-EV–treated mice were analyzed for the presence of CII-specific IgG1and IgG2a Abs. Interestingly, no obvious changes in the levels ofserum and synovial fluid CII-specific IgG1 Abs were observed ineither group (Fig. 7F). In contrast, synovial fluid levels of CII-specificIgG2a Abs were dramatically lower in Ad-IL-37–treated mice com-pared with Ad-EV–treated mice, whereas serum CII–specific IgG2aAb levels were not affected (Fig. 7G). Taken together, these resultsindicate that local expression of IL-37 contributes directly to thesuppression of joint inflammation and pathologic changes in CIA.

FIGURE 5. IL-37 inhibited murine Th17 cell pro-

liferation 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 per-

centage of CD4+IL-17+ cells was determined by flow

cytometry. The flow cytometric profiles are represen-

tative 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 cy-

tometry. The shaded line graph represents isotype

staining. Results represent four independent experi-

ments. (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 inde-

pendent experiments. *p , 0.05.

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–IL-

37 for 72 h, and IL-37 protein expression was deter-

mined by Western blot. (B) Infection efficiency of Ad-

EV 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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The anti-inflammatory effects of Ad–IL-37 are associated withdownregulated IL-17 and IL-17–triggering cytokines in thejoints of mice with CIA

Proinflammatory cytokines (IL-17, IL-6, and IL-1b) are implicatedin the pathogenesis of RA (2). Therefore, we asked whether IL-37inhibits the expression of these proinflammatory factors in vivo.Compared with Ad-EV–treated mice with CIA, IL-17 levels weresignificantly decreased in the synovial fluid of Ad–IL-37–treatedmice with CIA (Fig. 8A). IL-17–triggering cytokines (IL-6 andIL-1b) also were significantly reduced in the synovial fluid of Ad–IL-37–treated mice with CIA (Fig. 8A). Consistent with the abovefindings, the expression of IL-17 (Fig. 8B), IL-1b (Fig. 8C), andIL-6 (Fig. 8D) was dramatically downregulated in synovium andjoint cells from Ad–IL-37–treated mice with CIA. Importantly,our results also revealed that rhIL-37 could significantly decreaseIL-17 and IL-17–triggering cytokine (IL-1b and IL-6) mRNAexpression in joint cells from CIA mice in vitro (Fig. 8E). Theseresults suggest that local overexpression of IL-37 could curb IL-17and IL-17–triggering cytokine (IL-1b and IL-6) production injoints of mice with CIA.

DiscussionIL-37 was shown to be involved in autoimmune disease as an anti-inflammatory cytokine and an inhibitor of both innate and acquiredimmune responses (6, 8). However, the source of IL-37–express-ing cells in RA is unclear, and the effect of IL-37 in the patho-genesis of RA remains unknown. In this study, we providea detailed analysis of IL-37 expression in various immune cellsfrom 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 cyto-kines and the frequency of Th17 cells in CD4+ T cells and PBMCsin RA patients; these immunosuppressive effects of IL-37 appearto be independent of IL-1b. Additionally, injection of Ad–IL-37effectively reduced the clinical and histologic scores in CIA miceby downregulating IL-17 and IL-17–triggering cytokine produc-tion. The antiarthritic effects of IL-37 on the production of IL-17can be attributed, in part, to its inhibitory effects on Th17 cellproliferation but not Th17 cell differentiation.Recent data demonstrated that IL-37 expression was signifi-

cantly elevated in the plasma and synovium of patients with RA(8, 10). Consistent with these findings, our results also revealedhigher levels of IL-37 in PBMCs and serum from RA patientscompared with HCs. Additionally, we clearly observed that IL-37mRNA and protein levels were significantly higher in 26 patientswith active disease than in 23 patients with inactive disease and 33HCs. To the best of our knowledge, the monitoring of RA diseaseactivity is traditionally based on clinical observations and labo-ratory values, such as DAS28, CRP, ESR, RF, and anti-CCP. Al-though CRP concentrations and ESR are reliable biochemicalindicators of the acute-phase response in RA, neither is sufficientfor distinguishing among RA patients with active disease. Con-sistent with previous results (10), serum IL-37 correlated closelywith DAS28 and CRP. It is important to mention that serum IL-37level also correlated with ESR, but it lacked an association withother laboratory values (RF and anti-CCP). Given that IL-17 andIL-17–triggering cytokines (IL-1b and IL-6) are involved in RAinflammation responses (2), a significantly positive correlationwas observed between serum IL-37 levels and these three cyto-kines. It is worth noting that IL-1b and IL-6 are the main stim-

FIGURE 7. Local overexpression of IL-

37 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 intra-

articularly into the knee joint on days 17

and 23 after the first immunization. Mice

were killed on day 32 or later for experi-

mental 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–IL-

37– 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 ero-

sion 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 sy-

novial 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 hada significant correlation with the inflammation marker CRP andESR (20). Hence, these data suggest that IL-17 and IL-17–drivingcytokines enhance the acute-phase response, as well as stimulateanti-inflammatory cytokine IL-37 expression to downregulateexcessive inflammation during the pathogenic process of RA.Furthermore, our findings found that there was no associationbetween IL-37 and RF/anti-CCP, indicating the preponderantrole of T cells in the pathogenesis of RA, because RF and anti-CCP Ab production were mainly derived from plasma cells andB lymphocytes (21).The cumulative evidence suggested that the inflammatory re-

sponse induced the upregulation of IL-37, which exerts animmunosuppressive role in autoimmune diseases (6, 8). Aninteresting question that emerges from these findings is “What isthe source of IL-37 expression in the process of RA inflammatoryresponse?” Considering the crucial role of T and B cells in thepathogenesis of RA (1), we attempted to investigate whether T andB cells are critical for IL-37 expression in RA patients. Our resultsindicate that IL-37 is predominantly detected in CD3+ and CD4+

T cells, but not in CD8+ T cells or CD19+ B cells, from patientswith 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, accu-mulate mainly in RA periphery and joint and may be involved inthe pathogenesis of RA (1, 22). These data probably reflect thefact that, in RA periphery–activated immune cells, CD3+ and

CD4+ T cells are the mostly like source of IL-37. Therefore, IL-37–mediated anti-inflammatory effects could be mediated byCD3+ and CD4+ T cells. Despite all of this, the presence of im-mune cells and the expression of IL-37 may be affected by RAdisease activity. Further research is necessary to determine theexpression of IL-37 immune cells during the different stages ofRA disease activity.Many studies demonstrated that the balance of CD4+ T cell

subsets (Th1, Th2, and Th17) is involved in the pathogenesis ofRA via secretion of IFN-g, IL-4, and IL-17 (2, 5). In this study, weshowed that Th1- and Th17-polarizing conditions induced stableIL-37 expression, but the expression of IL-37 in CD4+ T cellspolarized under Th2 conditions was unaffected. Furthermore,Th1-secreted cytokine IFN-g and Th17-secreted cytokine IL-17were increased in CD4+ T cells polarized under Th1 and Th17conditions, respectively. It was shown that Th1-secreted cytokineIFN-g was highly effective in inducing IL-37, whereas Th2-secreted cytokine IL-4 plus GM-CSF inhibited constitutive IL-37 expression (8). Actually, as mentioned above (SupplementalFig. 1F), there was an obvious correlation between IL-37 andTh17-secreted cytokine IL-17. These observations suggest thatTh1/Th17 cytokines (IFN-g and IL-17) may play a part or a keyrole in inducing IL-37 production in CD4+ T cells. In contrast,Th2 cytokine IL-4 may provide an inhibitor signal for IL-37production 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 expressionin serum and synovial tissue from patients with RA was demon-

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 Ad-

EV or Ad–IL-37 were measured by ELISA. Ex-

pression 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 real-

time 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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strated in some studies (4). In accordance with previous data, wefound higher levels of IL-17 in serum and PBMCs from RApatients compared with HCs. One intriguing finding of our study isthat IL-17 production in CD4+ T cells and PBMCs from RApatients was significantly suppressed by rIL-37 treatment. Strik-ingly, IL-37 also markedly inhibited the percentage of IL-17+

CD4+ T cells from RA patients in vitro. It was shown recently thatcytokines IL-1b and IL-6 are very potent at inducing IL-17 se-cretion 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 andPBMCs from RA patients, IL-37 did not appear to exert its anti-inflammatory 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 anti-inflammatory role of IL-37 in IL-17 production in RA seems to beindependent of IL-1b. Although IL-1b is considered the criticalcytokine for IL-17 production in CD4+ T cells and Th17 celldifferentiation (23, 24), consistent with our results, IL-1 is se-lectively required for IL-17 secretion and Th17 cell induction(24), because IL-23 or IL-1 and IL-23, but not IL-1 alone, inducedpotent IL-17 gene transcription. Moreover, recent evidence sug-gests that, in addition to IL-1, Th17 cell differentiation is drivenby 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 speculatethat IL-37 exerts directly suppressive effects on IL-17 productioninvolved in regulating Th17 cell differentiation or proliferation. Instark contrast, our results suggest that IL-37 failed to significantlylimit the differentiation of Th17 cells, because there was no ob-vious alteration in the frequency of IL-17+CD4+ T cells in naiveCD4+ T cells treated with IL-37 under Th17 cell–differentiationconditions. Likewise, the transcription factor RORgt, which iscritical for Th17 cell generation (5), also was found to be largelyresistant to direct suppression by IL-37. Of great interest, IL-37dramatically limited Th17 cell proliferation in culture. Our presentstudy identified that in vitro administration of IL-37 inhibited IL-17 production that was associated with restriction of Th17 cellproliferation. Despite these results indicating that IL-37 has littleor no effect on Th17 cell differentiation directly, it is likely thatthe immunosuppressive effect of IL-37 on Th17 cell and IL-17production is through regulating dendritic cell (DC) function orphenotype. Actually, recent evidence suggests that IL-37 signifi-cantly suppressed Ag-specific adaptive immunity through the in-duction of tolerogenic DCs and the promotion of IL-10 expressionon DCs (27). Similarly, the presence of IL-37 markedly inhibitedDC function and Th17 cell differentiation–related cytokine pro-duction (e.g. IL-6 and TGF-b) (6, 8). Notably, DCs are the mainAPCs mediating Th17 responses by secreting anti-inflammatoryor proinflammatory cytokines, such as IL-6, TGF-b, and IL-10(28). These results indicate that IL-37 is capable of regulatingDC function and phenotype directly or indirectly and, thereby,inhibiting Th17- and IL-17–mediated inflammatory responsesin RA.In vivo, we attempted to confirm our findings of IL-37 as an anti-

inflammatory cytokine in mice with CIA by local intra-articularinjection of Ad–IL-37. As expected, overexpression of IL-37 inthe CIA model resulted in a delayed onset of disease and allevi-ated the severity of clinical symptoms. Furthermore, the antiar-thritic effect of IL-37 in CIA was associated with a reduction inIL-17– and IL-17–triggering cytokines (IL-1b and IL-6). Patho-genic Th17 cells secreting their signature proinflammatory cyto-kine, IL-17, were shown to promote inflammatory responses andto contribute to the pathogenesis of RA (3, 4). More importantly, itis generally regarded that IL-17 is the critical cytokine for in-

ducing proinflammatory cytokine IL-1b and IL-6 secretion inautoimmune diseases (4, 5). Therefore, it is plausible to suggestthat overproduction of IL-37 curbed RA disease activity and mightbe involved in suppressing the IL-17–mediated inflammatory re-sponse. Notably, the importance of IL-37 in modulating the in-flammatory response is also supported by recent data showing thatdecreased IL-37 expression in patients with Behcet’s diseasetriggers the Th17-inflammation response (29). Although the mo-lecular mechanism of IL-37 in autoimmune disease remains un-known, recent studies showed that IL-37 acts as an extracellularcytokine by binding IL-18R and IL-1R8 for its anti-inflammatoryproperties (30). In addition, IL-37 fails to inhibit IL-17–triggeringcytokine (IL-1b and IL-6) production and the MAPK signalingpathway in DCs from IL-1R8–deficient mice (30). A recent studyshowed that IL-17 expression was dramatically enhanced in IL-1R8–deficient mice (31). These results suggest that IL-37 maysuppress IL-17 and IL-17–triggering cytokine production in thedevelopment of RA via binding IL-1R8 and curbing the MAPKsignaling pathway in DCs. Nevertheless, further investigations andmanipulations of IL-37 signaling may improve therapeutic optionsfor RA patients.In summary, we demonstrate for the first time, to our knowledge,

that IL-37 levels were elevated in active RA and were associatedparticularly with Th17 cytokines and with Th17/Th1 differentiation.More importantly, the results reveal an anti-inflammatory effect ofIL-37 in human RA and the CIA model. IL-37 may mediate itsantiarthritic effects through inhibition of IL-17 and IL-17–triggeringcytokines and via suppression of Th17 cell proliferation. These datasuggest a possible therapeutic significance for IL-37 in RA.

AcknowledgmentsWe 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.

DisclosuresThe authors have no financial conflicts of interest.

References1. Firestein, G. S. 2003. Evolving concepts of rheumatoid arthritis. Nature 423:

356–361.2. McInnes, I. B., and G. Schett. 2007. Cytokines in the pathogenesis of rheumatoid

arthritis. Nat. Rev. Immunol. 7: 429–442.3. Gaffen, S. L. 2009. The role of interleukin-17 in the pathogenesis of rheumatoid

arthritis. Curr. Rheumatol. Rep. 11: 365–370.4. Hu, Y., F. Shen, N. K. Crellin, andW. Ouyang. 2011. The IL-17 pathway as a major

therapeutic target in autoimmune diseases. Ann. N. Y. Acad. Sci. 1217: 60–76.5. Miossec, P., and J. K. Kolls. 2012. Targeting IL-17 and TH17 cells in chronic

inflammation. Nat. Rev. Drug Discov. 11: 763–776.6. Boraschi, D., D. Lucchesi, S. Hainzl, M. Leitner, E. Maier, D. Mangelberger,

G. J. Oostingh, T. Pfaller, C. Pixner, G. Posselt, et al. 2011. IL-37: a new anti-inflammatory cytokine of the IL-1 family. Eur. Cytokine Netw. 22: 127–147.

7. Kumar, S., C. R. Hanning, M. R. Brigham-Burke, D. J. Rieman, R. Lehr,S. Khandekar, R. B. Kirkpatrick, G. F. Scott, J. C. Lee, F. J. Lynch, et al. 2002.Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production.Cytokine 18: 61–71.

8. Nold, M. F., C. A. Nold-Petry, J. A. Zepp, B. E. Palmer, P. Bufler, andC. A. Dinarello. 2010. IL-37 is a fundamental inhibitor of innate immunity. Nat.Immunol. 11: 1014–1022.

9. Ye, L., L. Ji, Z. Wen, Y. Zhou, D. Hu, Y. Li, T. Yu, B. Chen, J. Zhang, L. Ding,et al. 2014. IL-37 inhibits the production of inflammatory cytokines in peripheralblood mononuclear cells of patients with systemic lupus erythematosus: itscorrelation with disease activity. J. Transl. Med. 12: 69.

10. Zhao, P.-W., W.-G. Jiang, L. Wang, Z.-Y. Jiang, Y.-X. Shan, and Y.-F. Jiang.2014. Plasma levels of IL-37 and correlation with TNF-a, IL-17A, and diseaseactivity during DMARD treatment of rheumatoid arthritis. PLoS ONE 9: e95346.Available at: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095346.

11. Lopetuso, L. R., S. Chowdhry, and T. T. Pizarro. 2013. Opposing Functions ofClassic and Novel IL-1 Family Members in Gut Health and Disease. Front.Immunol. 4: 181.

The Journal of Immunology 9

by guest on April 3, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

12. Teng, X., Z. Hu, X. Wei, Z. Wang, T. Guan, N. Liu, X. Liu, N. Ye, G. Deng,C. Luo, et al. 2014. IL-37 ameliorates the inflammatory process in psoriasis bysuppressing proinflammatory cytokine production. J. Immunol. 192: 1815–1823.

13. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries,N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al. 1988.The American Rheumatism Association 1987 revised criteria for the classifi-cation of rheumatoid arthritis. Arthritis Rheum. 31: 315–324.

14. Aletaha, D., T. Neogi, A. J. Silman, J. Funovits, D. T. Felson, C. O. Bingham, III,N. S. Birnbaum, G. R. Burmester, V. P. Bykerk, M. D. Cohen, et al. 2010.2010 Rheumatoid arthritis classification criteria: an American College ofRheumatology/European League Against Rheumatism collaborative initiative.Arthritis Rheum. 62: 2569–2581.

15. van Tuyl, L. H., S. C. Vlad, D. T. Felson, G. Wells, and M. Boers. 2009. Definingremission in rheumatoid arthritis: results of an initial American College ofRheumatology/European League Against Rheumatism consensus conference.Arthritis Rheum. 61: 704–710.

16. Yang, M., J. Deng, Y. Liu, K. H. Ko, X. Wang, Z. Jiao, S. Wang, Z. Hua, L. Sun,G. Srivastava, et al. 2012. IL-10-producing regulatory B10 cells amelioratecollagen-induced arthritis via suppressing Th17 cell generation. Am. J. Pathol.180: 2375–2385.

17. Zhu, S., W. Pan, X. Song, Y. Liu, X. Shao, Y. Tang, D. Liang, D. He, H. Wang,W. Liu, et al. 2012. The microRNA miR-23b suppresses IL-17-associated au-toimmune inflammation by targeting TAB2, TAB3 and IKK-a. Nat. Med. 18:1077–1086.

18. Ye, L., Z. Wen, Y. Li, B. Chen, T. Yu, L. Liu, J. Zhang, Y. Ma, S. Xiao, L. Ding,et al. 2014. Interleukin-10 attenuation of collagen-induced arthritis is associatedwith suppression of interleukin-17 and retinoid-related orphan receptor gt pro-duction in macrophages and repression of classically activated macrophages.Arthritis Res. Ther. 16: R96.

19. Badolato, R., and J. J. Oppenheim. 1996. Role of cytokines, acute-phase pro-teins, and chemokines in the progression of rheumatoid arthritis. Semin. ArthritisRheum. 26: 526–538.

20. Rosu, A., C. M�arg�aritescu, A. Stepan, A. Musetescu, and M. Ene. 2012. IL-17patterns in synovium, serum and synovial fluid from treatment-naıve, earlyrheumatoid arthritis patients. Rom. J. Morphol. Embryol. 53: 73–80.

21. Zizzo, G., M. De Santis, S. L. Bosello, A. L. Fedele, G. Peluso, E. Gremese,B. Tolusso, and G. Ferraccioli. 2011. Synovial fluid-derived T helper 17 cells

correlate with inflammatory activity in arthritis, irrespectively of diagnosis. Clin.Immunol. 138: 107–116.

22. Holoshitz, J., F. Koning, J. E. Coligan, J. De Bruyn, and S. Strober. 1989. Iso-lation of CD42 CD82 mycobacteria-reactive T lymphocyte clones from rheu-matoid arthritis synovial fluid. Nature 339: 226–229.

23. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, and F. Sallusto. 2007.Interleukins 1beta and 6 but not transforming growth factor-beta are essential forthe differentiation of interleukin 17-producing human T helper cells. Nat.Immunol. 8: 942–949.

24. Sutton, C., C. Brereton, B. Keogh, K. H. Mills, and E. C. Lavelle. 2006. Acrucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells thatmediate autoimmune encephalomyelitis. J. Exp. Med. 203: 1685–1691.

25. Manel, N., D. Unutmaz, and D. R. Littman. 2008. The differentiation of humanT(H)-17 cells requires transforming growth factor-beta and induction of the nu-clear receptor RORgammat. Nat. Immunol. 9: 641–649.

26. Volpe, E., N. Servant, R. Zollinger, S. I. Bogiatzi, P. Hupe, E. Barillot, andV. Soumelis. 2008. A critical function for transforming growth factor-beta, in-terleukin 23 and proinflammatory cytokines in driving and modulating humanT(H)-17 responses. Nat. Immunol. 9: 650–657.

27. Luo, Y., X. Cai, S. Liu, S. Wang, C. A. Nold-Petry, M. F. Nold, P. Bufler,D. Norris, C. A. Dinarello, and M. Fujita. 2014. Suppression of antigen-specificadaptive immunity by IL-37 via induction of tolerogenic dendritic cells. Proc.Natl. Acad. Sci. USA 111: 15178–15183.

28. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, and S. Amigorena. 2002.Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev.Immunol. 20: 621–667.

29. Ye, Z., C. Wang, A. Kijlstra, X. Zhou, and P. Yang. 2014. A possible role forinterleukin 37 in the pathogenesis of Behcet’s disease. Curr. Mol. Med. 14: 535–542.

30. Li, S., C. P. Neff, K. Barber, J. Hong, Y. Luo, T. Azam, B. E. Palmer, M. Fujita,C. Garlanda, A. Mantovani, et al. 2015. Extracellular forms of IL-37 inhibitinnate inflammation in vitro and in vivo but require the IL-1 family decoy re-ceptor IL-1R8. Proc. Natl. Acad. Sci USA. 112: 2497–2502.

31. Bozza, S., T. Zelante, S. Moretti, P. Bonifazi, A. DeLuca, C. D’Angelo,G. Giovannini, C. Garlanda, L. Boon, F. Bistoni, et al. 2008. Lack of Toll IL-1R8exacerbates Th17 cell responses in fungal infection. J. Immunol. 180: 4022–4031.

10 IL-37 SUPPRESSES Th17 RESPONSE IN ARTHRITIS

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