Th17 Cytokines Regulate Osteoclastogenesis in Rheumatoid Arthritis

Th17 Cytokines Regulate Osteoclastogenesis in Rheumatoid Arthritis

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The American Journal of Pathology, Vol. -, No. -, - 2015

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Th17 Cytokines Regulate Osteoclastogenesis in Rheumatoid Arthritis Q26

Kyoung-Woon Kim,* Hae-Rim Kim,y Bo-Mi Kim,* Mi-La Cho,*z and Sang-Heon Leey From the Conversant Research Consorcium in Immunologic Disease,* Seoul St. Mary’s Hospital, College of Medicine, and the Rheumatism Research Center,z The Catholic University of Korea, Seoul; and the Division of Rheumatology,y Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Republic of Korea Accepted for publication July 30, 2015. Address correspondence to Sang-Heon Lee, M.D., Ph.D., Division of Rheumatology, Department of Internal Medicine, Konkuk University School of Medicine, 4-12 Hwayangdong, Gwangjin-gu, Seoul 143-729, Republic of Korea; or Mi-La Cho, Ph.D., Rheumatism Research Center, Catholic Institutes of Medical Science, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-040, Republic of Korea. E-mail: [email protected] or iammila@ catholic.ac.kr.

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This study determined the regulatory effect of type 17 helper T-cell (Th17) cytokines on osteoclastogenesis in rheumatoid arthritis (RA). The expression of IL-17 and receptor activator of NF-kB ligand (RANKL) was determined in synovial tissue, fibroblast-like synoviocytes (FLSs), and synovial fluids of RA patients using immunohistochemical staining, enzyme-linked immunosorbent assay, and real-time PCR. Th17 cytokineeinduced RANKL expression was studied in RA FLS by using real-time PCR, luciferase activity assays, and Western blot analysis. Human peripheral blood monocytes were cultured with macrophage colony-stimulating factor and Th17 cytokines, after which osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphataseepositive multinucleated cells. Osteoclastogenesis was also evaluated after human monocytes were co-cultured with IL-17e prestimulated FLS. There was significant correlation between RANKL and IL-17 levels in RA synovial fluid. IL-17, IL-21, and IL-22 increased the expression of Rankl mRNA in RA FLS, and the IL-17einduced RANKL expression decreased by the inhibition of Act1, tumor necrosis factor receptoreassociated factor 6, NF-kB, and activator protein-1. Th17 cytokines and IL-17eprestimulated FLS induced osteoclastogenesis from monocytes in the absence of exogenous RANKL. The osteoclastic effect was reduced by inhibition of tumor necrosis factor-a. Th17 cytokines have a dual effect on osteoclastogenesis in RA: direct induction of osteoclastogenesis from monocytes and up-regulation of RANKL production in RA FLS. This Th17 cytokine/RANKL axis could be a potential therapeutic target for bone destruction in RA. (Am J Pathol 2015, -: 1e14; http://dx.doi.org/10.1016/j.ajpath.2015.07.017)

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovitis and subsequent destruction of cartilage and bone. Osteoclasts are detected in synovial tissues and in the eroded bone surfaces, suggesting that osteoclastic bone resorption is involved in the pathogenesis of RA.1,2 Type 17 helper T cells (Th17), which are defined by selective IL-17 secretion, are considered a distinct lineage of CD4þ helper T cells, which are regulated by Th1 and Th2 cytokines.3e5 IL-17A is a dominant proinflammatory cytokine produced by type 17 helper T cells (Th17).6,7 IL-21, IL-22, and IL-26 are also produced by Th17 cells. In RA, IL-17A induces the production of proinflammatory mediators, such as IL-1 and tumor necrosis factor (TNF)-a from synovial fibroblasts, macrophages, and chondrocytes. Levels of IL-17 produced by CD4þ T cells in the synovium are significantly higher in patients with RA than in patients

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with osteoarthritis (OA).8,9 The major roles of IL-17 are to coordinate local inflammation through the up-regulation of proinflammatory and neutrophil-mobilizing cytokines and chemokines, including IL-6, granulocyte colony-stimulating factor, TNF-a, IL-1b, CXCL1 (KC), chemokine (C-C Q7 motif) ligand 2 (monocyte chemoattractant protein-1), CXCL2 (macrophage inflammatory protein-2), chemokine (C-C motif) ligand 7 (monocyte chemoattractant protein-3), and chemokine (C-C motif) ligand 20 (macrophage Supported by the Korea Healthcare Technology R&D Project grant Q2 A092258, the Ministry for Health, Welfare, and Family Affairs, Republic of Korea, the National Research Foundation of Korea grant, and Korean Government grants NRF-2010-0024198, NRF-2013R1A1A1008171, and Q3 NRF-2014R1A2A2A01007223. K.-W.K. and H.-R.K. contributed equally to this work, and M.-L.C. and S.-H.L. contributed equally as senior authors. Disclosures: None declared.

Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.07.017

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inflammatory protein-3A), as well as matrix metalloproteases, and to enable activated T cells to penetrate the extracellular matrix. IL-17A also induces receptor activator of NF-kB ligand (RANKL) expression by synovial fibroblasts and osteoblasts to drive bone erosion indirectly10 and to activate synovial macrophages to secrete TNF-a and IL-1b, two known osteoclastogenic factors.11 Synovial macrophages can be differentiated into fully functional bone-resorbing osteoclasts, and Th17-induced differentiation of synovial macrophages into osteoclasts represents an important cellular mechanism in bone destruction associated with RA.12,13 Act1 adaptor protein is required for the development of joint pathology in the collagen-induced arthritis (CIA) model.14 Act1 (alias CIKS) is an adaptor protein required for IL-17A signaling and has an expression level similar to that of the fibroblast growth factor/IL-17 receptor (SEFIR) domain. On IL-17 stimulation, Act1 is recruited to IL-17 receptors through its SEFIR domain and is required for IL17emediated expression of genes involved in inflammation. Interestingly, Act1 is also needed for IL-17edependent mRNA stability.15 Although the contribution of IL-17 in osteoclastogenesis using cultures of human monocytes alone has been documented in earlier studies,16 the intracellular signal transduction pathway for RANKL production and osteoclast differentiation by IL-17 remains uncertain. Herein, we investigated the effect of Th17 cytokines on RANKL production in fibroblast-like synoviocytes (FLSs) and osteoclast differentiation from peripheral blood monocytes in RA, and we determined the signal transduction pathways mediating IL-17einduced osteoclastogenesis. We found that Th17 cytokines, especially IL-17, regulated osteoclastogenesis through both direct and indirect pathways. Indirectly, IL-17 induced RANKL production of RA FLS and, directly, it induced osteoclast differentiation from osteoclast precursors. Act1/TNF receptoreassociated factor (TRAF) 6/NF-kB and activator protein (AP)-1 mediated IL-17einduced RANKL production and osteoclast differentiation.

Isolation of FLS FLSs were isolated by enzymatic digestion of synovial tissues obtained from RA and OA patients undergoing total knee replacement surgery, as described previously.18

Reagents Recombinant IL-17, IL-21, IL-22, receptor activator of NFkB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (Minneapolis, MN). Pyrrolidine dithiocarbamate, curcumin, and parthenolide were obtained from Sigma (St. Louis, MO). Q9 LY294002, SB203580, SP600125, and AG490 were obtained from Calbiochem (Schwalbach, Germany). TRAF6 inhibitor peptide was purchased from IMGENEX (San Diego, CA).

Immunohistochemistry of RA Synovium Immunohistochemical staining for RANKL was performed with sections of synovium. Briefly, synovium was obtained from patients with RA and OA, fixed with 4% paraformaldehyde solution overnight at 4 C, dehydrated with alcohol, washed, embedded in paraffin, and divided into sections (7 mm thick). Sections were depleted of endogenous peroxidase activity by adding methanolic hydrogen peroxide and blocked with normal serum for 30 minutes. After overnight incubation at 4 C with polyclonal antihuman RANKL antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the samples were incubated with the secondary antibody, biotinylated anti-rabbit IgG, for 20 minutes, and then incubated with streptavidin-peroxidase complex (Vector, Peterborough, UK) for 1 hour, followed by incubation with 3,30 -diaminobenzidine (Dako, Glostrup, Denmark) for 5 minutes. The sections were counterstained with hematoxylin. Samples were finally imaged with an Olympus (Tokyo, Japan) photomicroscope.

Enzyme-Linked Immunosorbent Assay

Materials and Methods Patients Samples of RA synovium were isolated from seven patients with RA and five patients with OA (age range, 32 to 70 years; mean age, 56.6  4.7 years), who were undergoing total knee replacement surgery. Synovial fluid was obtained from 19 RA patients who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association)17 and 19 patients who had symptomatic knee OA. Informed consent was obtained from all patients, and the experimental protocol was approved by the Konkuk University School of Medicine (Seoul, Republic of Korea) Human Research Ethics Committee.

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In brief, a 96-well plate (Nunc) was coated with 4 mg/mL Q10 monoclonal antibodies against soluble RANKL (sRANKL) and IL-17 (R&D Systems) at 4 C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin/ 0.05% Tween 20 for 2 hours at room temperature (22 C to 25 C), test samples and the standard recombinant sRANKL and IL-17 (R&D Systems) were added to the 96-well plate and incubated at room temperature for 2 hours. Plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/mL biotinylated mouse monoclonal antibodies against sRANKL and IL-17 (R&D Systems) for 2 hours at room temperature. After washing, streptavidinealkaline phosphateehorseradish peroxidase conjugate (Sigma) was incubated for 2 hours, then washed again and incubated with 1 mg/mL p-nitrophenyl phosphate

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Th17 Cytokines and Osteoclastogenesis Q1 Table 1

Primers Used for PCR

Primer name Rankl b-Actin Gapdh Act1 Traf6 Traf3 Trap Rank Cathepsin K Ctr Mmp9

Forward primer 0

Reverse primer 0

5 -ACCAUCATCAAAATCCCAAG-3 50 -GGACTTCGAGCAAGAGATGG-30 50 -CGATGCTGGGCGTGAGTAC-30 50 -GCATTCCTGTGGAGGTTGAT-30 50 -CCAATTTGCACATTCAG-30 50 -GGCCGTTTAAGCAGAAAGTG-30 50 -GACCACCTTGGCAATGTCTCTG-30 50 -GCTCTAACAAATGTGAACCAGGA-30 50 -TGAGGCTTCTCTTGGTGTCCATAC-30 50 -TGGTGCCAACCACTATCCATGC-30 50 -CGCAGACATCGTCATCCAGT-30

50 -CCCCAAAGTATGTTGCATCC-30 50 -TGTGTTGGGGTACAGGTCTTTG-30 50 -CGTTCAGCTCAGGGATGACC-30 50 -GTCTCCGGAGGAATTGTGAA-30 50 -GGGCCAACATTCTCATGTGT-30 50 -ACAGTTTGGGCCACAAAGAC-30 50 -TGGCTGAGGAAGTCTCTGAGTTG-30 50 -GCCTTGCCTGTATCACAAACT-30 50 -AAAGGGTGTCATTACTGCGGG-30 50 -CACAAGTGCCGCCATGACAG-30 50 -GGATTGGCCTTGGAAGATGA-30

Act, ---; Ctr, ---; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Mmp, matrix metalloprotease; Rankl, receptor activator of NF-kB ligand; Traf, tumor necrosis factor receptoreassociated factor; Trap, tartrate-resistant acid phosphatase.

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(Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 mol/L NaOH, and the optical density of each well was read at 405 nm. The lower limit of sRANKL and IL-17 detection was 10 pg/mL. Recombinant human sRANKL and IL-17 diluted in culture medium were used as a calibration standard, ranging from 10 to 2000 pg/mL. A standard curve was drawn by plotting optical density against the log of the concentration of recombinant cytokines, and used for determination of sRANKL and IL-17 concentrations in test samples.

Dual-Luciferase Assay

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RANKL promoter activity was assessed by measurements of luciferase expression in RA FLS with transient transfection of appropriate cDNA constructs and luciferase reporter vectors (namely, pGL3-704) to 111. Another reporter plasmid, Renilla luciferase (Promega, Madison, WI), was cotransfected as an internal transfection control. RANKL transcription was evaluated with pGL3-RANKL-luc plasmid containing the luciferase gene under the control of the RANKL-inducible promoter. Transient transfection of RA FLS by FuGENE HD (Promega) was previously described.11 Briefly, cells were seeded in 6-well plates at 2  105 cells per well 1 day before transfection. Transfection was performed according to the manufacturer’s recommendations using 1 mg of pGL3-RANKL reporter plasmids and 1 mg of pRLTk control plasmid, 24 hours after transfection cells were stimulated or left unstimulated for 24 hours. After 24 hours, cells were lysed by 500 mL of Passive Lysis Buffer 1 supplied by Promega. Luciferase assays were performed using the dual-luciferase reporter assay kit (Promega).

Real-Time PCR FLSs were stimulated with various concentrations of IL-17 (0.1, 1, 10, 20, and 50 ng/mL). For signal pathway analysis of RANKL, FLSs were incubated in the presence or

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absence of 10 mmol/L parthenolide, 10 mmol/L curcumin, 20 mmol/L Ly294002, 10 nmol/L SB203580, 1 mmol/L SP600125, or 50 mmol/L AG490 for 1 hour before the addition of IL-17. After incubation for 72 hours, mRNA was extracted using RNAzol B (Biotex Laboratories, Houston, TX), according to the manufacturer’s instructions. Reverse transcription of 2 mg of total mRNA was performed at 42 C using the Superscript reverse transcription system (Takara, Shiga, Japan). PCR was performed in a 20mL final volume in capillary tubes in a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). The reaction mixture contained 2 mL of LightCycler FastStart DNA MasterMix for SYBR Green I (Roche Diagnostics), 0.5 mmol/L of each primer, 4 mmol/L MgCl2, and 2 mL of template DNA. All capillaries were sealed, centrifuged at 500 X g for 5 seconds, and then amplified in a LightCycler instrument, with activation of polymerase (95 C for 10 minutes), followed by 45 cycles of 10 seconds at 95 C, 10 seconds at 60 C (b-actin) and at 59 C (Rankl ), and 10 seconds at 72 C. The temperature transition rate was 20 C/ second for all steps. The double-stranded PCR product was measured during the 72 C extension step by detection of fluorescence associated with the binding of SYBR Green I to the product. Fluorescence curves were analyzed with LightCycler software version 3.0 (Roche Diagnostics). The LightCycler was used to quantify Rankl mRNA. The relative expression level of each sample was calculated as the level of Rankl normalized to the endogenously expressed b-actin gene. Melting curve analysis was performed immediately after the amplification protocol under the following conditions: 0 second (hold time) at 95 C, 15 seconds at 71 C, and 0 second (hold time) at 95 C. The rate of temperature change was 20 C/second, except for 0.1 C/ second in the final step. The melting peak generated represented the amount of specific amplified product. The crossing point was defined as the maximum of the second derivative from the fluorescence curve. Negative controls were also included and contained all of the elements of the reaction mixture except template DNA. All samples were processed in duplicate.

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Expression of IL-17 and receptor activator of NF-kB ligand (RANKL) in synovial tissues and fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients. A: Immunohistochemical staining of IL-17, Act1, RANKL, and RANK in the synovial tissues. There is more intense expression of IL-17, Act1, RANKL, and RANK in RA synovial tissues compared with osteoarthritis (OA) synovial tissues. The figures are representative of three independent experiments. B: RA FLS, but not OA FLS, constitutively expresses RANKL protein, which was determined by Western blot analysis. The amount of protein expression was normalized by b-actin. The figures are representative of three independent experiments. C: Synovial fluid levels of IL-17 and soluble RANKL (sRANKL) in RA patients were measured using an enzyme-linked immunosorbent assay. D: RA FLSs were transfected with 1 mg of pGL3-RANKL reporter plasmids and 1 mg of pRLTk control plasmid. Both firefly and renilla luminescences were measured after 24 hours’ incubation. Data represent means  SEM of three independent experiments (B and D). n Z 19 (C). ***P < 0.005. Original magnification, 400 (A). H&E, hematoxylin and eosin.

Figure 1

RT-PCR FLSs were incubated with various concentrations of IL-17, IL-21, or IL-22. After incubation for 72 hours, mRNA was extracted using RNAzol B (Biotex Laboratories), according to the manufacturer’s instructions. Reverse transcription of 2 mg total mRNA was performed at 42 C using the Superscript reverse transcription system (Takara). PCR amplification of cDNA aliquots was performed by adding 2.5 mmol/L dNTPs, 2.5 U Taq DNA polymerase (Takara), and 0.25 mmol/L of sense and antisense primers. The reaction was performed in PCR buffer (1.5 mmol/L MgCl2, 50

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mmol/L KCl, and 10 mmol/L Tris-HCl, pH 8.3) in a total volume of 25 mL. The following sense and antisense primers for each molecule were used (50 /30 ) (Table 1). Reactions ½T1 were processed in a DNA thermal cycler (PerkinElmer Cetus, Wellesley, MA). PCR products were run on a 2% agarose gel and stained with ethidium bromide. Results are expressed as the ratio of Rankl (forward primer, 50 ACCAGCATCAAAATCCCAAG-30 ; reverse primer, 50 CCCCAAAGTATGTTGCATCC-30 ) and PCR product relative to Gapdh (forward primer, 50 -CGATGCTGGGCGTGAGTAC-30 ; reverse primer, 50 -CGTTCAGCTCAGGGATGACC-30 ).

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Th17 Cytokines and Osteoclastogenesis

Western Blot Analysis

hours later. Act1 and control were purchased from Dharmacon Inc. (Chicago, IL).

FLSs were incubated with IL-17. After incubation for 72 hours, whole-cell lysates were prepared from approximately 2  105 cells by homogenization in the lysis buffer, and centrifuged at Q13 14,000  rpm for 15 minutes. The protein concentration in the supernatant was determined using the Bradford method (BioRad, Hercules, CA). Protein samples were separated onto 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membrane was preincubated with 0.5% skim milk in 0.1% Tween 20 in Tris-buffered saline at room temperature for 2 hours. The primary antibody to RANKL (R&D Systems), diluted 1:1000 in 5% bovine serum albumine0.1% Tween 20/Tris-buffered saline, was added and incubated for overnight at 4 C. The membrane was washed four times with 0.1% Tween 20 in Tris-buffered saline, and horseradish peroxidaseeconjugated secondary antibody was added and incubated for 1 hour at room temperature. After 0.1% Tween 20 in Tris-buffered saline washing, hybridized bands were detected using the electrochemiluminescence detection kit Q14 Q15 and Hyperfilm-ECL reagents (Amersham Pharmacia).

Isolation of Peripheral Blood Monocytes Peripheral blood mononuclear cells were separated by FicollHypaque (Sigma Chemicals, Poole, Dorset, UK) density gradient centrifugation from the buffy coats obtained from healthy volunteers. The cells were washed three times with sterile phosphate-buffered saline and resuspended in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 1% penicillin-streptomycin, henceforth called complete medium. Freshly isolated peripheral blood mononuclear cells were incubated at 37 C in complete medium and allowed to adhere for 45 minutes. The nonadherent cells were removed, and the adherent cells were washed with sterile phosphate-buffered saline, harvested with a rubber policeman, and stained with Q16 the monocyte-specific anti-CD14 monoclonal antibody to assess the purity of the preparation. Of the isolated cells, 90% expressed CD14. The osteoclast precursors were prepared using the monocytes-enriched fraction from the peripheral blood.

Magnetofection of Oligonucleotides

Osteoclast Differentiation

siRNAs were transfected using magnetofection, as described previously,19,20 and the assays were performed 24

RA synovial fibroblasts were pretreated with 10 ng/mL rhIL-17 for 3 days, after which monocytes were added to Q17

Figure 2 The expression of receptor activator of NF-kB ligand (RANKL) is up-regulated by type 17 helper T-cell cytokines in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). A: RA FLSs were cultured for 72 hours with 0 to 50 ng/mL IL17, and Rankl mRNA expression was determined using real-time PCR. The data were expressed as the relative mRNA expression of Rankl/b-actin. B: RA FLSs were cultured with 50 ng/mL of IL-17 for 72 hours, and the expression of RANKL protein in cell lysates was determined using Western blot analysis. The amount of RANKL expression was normalized by b-actin. The figures are representative of three independent experiments. C: RA FLSs were cultured with 50 ng/mL of IL-17 for 72 hours, and the RANKL concentration of cultured media was determined using an enzyme-linked immunosorbent assay. D: RA FLSs were cultured for 72 hours with IL-21 or IL-22, and Rankl mRNA expression was determined using real-time PCR. The data were expressed as the relative mRNA expression of Rankl/b-actin. The figures are representative of three independent experiments. Data represent means  SEM of three independent experiments (). n Z 3 (D). **P < 0.01, ***P < 0.005. sRANKL, soluble RANKL.

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each well along with the different signal inhibitors, including 20 mmol/L LY294002, 50 mmol/L AG490, 10 mmol/L SB203580, 10 mmol/L parthenolide, or 10 mmol/L curcumin. As described above, the isolated human monocytes (5  104 cells per well) were added to the stimulated fibroblasts with fresh media. The cells were co-cultured for 3 weeks in a-minimal essential medium and 10% heatinactivated fetal bovine serum in the presence of 25 ng/ mL of rhM-CSF. The medium was changed on day 3 and then every other day. The addition of rhRANKL protein, prepared as described previously,21 was used as the positive control. On day 21, tartrate-resistant acid phosphatase (TRAP)epositive cells were identified using a leukocyte acid phosphatase kit, according to the manufacturer’s protocol (Sigma-Aldrich).22

Bone Resorption Assay Q19

We performed an in vitro resorption pit assay using a bone resorption assay kit (Cosmo Bio Co, Ltd). Monocytes were cultured on a bone-coating plate with M-CSF in the presence or absence of various concentrations of Th17 cytokines (IL-17, IL-21, and IL-22) for 14 days. The cells were removed from the bone-coating plate by wiping the surface. The numbers of pit formed by bone resorption on the plate were counted.

Statistical Analysis Data are expressed as the means  SEM. Statistical analysis was performed using one-way analysis of variance, followed by the Dunnett’s multiple-comparison test for comparison of more than two experimental groups. P < 0.05 was considered statistically significant.

Results Expression of IL-17, Act1, RANKL, and RANK in RA Synovial Tissues IL-17, Act1, RANKL, and RANK, identified by immunohistochemical staining, were expressed intensively in RA synovial tissues, compared with OA synovium (Figure 1A). ½F1 Specifically, IL-17, Act1, RANKL, and RANK were abundantly expressed in the lining and sublining of RA synovium, whereas their expression was faint in OA synovium. The basal expression of RANKL protein was higher in RA FLS than in OA FLS, as determined by Western blot analysis (Figure 1B). Synovial fluid IL-17 (RA versus OA, 254.7  49.9 versus 70.1  98.2 pg/mL; P < 0.001) and sRANKL (RA versus OA, 280.4  199.9 versus 215.6  105.0 pg/mL; P < 0.01) levels were much higher in 19 RA patients than in 19 OA patients. Synovial sRANKL and synovial IL-17 levels were

Figure 3

The IL-17einduced receptor activator of NF-kB ligand (RANKL) expression is mediated through Act1 and tumor necrosis factor receptoreassociated factor (TRAF) 6 pathways. A: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) were cultured for 72 hours with IL-17, and the expression of Act1 was determined using real-time PCR. The data were expressed as the relative mRNA expression of Act1/b-actin. B: RA FLSs were cultured with 50 ng/mL of IL-17 and Act1 siRNA or TRAF6 inhibitor peptide for 72 hours, and the mRNA expression of Act1, Traf6, and Traf3 was determined by real-time PCR. The amount of target gene expression was normalized by b-actin. Data are shown as the relative mRNA expression of target/b-actin. C: The protein expression of Act1 and TRAF6 by IL-17 was determined by Western blot analysis. The figures are representative of three independent experiments. The data are shown as target protein/b-actin ratio. D: IL-17einduced Rankl expression in RA FLS, which was determined by real-time PCR. The data were expressed as the relative mRNA expression of Rankl/b-actin. Data represent means  SEM of three independent experiments (AeD). *P < 0.05, **P < 0.01, and ***P < 0.005.

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Th17 Cytokines and Osteoclastogenesis

correlated in 19 RA patients (r2 Z 0.48, P < 0.05) (Figure 1C). The patients included 4 men and 15 women, with a mean age of 50.4  1.5 years (range, 23 to 77 years) and a mean disease duration of 71.5  8.2 months (range, 3 to 240 months). To investigate RANKL-promoting activity by IL-17, we performed a pGL3-luciferase reporter gene assay using pGL3-RANKL Luc and Renilla Luc transfection. IL-17 increased RANKL activity by 105-fold compared with the unstimulated condition (Figure 1D).

Th17 Cytokines Stimulate RA FLS to Express and Produce RANKL After RA FLSs were stimulated with various dosages of IL17, the expression of Rankl mRNA was determined using real-time PCR. IL-17 induced the expression of Rankl ½F2 mRNA in a dose-dependent manner (Figure 2A). No additive effect was observed on RANKL expression after stimulation with a combination of IL-17 and other proinflammatory cytokines, such as TNF-a and IL-1b (data not shown). Neither a cytotoxic effect nor a proliferative effect was observed on RA FLS at the experimental dosages of IL17 (data not shown). The expression and production of

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807 808 Figure 4 Downstream signal pathways medi809 ating IL-17einduced receptor activator of NF-kB 810 ligand (RANKL) expression in rheumatoid arthritis 811 (RA) fibroblast-like synoviocytes (FLSs). A: RA FLSs 812 were pretreated with 20 mmol/L of LY294002 (LY), 813 10 nmol/L of SB203580 (SB), 1 mmol/L of SP600125 (SP), 1 mmol/L of AG490 (AG), 10 mmol/ 814 L of parthenolide (PA), 0.1 mmol/L of BAY11-7085 815 (BA), 10 mmol/L of curcumin (CU), or 1 mmol/L of 816 SR11302 (SR) for 1 hour, and then they were 817 cultured with 50 ng/mL of IL-17 for 72 hours. 818 Rankl mRNA was extracted and quantified using 819 quantitative real-time PCR. The amount of Rankl 820 expression was normalized by b-actin. The data 821 were expressed as the relative mRNA expression of 822 Rankl/b-actin. B: RA FLSs were cultured with IL-17 823 and 10 mmol/L of pyrrolidine dithiocarbamate (PDTC) or 10 mmol/L of curcumin, and then the 824 RANKL concentration was measured in the cultured 825 media using enzyme-linked immunosorbent assay. 826 C: RA FLSs were pretreated with PDTC or curcumin 827 for 1 hour, and then cultured with 50 ng/mL of IL828 17 for 72 hours. Rankl mRNA was extracted and 829 quantified using RT-PCR. The data were expressed 830 as the relative mRNA expression of Rankl/b-actin. 831 D: RA FLSs were cultured with 50 ng/mL of IL-17 832 and then the phosphorylation of IkBa and c-Jun 833 was assessed in cell lysates using Western blot analysis. The figures are representative of three 834 independent experiments. The data were expressed 835 as the relative protein expression of targets/b836 actin. Data represent the means  SEM of three 837 independent experiments (AeD). ***P < 0.005. 838 sRANKL, soluble RANKL. 839 840 841 842 RANKL protein were also increased with IL-17 stimulation 843 in RA FLS, as determined by Western blot analysis 844 (Figure 2B) and enzyme-linked immunosorbent assay in 845 culture media (Figure 2C). After RA FLSs were stimulated 846 with other Th17 cytokines, IL-21 and IL-22, the expression 847 of Rankl mRNA was determined using RT-PCR. IL-21 and 848 IL-22 also increased the expression of Rankl mRNA in a 849 dose-dependent manner (Figure 2D). 850 851 852 Act1 and TRAF6 Mediate IL-17eInduced RANKL 853 Expression 854 855 In a previous study, IL-17 was found to increase Act1 ac856 tivity in human fibroblasts, the biological function of which 857 is TRAF6 dependent.23 To determine the intracellular 858 pathways of IL-17einduced RANKL expression in RA 859 FLS, we explored the regulatory effect of IL-17 on Act1 860 activation. IL-17 increased the expression of Act1 mRNA in 861 RA FLS in a dose-dependent manner (Figure 3A). Then, by ½F3 862 using Act1 siRNA, the expression of TRAF6 and TRAF3 863 was observed. Act1 siRNA blocked both IL-17einduced 864 865 Act1 and TRAF6 expression, and TRAF6 inhibitor peptide 866 decreased TRAF6 expression (Figure 3, B and C). IL867 17einduced Rankl expression was significantly decreased 868

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869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 Figure 5 Type 17 helper T-cell (Th17) cytokines differentiate peripheral blood monocytes into osteoclasts. Human peripheral blood CD14þ monocytes were 895 isolated and cultured with 25 ng/mL of macrophage colony-stimulating factor and Th17 cytokines, IL-17, IL-21, or IL-22, and the osteoclast differentiation 896 was determined by counting tartrate-resistant acid phosphatase (TRAP)þ multinucleated cells and bone resorption assay. The cultures were stained with TRAP, 897 and the number of TRAPþ multinucleated cells was counted. The figures are representative of three independent experiments. *P < 0.05, **P < 0.01. 898 899 with Act1 and TRAF6 inhibition (Figure 3D). This result intracellular signaling molecules by IL-17 in RA FLS, the 900 suggests that IL-17einduced RANKL expression is mediphosphorylation of IkB-a and c-Jun was evaluated by Western 901 902 ated through an Act1 and TRAF6 pathway. blot analysis. The activation of NF-kB was analyzed by 903 monitoring the phosphorylation of IkB-a, and the activation of 904 AP-1 was analyzed by monitoring the phosphorylation of cIL-17eInduced RANKL Expression Is Mediated by NF905 Jun. IL-17 increased the phosphorylation of IkB-a and c-Jun kB and AP-1 Signaling Pathways 906 in RA FLS, and concomitantly increased the production of 907 To determine the downstream signaling pathways mediating RANKL, whereas the total protein levels of IkB-a and c-Jun 908 the up-regulation of RANKL by IL-17, we used 20 mmol/L of remained unchanged (Figure 4D). 909 LY294002, 10 mmol/L of SB203580, 1 mmol/L of SP600125, 910 50 mmol/L of AG490, 10 mmol/L of parthenolide, and 0.1 The Effect of Th17 Family Cytokines on Osteoclast 911 mmol/L of BAY11-7085, 10 mmol/L of curcumin, and 1 mmol/ Differentiation from Monocytes 912 L of SR11302 that antagonize the activation of phosphatidy913 linositol 3-kinase, p38 mitogen-activated protein kinase Human peripheral blood monocytes can differentiate into 914 915 Q20 (MAPK), c-Jun N-terminal kinase, STAT3, NF-kB, and AP-1, TRAP-positive multinucleated OCs in the presence of Q21 916 respectively. After RA FLSs were cultured with IL-17 and the RANKL and M-CSF.10 To evaluate whether Th17 cyto917 various signal inhibitors, expression of Rankl mRNA by FLS kines also have a role in the differentiation into OCs, human 918 was evaluated by RT-PCR and real-time PCR. IL-17einduced monocytes isolated from peripheral blood were cultured 919 Rankl expression was significantly decreased after inhibiting with IL-17, IL-21, IL-22, and M-CSF in the absence of 920 exogenous RANKL. Under the effects of IL-17, IL-21, ½F4 NF-kB and AP-1 activities (Figure 4A). However, the 921 IL-22, and M-CSF, TRAP-positive multinucleated cells (ie, blockage of phosphatidylinositol 3-kinase, p38 MAPK, c-Jun 922 OCs) differentiated from monocytes (P < 0.05). However, N-terminal kinase, and STAT3 showed no effect on IL923 no significant additive effect of IL-17 with other Th17 17einduced RANKL expression. The expression of RANKL 924 cytokines was observed (Figure 5). protein in RA FLS and the production of RANKL in culture ½F5 925 were also decreased by inhibition of NF-kB and AP-1 activFor determining the mediation of RANKL and TNF-a in 926 927 ities (Figure 4, B and C). These results suggest that IL-17 the IL-17einduced osteoclastogenesis, after neutralization 928 regulates RANKL expression in RA FLS via NF-kBe and of RANKL and TNF-a, the osteoclastogenesis was 929 AP-1emediated pathways. To confirm the activation of these assessed. RANKL and TNF-a induced osteoclastogenesis 930

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Th17 Cytokines and Osteoclastogenesis

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993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 Figure 6 Type 17 helper T-cell (Th17) cytokines differentiate monocytes into osteoclasts without exogenous receptor activator of NF-kB ligand (RANKL). 1027 Peripheral blood CD14þ monocytes were cultured with 30 ng/mL RANKL, 10 ng/mL tumor necrosis factor (TNF)-a, or 50 ng/mL IL-17 in the presence of 1028 macrophage colony-stimulating factor. Monocytes were cultured with 10 ng/mL anti-TNF or anti-RANKL in the presence of IL-17 and M-CSF. The figures are representative of three independent experiments. *P < 0.05, ***P < 0.005. 1029 1030 1031 with a similar effect of IL-17, and the inhibition of RANKL Mmp9, was also decreased after treatment with p38 MAPK, 1032 and TNF-a reduced the IL-17einduced osteoclastogenesis. NF-kB, and AP-1 inhibitors (P < 0.05) (Figure 7B). This 1033 A bone-resorbing assay showed that the neutralization of result suggests that IL-17 differentiates osteoclast precursors 1034 TNF-a decreased IL-17einduced bone-resorbing activity, into mature osteoclasts in the absence of RANKL or osteo1035 but the effect was not statistically significant (Figure 6 blasts and that this osteoclastogenesis is mediated by p38 ½F6 ). 1036 MAPK, NF-kB, and AP-1 pathways. To analyze intracellular signaling pathways mediating IL1037 17einduced OC differentiation, monocytes were cultured 1038 1039 with IL-17 and different signal inhibitors. Adhesion of RA FLSs Pre-Exposed to IL-17 Promote Osteoclast 1040 monocytes and differentiation into TRAP-positive OCs were Differentiation 1041 significantly decreased after culture with p38 MAPK, NF-kB, 1042 ½F7 and AP-1 inhibitors (P < 0.05) (Figure 7A). We performed an RA FLSs can induce osteoclast differentiation when they 1043 in vitro resorption pit assay using a bone resorption assay kit are co-cultured with peripheral blood monocytes. In the 1044 (Cosmo Bio Co, Ltd). Many resorption pits were generated in clinical scenario, RA FLSs might be exposed and activated 1045 wells with Th17 cytokine-treated cells. In contrast, curcumin by many proinflammatory cytokines before they contact 1046 treatment inhibited formation of resorption pits in the ILosteoclast precursors. We studied peripheral blood CD14þ 1047 17etreated cells. The number of resorbed pits revealed >50% monocytes that were co-cultured with TNF-ae or IL1048 inhibition by 10 mmol/L curcumin. Together, these results 17eprestimulated RA FLS and M-CSF. Without addition of 1049 suggest that curcumin, an AP-1 inhibitor, exerts an antiexogenous RANKL, the monocytes differentiated into 1050 1051 osteoclastogenic effect on bone marrowederived precursor multinucleated TRAP-positive multinucleated cells, osteo1052 cells, which results in reduced bone resorption. The expression clasts, in contrast to that observed in co-culture with unsti1053 of osteoclast markers, such as Trap, Rank, Ctsk, Calcr, and mulated FLSs (P < 0.05). The osteoclast differentiation 1054

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Figure 7

Intracellular signaling pathways involved in type 17 helper T-cell (Th17) cytokineinduced osteoclastogenesis. A: Peripheral blood CD14þ monocytes were cultured with macrophage colony-stimulating factor and 50 ng/mL of IL-17 in the presence of signal inhibitors, such as 20 mmol/L of LY294002 (LY), 10 mmol/L of SB203580 (SB), 50 mmol/L of AG490 (AG), 10 mmol/L of parthenolide (PA), or 10 mmol/L of curcumin (CU). The figures are representative of three independent experiments. B: The expression of tartrate-resistant acid phosphatase (Trap), receptor activator of NF-kB (Rank), cathepsin K (Ctsk), calcitonin receptor (Calcr), and matrix metalloprotease (Mmp) 9 of the osteoclasts was determined using real-time PCR. The amount of mRNA expression was normalized by b-actin. The data were expressed as the relative mRNA expression of target/b-actin. Data are presented as means  SEM of three separate experiments (A and B). *P < 0.05, **P < 0.01, and ***P < 0.005.

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culturing with IL-17estimulated RA FLS was decreased with the inhibition of osteoprotegerin and TNF-a (Figure 8). After inhibition of p38 MAPK, STAT3, NF-kB, and AP-1 activities, differentiation into OCs was significantly decreased (P < 0.05) (Figure 9A). The expression of Trap, Ctsk, and Calcr was significantly decreased after treatment with parthenolide and curcumin (P < 0.05) (Figure 9B).

Discussion Periarticular osteopenia, joint space narrowing, and bony erosions are characteristic of bone involvement in RA.24

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Dysregulated osteoclastogenesis and production of tissuedegrading molecules in response to inflammatory stimuli result in the destruction of cartilage and bones.25 Various inflammatory cytokines and immune cells are involved in the bony destructive process of RA. Among the destructive proteinases or cytokines produced by RA synovial and immune cells, RANKL is most important for bony destruction in RA.25 Activated CD4þ T cells and FLSs play a key role in osteoclastogenesis.26 They express RANKL and differentiate osteoclast precursors into osteoclasts in vitro. However, the regulatory role of CD4þ T cells in osteoclastogenesis is still controversial. Both Th1 cytokines (ie, IL-12 and IL-18) and Th2 cytokines (ie, IL-4 and IL-10)

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Th17 Cytokines and Osteoclastogenesis

IL-17eprestimulated rheumatoid arthritis (RA) fibroblastlike synoviocytes (FLSs) induce osteoclast differentiation. RA FLSs were pretreated with 50 ng/mL of IL-17 for 3 days, and then monocytes were co-cultured with the IL-17epretreated RA FLS for 3 weeks in a-minimal essential medium containing 10% horse serum with 100 ng/mL macrophage colony-stimulating factor. The osteoclast differentiation was determined by counting tartrate-resistant acid phosphatase (TRAP)þ multinucleated cells. The figures are representative of three independent experiments. ***P < 0.005. OPG, osteoprotegerin; RANKL, receptor activator of NF-kB ligand.

Figure 8

generally inhibit osteoclastogenesis. Interferon-g, a representative Th1 cytokine, strongly inhibits osteoclastogenesis, and normal Th1 cells do not induce osteoclastogenesis. Therefore, activated CD4þ T cells can induce osteoclastogenesis indirectly through the up-regulation of other osteoclastogenic cytokines. In addition to RANKL, FLSs produce TNF-a, IL-1, IL-6, IL-17, and prostaglandin E2, which stimulate osteoclast activation and differentiation. IL-17 induces the production of proinflammatory mediators, such as IL-1 and TNF-a from several synovial cells, including synovial fibroblasts, macrophages, and chondrocytes.27,28 IL-17 also induces RANKL expression, which is crucial for osteoclastogenesis and bone resorption,29 and directly stimulates osteoclast differentiation in vitro. IL-17 promotes cartilage destruction and bone erosion in experimental arthritis models.10,30e33 IL-17 is an important mediator in the development of arthritis in CIA, and blockage of IL-17 by neutralizing antibodies reduces the development and severity of CIA. In this study, IL-17 level correlated with RANKL level in RA synovial fluid. The correlation of their synovial levels was significant, and IL-17 increased RANKL activity in a luciferase assay in RA FLS. These results suggest that IL-17 regulates RANKL production in FLSs of RA. Apart from the activities of IL-17, there is evidence that other Th17 cytokines regulate RANKL production and osteoclast differentiation in RA. Recently, we showed that IL-22 is upregulated in RA synovial tissues, and it induces RANKL expression in RA FLS and osteoclastogenesis through the activation of the JAK2/STAT3 and p38 MAPK/NF-kB pathways.34 IL-21 is also up-regulated in synovial tissues

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and sera of RA patients, and it induces RANKL expression in FLS and enhances in vitro osteoclastogenesis.35 Although IL-21 is produced by Th17 cells, it also critically regulates Th17 cell development.36,37 The major source of IL-17 in the CIA model is a subset of CD4þ T cells, named Th17 cells. Th17 cells are essential to bone destruction in RA, and antieIL-17 therapy and genetic IL-17 deficiency ameliorate disease severity and bone damage by reducing the number of osteoclasts in CIA.31 IL17 and RANKL are highly expressed in RA synovial fluid and tissues, and they enhance osteoclastogenesis in vitro.23 IL-17 differentiates human peripheral blood monocytes into osteoclasts in the absence of osteoblasts or soluble RANKL, and TNF-a and RANKL synergistically induce osteoclastogenesis.16 Because RANKL is a key molecule for osteoclast activation and differentiation, we determined the ability of three different Th17 cytokines, IL-17, IL-21, and IL-22, to induce RANKL in FLS. In this study, Th17 cytokines induced RANKL expression in RA FLS, and IL-17 and IL-22 showed similar effects on the induction of RANKL expression, with no additive effect. IL-21 showed a lesser effect on RANKL induction than the other two Th17 cytokines. This result suggests that IL-17 and IL-22 are major cytokines for the induction of RANKL and the stimulation of osteoclast differentiation, and that IL-21 plays an accessory role in osteoclastogenesis by regulating Th17 cell differentiation. Next, we determined Th17 cytokine-induced osteoclast differentiation through two approaches. First, peripheral blood monocytes were cultured with M-CSF and three Th17 cytokines in the absence of RANKL. The Th17 cytokines

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Figure 9 Intracellular signaling pathways involved in the induction of osteoclastogenesis by IL-17estimulated rheumatoid arthritis (RA) synovial fibroblasts [fibroblast-like synoviocytes (FLSs)]. A: Peripheral blood CD14þ monocytes were cultured with macrophage colony-stimulating factor and IL-17eprestimulated RA FLS in the presence of signal inhibitors, such as 10 mmol/L of SB203580 (SB), 50 mmol/L of AG490 (AG), 10 mmol/L of parthenolide (PA), 10 mmol/L of curcumin (CU), or antiereceptor activator of NF-kB ligand (RANKL). The figures are representative of three independent experiments. B: The expression of tartrate-resistant acid phosphatase (Trap), Rank, cathepsin K (Ctsk), calcitonin receptor (Calcr), and matrix metalloprotease (Mmp) 9 of osteoclasts was determined using real-time PCR. The amount of mRNA expression was normalized by b-actin. The data were expressed as the relative mRNA expression of target/b-actin. Data are presented as means  SEM of three separate experiments (A and B). **P < 0.01, ***P < 0.005.

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significantly induced osteoclast differentiation from monocytes. Similar to the pattern by which they induced RANKL expression, the effects of IL-17 and IL-22 were stronger than the effect of IL-21 in osteoclastogenesis; however, no additive effect of these cytokines was observed. Because Th17 cytokines share the same receptors as osteoclast precursors, they might not have an additive effect. The osteoclastogenic effect of IL-17 was similar to that of RANKL and TNF-a, suggesting that IL-17 is one of the most effective inducers for osteoclastogenesis. The neutralization of TNF-a reduced the IL-17einduced osteoclastogenesis because there are different receptors and independent pathways among the cytokines and their reciprocal activations. These data suggest that TNF-a can mediate the IL17einduced osteoclastogenesis, it has a stronger effect than IL-17, or it plays at the most final process than IL-17 or RANKL. Second, we exposed FLSs to IL-17 before they were co-cultured with osteoclast precursors. RA FLS can induce osteoclastogenesis when cultured with monocytes.26 In a real clinical setting, FLSs are exposed and responsive to

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many inflammatory cytokines and chemokines; hence, we designed the co-culture system of monocytee and cytokinese pre-exposed FLS to mimic this milieu. When monocytes were cultured with IL-17epreexposed FLS, more osteoclasts differentiated than that observed when monocytes were cultured with cytokine-unexposed FLS. This result suggests that IL-17 confers FLS with the potential to induce osteoclastogenesis. The inhibition of TNF-a or osteoprotegerin reduced the osteoclast differentiation culturing with IL-17eprestimulated RA FLS, and the inhibitory effect of antieTNF-a was more potent than antiRANKL or osteoprotegerin. The adaptor molecule known as Act1 is a critical mediator of IL-17 receptor family signaling.28,38 On IL-17 stimulation, Act1 is recruited to IL-17 receptors through its SEFIR domain, and it is required for IL-17emediated expression of genes involved in inflammation. Act1deficient mice are protected from CIA development, resulting from the prevention of neutrophil infiltration into the synovium, bone erosion, and cartilage damage and

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Th17 Cytokines and Osteoclastogenesis reduction of type II collagen-specific antibody production. These results suggest that Act1 is a critical mediator in the pathogenesis of RA animal modes.14 TRAF6 has a key role in RANK signaling in osteoclastogenesis, and TRAF6 deficiency results in compromised differentiation and activation of osteoclasts.39 TRAF6 is overexpressed in RA synovium, and synovial TRAF6 is correlated with synovial score and inflammatory cell infiltration.40 TRAF3 is a proximal negative regulator of IL-17 receptor signaling. TRAF3 has been shown to greatly suppress IL-17einduced NF-kB and MAPK activation and the subsequent production of inflammatory cytokines and chemokines.41 In this study, Act1 was induced by IL-17 stimulation in RA FLS in a dose-dependent manner, and when Act1 was blocked by siRNA, the IL-17einduced RANKL expression was completely inhibited. TRAF6 was also decreased with the blockage of Act1. These results show that the Act1/TRAF6 pathway is essential in IL-17einduced RANKL expression of RA FLS and that TRAF6 is a downstream molecule of Act1. In addition, the blockage of NF-kB and AP-1 decreased IL-17einduced RANKL expression in RA FLS. This result suggests that up-regulation of RANKL by IL-17 is mediated through the Act1/TRAF6 pathway upstream and the NF-kB and AP-1 pathways downstream. In summary, Th17 cytokines regulate osteoclastogenesis in RA through the up-regulation of RANKL expression in FLS and the direct induction of osteoclast differentiation. The IL-17einduced RANKL expression and osteoclastogenesis were mediated by the Act1/TRAF6/NF-kB and AP1 pathways and TNF-a. The blockage of this IL-17 and RANKL axis could be a new therapeutic target for bone destruction in RA.

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Acknowledgments K.-W.K. designed and performed the experiment and analyzed the data, K.-W.K. and H.-R.K. wrote the manuscript, B.-M.K. performed the experiments and analyzed the data, and S.-H.L. and M.-L.C. supervised the project.

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References 1. Kotake S, Sato K, Kim KJ, Takahashi N, Udagawa N, Nakamura I, Yamaguchi A, Kishimoto T, Suda T, Kashiwazaki S: Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. J Bone Miner Res 1996, 11:88e95 2. Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS, Goldring SR: Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis. Am J Pathol 1998, 152:943e951 3. Suryani S, Sutton I: An interferon-gamma-producing Th1 subset is the major source of IL-17 in experimental autoimmune encephalitis. J Neuroimmunol 2007, 183:96e103 4. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S, Frosali F, Giudici F, Romagnani P, Parronchi P, Tonelli F, Maggi E, Romagnani S:

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