ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 5, May 2014, pp 1195–1207 DOI 10.1002/art.38313 © 2014, American College of Rheumatology

Halofuginone Ameliorates Autoimmune Arthritis in Mice by Regulating the Balance Between Th17 and Treg Cells and Inhibiting Osteoclastogenesis Mi-Kyung Park,1 Jin-Sil Park,1 Eun-Mi Park,1 Mi-Ae Lim,1 Sung-Min Kim,1 Dong-Gun Lee,1 Seung-Ye Baek,1 Eun-Ji Yang,1 Jung-Won Woo,2 Jennifer Lee,1 Seung-Ki Kwok,1 Ho-Youn Kim,1 Mi-La Cho,3 and Sung-Hwan Park1 Osteoclast differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase (TRAP)–positive multinucleated cells and area of resorbed bone. Results. Treatment with halofuginone suppressed the development of autoimmune arthritis and reciprocally regulated Th17 cells and FoxP3ⴙ Treg cells. These effects of halofuginone on Th17 differentiation involved increased signaling of ERK and reduction of STAT-3 and NF-ATc1 expression. Furthermore, halofuginone induced the expression of indoleamine 2,3-dioxygenase (IDO) in dendritic cells, leading to reduced production of Th17 cells. In addition, halofuginone prevented the formation and activity of osteoclasts through suppression of transcription factors, such as activator protein 1 and NF-ATc1, and inhibited cell cycle arrest by the committed osteoclast precursors via expression of Ccnd1 encoding cyclin D1. Conclusion. Taken together, our results suggest that halofuginone is a promising therapeutic agent for the treatment of Th17 cell–mediated inflammatory diseases and bone diseases.

Objective. The small molecule halofuginone has been shown to inhibit fibrosis, angiogenesis, and tumor progression. This study was undertaken to evaluate the effects of halofuginone in preventing autoimmune arthritis in mice. Methods. The effects of halofuginone on joint diseases were assessed by clinical scoring and histologic analysis. Protein expression levels were confirmed by immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry, and/or Western blotting. The expression levels of messenger RNA (mRNA) for various molecules were determined by real-time polymerase chain reaction (PCR). Proliferation of osteoclast precursors was assessed by bromodeoxyuridine uptake. Supported by the Ministry of Education, Science, and Technology, Republic of Korea through funding to the National Research Foundation of Korea (Basic Science Research Program grant number 2005-0048480) and by the Ministry for Health, Welfare, and Family Affairs, Republic of Korea (Korea Health Technology R&D Project grant number A092258). 1 Mi-Kyung Park, PhD, Jin-Sil Park, PhD, Eun-Mi Park, MS, Mi-Ae Lim, BS, Sung-Min Kim, BS, Dong-Gun Lee, MS, Seung-Ye Baek, BS, Eun-Ji Yang, BS, Jennifer Lee, MD, Seung-Ki Kwok, MD, PhD, Ho-Youn Kim, MD, PhD, Sung-Hwan Park, MD, PhD: Catholic University of Korea, Seoul, South Korea; 2Jung-Won Woo, PhD: Seoul St. Mary’s Hospital, Seoul, South Korea; 3Mi-La Cho, PhD: Catholic University of Korea and Seoul St. Mary’s Hospital, Seoul, South Korea. Drs. M.-K. Park and J.-S. Park and Ms E.-M. Park contributed equally to this work. Drs. M.-L. Cho and S.-H. Park contributed equally to this work. Address correspondence to Mi-La Cho, PhD, Rheumatism Research Center, Catholic University of Korea, 222 Banpo-Daero, Seocho-gu, Seoul 137-701, South Korea (e-mail: iammila @catholic.ac.kr); or to Sung-Hwan Park, MD, PhD, Division of Rheumatology, Department of Internal Medicine, School of Medicine, Catholic University of Korea, Seoul St. Mary’s Hospital, 505 Banpo-dong, Seocho-gu, Seoul 137-701, South Korea (e-mail: [email protected]). Submitted for publication March 5, 2013; accepted in revised form December 5, 2013.

Rheumatoid arthritis (RA) is characterized by inflammation of synovial joints, with CD4⫹ T cell infiltration and synovial hyperplasia, leading to severe bone destruction mediated by osteoclasts (1). The cause of RA is largely unknown. However, substantial evidence has emerged supporting a role for immune cells, including T cells, macrophages, and fibroblasts, as well as inflammatory cytokines in the initiation and progression of RA (2). For many years, RA has been recognized as a Th1-mediated disease. However, experimental data have indicated that interleukin-17 (IL-17) plays a pivotal role in the pathogenesis of RA. In vitro human osteo1195

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clastogenesis by IL-17 can occur via RANKL induction of osteoblasts via IL-17 signaling (3), but the Th1-related cytokine interferon-␥ (IFN␥) inhibits osteoclast differentiation (4). Proinflammatory Th17 cells and immunosuppressive Treg cells functionally antagonize each other in RA. For example, Th17 cells were shown to be implicated in the initiation and maintenance of inflammation in collagen-induced arthritis (CIA) (5), while Treg cells from the joints of RA patients were found to inhibit the secretion of IL-17 from Teff cells (6). Therefore, altering the balance between Th17 and Treg cells is a promising approach in the treatment of RA. Joint damage is one of the most characteristic features of RA, in addition to the imbalance between Th17 and Treg cell subsets. There is increasing evidence that Th17 cells play a pathogenetic role in the abnormal cartilage destruction associated with RA (7). RANKL acts through its receptor RANK to initiate a signaling cascade that is crucial for osteoclast differentiation and activation, and also blocks cell cycle progression of osteoclast precursors, thereby coordinating cell cycle withdrawal with cell differentiation (8). Importantly, RANKL specifically and potently induces expression of the transcription factor NF-ATc1 via the tumor necrosis factor receptor–associated factor 6 (TRAF6) and c-Fos pathways, triggering a sustained NF-ATc1–dependent transcriptional program during osteoclast differentiation (9). Functional NF-AT sites have been identified in the genes encoding cathepsin K, tartrate-resistant acid phosphatase (TRAP), osteoclastassociated receptor (OSCAR), calcitonin receptor (CTR), and ␤3 integrin. Thus, NF-ATc1 regulates several osteoclast-specific genes in cooperation with other transcription factors, such as activator protein 1 (AP-1), PU.1, microphthalmia-associated transcription factor (MITF), and CREB (10,11). Aberrant immune response and osteoclastmediated bone destruction are connected in complex ways, and potentially effective therapeutic strategies for RA are considered to require an understanding of the relationship between bone and the immune system in arthritis by focusing mainly on osteoclasts and inflammation in joints. Although therapeutic intervention can effectively reduce inflammation, it frequently does not eliminate it, and so, some bone loss continues. Therefore, the ideal treatment for autoimmune diseases such as RA should involve control of joint inflammation and simultaneously, should minimize joint damage. Halofuginone is a synthetic, halogenated derivative of febrifugine, a natural plant alkaloid that was originally extracted from the roots of blue evergreen

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hydrangea. Halofuginone is a relatively small molecule and is well known to demonstrate antimalarial activity (12). Halofuginone is a potent inhibitor of type I collagen synthesis and extracellular matrix formation via the inhibition of transforming growth factor ␤ (TGF␤) signaling, and the enzymatic activity of matrix metalloproteinase 2 (MMP-2) (13). Its main clinical efficacy is in scleroderma, chronic graft-versus-host disease, and Kaposi’s sarcoma (14,15). It has recently been reported to selectively inhibit murine Th17 cell differentiation through activation of the amino acid starvation response without affecting maturation of other T cell lineages. Halofuginone effectively delays the onset and reduces the severity of experimental autoimmune encephalomyelitis (EAE) as Th17-dominant disease (16). However, there have been no previous reports regarding the therapeutic efficacy of halofuginone in RA, including inflammation and osteogenic differentiation. Given the capacity of halofuginone to dampen autoimmune diseases such as EAE, we hypothesized that it may also suppress disease progression in CIA. MATERIALS AND METHODS Mice. Male DBA/1J (H2q) mice (6–8 weeks old) were purchased from Orient Bio. IL-10–knockout mice on an H2q background were kindly provided by Dr. Andrew H. Kang (University of Tennessee, Memphis, TN). The animals were treated in accordance with the regulations of the Catholic Ethics Committee of the Catholic University of Korea, in conformity with the National Institutes of Health guidelines. Induction and evaluation of the CIA model. The induction and evaluation of CIA have been described previously (17). On day 7 after the first immunization, each arthritic mouse in the group was injected intraperitoneally with 500 ␮g/kg of halofuginone (P&S Chemicals) and 10% DMSO (vehicle control) 3 times a week for 9 weeks. Immunofluorescence microscopy. Frozen spleens from mice with CIA were immunostained with PerCP–Cy5.5– or Alexa Fluor 488–conjugated anti-CD4, phycoerythrin (PE)– conjugated anti-FoxP3, fluorescein isothiocyanate (FITC)– conjugated anti–IL-17, allophycocyanin (APC)–conjugated anti-CD25 (all from eBioscience), PE-conjugated anti– pSTAT-3 (Y705 and S727), and Alexa Fluor 488–conjugated anti–pSTAT-5 (Y694) monoclonal antibodies (all from BD Biosciences). Western blot analysis. Proteins were detected using antibodies against NF-ATc1 and phospho-Smad2/3 (both from Santa Cruz Biotechnology), STAT-3, STAT-5, ERK, pSTAT-3, pSTAT-5, and phospho-ERK (all from Cell Signaling Technology), or ␤-actin (Sigma). TRAP staining of differentiated osteoclasts and calcium phosphate resorption assays. Osteoclast-like cells were assayed for TRAP activity using a leukocyte acid phosphatase kit according to the recommendations of the manufacturer

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Figure 1. Suppression of the development of collagen-induced arthritis (CIA) by halofuginone (HF) treatment. A and B, Beginning 1 week after the induction of CIA, mice received 3 consecutive intraperitoneal injections of halofuginone (500 ␮g/kg) at 3-day intervals. A, Reduction in the arthritis score in mice with CIA treated with halofuginone. B, Histologic examination of joints from mice with CIA treated with vehicle or halofuginone. Specimens were stained with hematoxylin and eosin (H&E), Safranin O, or toluidine blue. C–F, Interleukin-10 (IL-10)–knockout mice were injected with type II collagen (CII) and Freund’s complete adjuvant. Beginning on day 7, mice received halofuginone (500 ␮g/kg) or vehicle for 9 weeks. C, Arthritis scores in mice with CIA treated with vehicle or halofuginone, determined as described in Materials and Methods. D, Histologic examination of knee joints and paws from mice with CIA treated with vehicle or halofuginone. Specimens were stained with H&E, Safranin O, or toluidine blue. E, Staining of mouse tissue sections with anti–IL-1␤, anti–tumor necrosis factor ␣ (anti-TNF␣), anti–IL-6, or anti–IL-17 antibodies. Cells stained with each antibody are shown in brown. Serum IL-6 and IL-17 levels were measured by enzyme-linked immunosorbent assay. F, CII-specific total IgG, IgG1, and IgG2a concentrations in mouse serum. Values are the mean ⫾ SEM (n ⫽ 5 mice per group). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001.

(Sigma), omitting counterstaining with hematoxylin. Osteoclasts were defined as cells with more than 3 nuclei. Resorption

pits were visualized by hematoxylin staining and counted with Olympus MicroSuite image analysis software.

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Cell cycle analysis. Cell cycle distribution was evaluated by staining cells using a bromodeoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) labeling kit (BrdU Flow Kit; BD Biosciences) according to the recommendations of the manufacturer (BD Biosciences). Osteoclast precursors were cultured with or without soluble RANKL. At 24 hours of culture, cells were labeled with 5 ␮M BrdU for 2 hours. BrdU incorporation and DNA content (7-AAD) were analyzed by flow cytometry. Statistical analysis. Statistical significance was determined by Student’s 2-tailed t-test or one-way analysis of variance with Bonferroni correction for multiple comparisons. In all analyses, P values less than 0.05 were considered significant.

RESULTS Amelioration of the development and progression of inflammatory arthritis by halofuginone treatment. To clarify the effects of halofuginone on disease activity and progression in CIA, DBA/1J mice were immunized with type II collagen (CII) and treated with halofuginone. Intraperitoneal injection of halofuginone (500 ␮g/kg) reduced the arthritis score compared with injection of vehicle (Figure 1A). Regarding the histopathologic features of the joint in arthritis, halofuginonetreated mice exhibited a lower degree of inflammation and cartilage destruction than vehicle-treated mice, as determined on day 50 after the first halofuginone injection (Figure 1B). Also, treatment with halofuginone reduced the expression of proinflammatory cytokines and the serum levels of CII-specific IgG, IgG1, and IgG2a (results are available online at http://www.rhrc. re.kr/bbs/board.php?bo_table⫽b_02_05&wr_id⫽95877 &sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). Next, we started halofuginone administration on day 28 after immunization to investigate its therapeutic effect. Halofuginone ameliorated the degree of arthritis in mice with established disease, indicating that halofuginone had therapeutic effects as well as preventive effects (results are available online at http://www.rhrc.re.kr/bbs/ board.php?bo_table⫽b_02_05&wr_id⫽95877&sca⫽&sfl ⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). It is known that IL-10–knockout mice exhibit higher rates of clinical signs and more severe knee and paw injury than wildtype mice (18). We therefore investigated whether treatment with halofuginone would suppress the severity of RA in CII-immunized IL-10–knockout mice. Mice treated with halofuginone showed complete suppression of the severity of CIA as well as lower degrees of inflammation and cartilage damage than vehicle-treated mice (Figures 1C and D).

We also performed immunohistochemical analysis of inflammatory cytokines in the joint tissue of mice treated with vehicle or halofuginone. The levels of proinflammatory cytokines were decreased in the joints of mice treated with halofuginone as compared to vehicle-treated controls. Consistent with the results of immunohistochemical analysis, IL-6 and IL-17 levels in serum were reduced by halofuginone treatment in a dose-dependent manner (Figure 1E). In addition, CIIspecific IgG and IgG2a levels were lower in serum from mice treated with halofuginone than in vehicle-treated control mice as early as 2 weeks after initiation of treatment (Figure 1F). Although in the early stages of treatment the serum level of IgG1 was similar in both control and halofuginone-treated mice, at 6 weeks, the IgG1 level was also significantly lowered in halofuginone-treated mice. These results indicate that halofuginone can suppress the development and progression of inflammatory arthritis, accompanied by blocking of joint inflammation and reduction of autoreactive B cell responses. Reciprocal regulation of Th17 and Treg cells in CIA by halofuginone. Pathogenic autoimmunity can be controlled by the balance of Th17 and Treg cells. Sundrud and colleagues reported that halofuginone specifically inhibits the Th17-associated autoimmune inflammatory disease EAE (16). We evaluated the population of Th17 and Treg cell subsets in the ex vivo splenocytes of mice with CIA using flow cytometry. As shown in Figure 2A, the mice treated with halofuginone had decreased expression of IL-17 and increased expression of FoxP3. This reciprocal regulation of Th17 and Treg cells was also observed in mice that had been treated with halofuginone beginning on day 28 after immunization (results are available online at http://www. rhrc.re.kr/bbs/board.php?bo_table⫽b_02_05&wr_id⫽ 95877&sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). Because STATs are central transcription factors of Th17 and Treg cell–dependent autoimmune processes, we analyzed the phosphorylation of STAT-3 and STAT-5 in the ex vivo splenocytes of mice with CIA treated with halofuginone. Halofuginone-treated mice showed decreased expression of pSTAT-3 (Y705) and increased expression of pSTAT-5 (Y694) in the ex vivo splenic CD4⫹ T cells (Figure 2B). (Additional results are available online at http://www.rhrc.re.kr/bbs/board. php?bo_table⫽b_02_05&wr_id⫽95877&sca⫽&sfl⫽&stx ⫽&sst⫽&sod⫽&spt⫽0&page⫽0). As expected, confocal microscopy showed that halofuginone treatment decreased the number of Th17 cells and CD4⫹pSTAT-3 (Y705)⫹ T cells and increased the number of Treg cells and CD4⫹pSTAT-5 (Y694)⫹ cells in spleen tissue from

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Figure 2. Simultaneous decrease in Th17 effector cells and increase in FoxP3⫹ Treg cells in mice with collagen-induced arthritis (CIA) treated with halofuginone (HF). A and B, Beginning 1 week after the induction of CIA, mice received 3 consecutive intraperitoneal injections of halofuginone (500 ␮g/kg) at 3-day intervals. Fifty days after the first intraperitoneal injection, the proportions of splenic Th17 and Treg cells (A) and pSTAT-3 and pSTAT-5 in splenic CD4⫹ T cells (B) were assessed ex vivo by intracellular fluorescence-activated cell sorting. C and D, Interleukin-10 (IL-10)–knockout mice were injected with type II collagen and Freund’s complete adjuvant. Beginning on day 7, mice received halofuginone (500 ␮g/kg) or vehicle for 9 weeks. Confocal microscopy of CD4⫹pSTAT-3⫹ and CD4⫹FoxP3⫹pSTAT-5⫹ cells in mouse spleen tissue was performed (C). Each confocal image is representative of 4 fields of view. Insets show higher-magnification views of double- or triple-positive cells. Expression of IL-17 and FoxP3 was measured in mouse splenocytes (top) and draining lymph node cells (bottom) cultured with phorbol myristate acetate and ionomycin for 4 hours (D). Expression levels were determined by real-time polymerase chain reaction and were normalized to that of ␤-actin. Bars show the mean ⫾ SEM (n ⫽ 5 mice per group). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001.

CII-immunized IL-10–knockout mice (Figure 2C). In addition, halofuginone-treated mice showed decreased expression of IL-17 and increased expression of FoxP3 in both the spleen and draining lymph nodes (Figure 2D). Taken together, these results suggest that halofuginone contributes to the inhibition of Th17 cell differentiation and simultaneously enhances the induction of FoxP3 expression in the development of CIA. Activation of ERK signaling on Th17 differentiation after halofuginone treatment. To confirm the regulatory role of halofuginone in Th17 differentiation,

we investigated changes in FoxP3 induction upon Th17 differentiation. Consistent with previous findings (16), halofuginone selectively inhibited IL-17 production in Th17 cells without affecting IFN␥ or IL-4 secretion by Th1 and Th2 cells, respectively (Figure 3A). The inhibition of IL-17 production by halofuginone was not related to either cell toxicity or IL-2 production (results are available online at http://www.rhrc.re.kr/bbs/board.php?bo_ table⫽b_02_05&wr_id⫽95877&sca⫽&sfl⫽&stx⫽&sst⫽ &sod⫽&spt⫽0&page⫽0). Interestingly, halofuginonetreated Th17 cells showed reciprocally increased FoxP3

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Figure 3. Halofuginone (HF) directly activates ERK signaling in mouse Th17 cells and indirectly inhibits interleukin-17 (IL-17) production by inducing indoleamine 2,3-dioxygenase (IDO). A–C, Splenic CD4⫹ T cells were cultured under Th17-, Th1-, and Th2-polarizing conditions for 72 hours in the presence or absence of halofuginone. A, Frequencies of CD4⫹IL-17A⫹ cells and CD4⫹CD25⫹FoxP3⫹ cells, assessed by intracellular flow cytometry, and levels of IL-17, interferon-␥ (IFN␥), and IL-4 in supernatants, measured by enzyme-linked immunosorbent assay (ELISA). B, Levels of IL-17, CCR6, suppressor of cytokine signaling protein 3 (SOCS-3), and FoxP3, assessed by real-time polymerase chain reaction (PCR). C, Western blot analysis. Cells were lysed with antibodies specific for ERK, pERK, STAT-3, pSTAT-3, STAT-5, pSTAT-5, pSmad2/3, and NF-ATc1. D, Levels of IDO in CD11C⫹ dendritic cells (DCs) and IL-17 in supernatants. CD11C⫹ DCs or CD4-depleted splenocytes were cultured with 2, 5, or 10 ng/ml of halofuginone or with 100 ng/ml of lipopolysaccharide (LPS) alone for 48 hours. Murine IDO mRNA expression in CD11C⫹ DCs was determined by reverse transcriptase–PCR and normalized to that of ␤-actin. CD4⫹ T cells were cultured under Th17 conditions for 48 hours and subsequently cocultured with pretreated CD4-depleted splenocytes for 48 hours under anti-CD3 stimulation. IL-17 levels in supernatants were determined by ELISA. Bars in A, B, and D show the mean ⫾ SEM. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. 1-MT ⫽ 1-methyltryptophan. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10. 1002/art.38313/abstract.

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and suppressor of cytokine signaling 3 expression and simultaneously decreased IL-17 and CCR6 levels (Figure 3B). To gain insight into the signaling events involved in the reciprocal effects of halofuginone on Th17 differentiation, we analyzed the expression of ERK kinases and STAT-5, which limit Th17 differentiation (19,20), as well as the activation of STAT-3, NF-ATc1, and Smad2/3, which can induce Th17 differentiation (21). Although there were no detectable differences in pSTAT-5 levels between halofuginone-treated and untreated cells, halofuginone markedly enhanced ERK phosphorylation, but showed only a slight increase in STAT-5 phosphorylation at 60 minutes, and concomitantly reduced STAT-3 phosphorylation and NF-ATc1 in cells differentiated under Th17-polarizing conditions (Figure 3C) (additional results are available online at http://www.rhrc.re.kr/bbs/board.php?bo_table⫽b_02_05 &wr_id⫽95877&sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽ 0&page⫽0). Thus, the reciprocal effect of halofuginone on Th17 cell differentiation likely involves elevated activation of ERK and, in part, reduction of STAT-3 and NF-ATc1 signals. Inhibition of IL-17 production in mouse Th17 cells by halofuginone-mediated indoleamine 2,3dioxygenase (IDO) induction. Upon interaction with dendritic cells (DCs), CD4⫹ T cells can differentiate into a variety of effector and regulatory subsets. The differentiating or committed Th cells are also controlled by various cytokines and molecules produced by DCs. We examined whether halofuginone treatment induces IDO as a key negative regulator of immune responses in DCs. As expected, lipopolysaccharide (LPS) induced IDO expression. Halofuginone also significantly increased IDO expression in DCs (Figure 3D). To examine the regulatory properties of IDO induced by halofuginone in Th17 differentiation, splenocytes were treated with halofuginone in the presence or absence of 1-methyltryptophan (1-MT) and then cocultured with Th17 cells. The results showed that halofuginonetreated DCs inhibited IL-17 production from Th17 cells, and IL-17 was recovered when IDO was blocked with 1-MT (Figure 3D). We also observed that either Th1 or Th2 cells were not affected by halofuginone-treated DCs (results are available online at http://www.rhrc.re.kr/bbs/ board.php?bo_table⫽b_02_05&wr_id⫽95877&sca⫽&sfl ⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). Taken together, these data demonstrated that halofuginone induced the expression of IDO in DCs and, thus, regulated the function of DCs to drive differentiation of CD4⫹ T cells into Th17 cells.

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Figure 4. Suppression of osteoclastogenesis in mice with collagen-induced arthritis (CIA) treated with halofuginone (HF). A and B, Beginning 1 week after the induction of CIA, mice received 3 consecutive intraperitoneal injections of halofuginone (500 ␮g/kg) at 3-day intervals. A, Tartrate-resistant acid phosphatase (TRAP) antibody staining of tissue sections from the joints of mice with CIA 50 days after the first intraperitoneal injection of halofuginone. Sections were counterstained with hematoxylin. B, Induction of osteoclast differentiation in bone marrow cells from mice with CIA treated with vehicle or halofuginone, using macrophage colony-stimulating factor (M-CSF; 10 ng/ml) and RANKL (25 ng/ml). Multinucleated TRAP⫹ cells were enumerated by light microscopy. C–E, Interleukin-10 (IL-10)–knockout mice were injected with type II collagen and Freund’s complete adjuvant. Beginning on day 7, mice received halofuginone (500 ␮g/kg) or vehicle for 9 weeks. C, TRAP staining of tissue sections from the joints of mice with CIA. Sections were counterstained with hematoxylin. D, Osteoclasts in bone marrow cells from vehicleor halofuginone-treated arthritic mice. Cells were cultured with 10 ng/ml M-CSF and 25 ng/ml RANKL. After 7 days, osteoclasts were identified by staining for TRAP. E, Expression levels of TRAP, NF-ATc1, ␤3 integrin, osteoclast-associated receptor (OSCAR), cathepsin K, and matrix metalloproteinase 9 (MMP-9), assessed by real-time polymerase chain reaction using total RNA. Bars show the mean ⫾ SEM (n ⫽ 5 mice per group). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01.

Suppression of joint destruction in mice with CIA treated with halofuginone. Increased bone resorption mediated by osteoclasts causes bone erosion in RA. To examine the effects of halofuginone on osteoclast formation, we performed TRAP staining on tissue sections from the joints of mice with CIA treated with halofuginone or vehicle. As shown in Figure 4A, the joints of halofuginone-treated mice showed reduced formation of osteoclasts compared with those of vehicletreated mice. We next examined the capacity of bone marrow

cells from vehicle- or halofuginone-treated mice to undergo osteoclastogenesis in vitro. The bone marrow cells from halofuginone-treated mice showed reduced osteoclastogenesis compared with those from vehicletreated mice (Figure 4B). This reduced osteoclastogenesis was consistently observed in mice with CIA that received halofuginone after the establishment of arthritis (results are available online at http://www.rhrc.re. kr/bbs/board.php?bo_table⫽b_02_05&wr_id⫽95877 &sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). The joints from CII-immunized IL-10–knockout mice treated

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Figure 5. Halofuginone down-regulates activator protein 1, NF-ATc1, and cyclin D1 during osteoclastogenesis. A, TRAP staining for osteoclasts and analysis of bone resorption in bone marrow cells from DBA/1J mice. Cells were cultured with 10 ng/ml of M-CSF and 50 ng/ml of RANKL in the presence or absence of halofuginone. After 5 days, osteoclasts were identified by staining for TRAP. To examine bone resorption activity, bone marrow cells were placed on dentin slices and cultured as described above. After 21 days, slices were stained with hematoxylin. B, Expression levels of TRAP, NF-ATc1, ␤3 integrin, OSCAR, cathepsin K, and MMP-9, assessed by real-time polymerase chain reaction (PCR). C, Expression levels of c-Fos, JunB, and Jun dimerization protein 2 (JDP-2), assessed by real-time PCR in bone marrow cells treated for 4 days as described in A. D, Cell cycle distribution of bone marrow cells treated for 4 days as described in A. Cell cycle distribution was analyzed by flow cytometry after coupled staining with antibromodeoxyuridine (anti-BrdU)–fluorescein isothiocyanate and 7-aminoactinomycin D (7-AAD)–PerCP–Cy5.5. A representative fluorescence-activated cell sorting plot of bone marrow cells in G0/G1 or S cell cycle phases based on incorporation of BrdU and 7-AAD is shown. The expression levels of Ccnd1 were assessed by real-time PCR. The levels of mRNA expression were normalized to that of ␤-actin. Bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 4 for other definitions.

with halofuginone showed a notable decrease in the number of TRAP⫹ osteoclasts compared with the joints from vehicle-treated mice (Figure 4C). The bone marrow cells from the control group differentiated into mature TRAP⫹ multinucleated osteoclasts, while those from the halofuginone-treated mice showed reduced formation and numbers of TRAP⫹ multinucleated cells (Figure 4D). The expression levels of osteoclastogenesisrelated molecules, such as TRAP, NF-ATc1, ␤3 integrin, OSCAR, cathepsin K, and MMP-9, were markedly decreased in halofuginone-treated mice (Figure 4E),

suggesting that halofuginone may have had beneficial effects on CIA progression by down-regulating the differentiation of osteoclasts. Halofuginone inhibits RANKL-mediated osteoclast differentiation by repressing AP-1 and NF-ATc1. To examine the effects of halofuginone on RANKLinduced osteoclastogenesis, we cultured osteoclast precursors with macrophage colony-stimulating factor (MCSF) and RANKL in the presence or absence of halofuginone. Halofuginone effectively inhibited the differentiation of osteoclasts and bone resorption in a

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Figure 6. Halofuginone inhibits Th17 differentiation as well as osteoclastogenesis in humans. A, Frequencies of CD4⫹IL-17⫹ cells and CD4⫹CD25⫹FoxP3⫹ cells, assessed by intracellular flow cytometry, and levels of IL-17A, IL-17F, and IL-22, assessed by real-time polymerase chain reaction (PCR). Human CD4⫹ T cells were cultured under Th17-polarizing conditions for 72 hours in the presence or absence of halofuginone. Cells were stained with antibodies against IL-17 and FoxP3. Cells were harvested and total RNAs were isolated for PCR. B, TRAP staining of human monocytes that were cultured with 25 ng/ml of M-CSF and 30 ng/ml of RANKL in the presence or absence of halofuginone for 9 days. To examine bone resorption activity, bone marrow cells were placed on dentin slices and cultured as described above. After 21 days, slices were stained with hematoxylin. C, Levels of TRAP, NF-ATc1, calcitonin receptor, cathepsin K, MMP-9, and RANK, assessed by real-time PCR. The mRNA expression levels were normalized to that of ␤-actin. Bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 4 for other definitions.

dose-dependent manner (Figure 5A). To characterize the molecular mechanisms associated with osteoclast formation in halofuginone-treated osteoclast precursors, we compared the expression of osteoclast-specific genes and transcription factors in the presence or absence of halofuginone. Consistent with in vivo observations, halofuginone inhibited RANKL-induced osteoclast differentiation, including osteoclast-specific genes (TRAP, ␤3 integrin, OSCAR, cathepsin K, and MMP-9) in a dosedependent manner (Figure 5B). Furthermore, we demonstrated that the addition of halofuginone downregulated the expression of the transcription factors

NF-ATc1, Jun dimerization protein 2 (JDP-2), c-Fos, and JunB in osteoclasts but had no effect on the expression of JunD (Figures 5B and C and data not shown). Thus, halofuginone inhibits osteoclastogenesis by suppressing expression of the AP-1 family members JDP-2, c-Fos, and JunB, as well as NF-ATc1, and subsequently suppresses both the formation and activity of osteoclasts. Halofuginone regulation of the G0/G1 cell cycle during osteoclastogenesis. Recent studies have shown that cell cycle arrest of committed osteoclast precursors during differentiation is associated with the down-

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regulation of cyclins (22). Therefore, using 7-AAD and BrdU staining, we examined the effects of halofuginone on cell cycle progression in osteoclast precursors. M-CSF/RANKL stimulation resulted in a decrease in the number of cells in S phase, as compared to M-CSF alone. The addition of halofuginone in the presence of M-CSF/RANKL increased the percentage of cells transitioning from G0/G1 to S phase (Figure 5D). Furthermore, we found that the expression of Ccnd1, which encodes cyclin D1, a major regulator of cell cycle progression, was increased directly by halofuginone (Figure 5D), suggesting that halofuginone inhibits the differentiation of osteoclast precursors by causing the cell cycle to enter the S phase. Halofuginone and the role of starvation response. To investigate whether the starvation response was associated with the effect of halofuginone on Th17/ Treg cell modulation, IDO expression, and osteoclastogenesis regulation, we treated the cells with a specific inhibitor of eukaryotic translation-initiation factor 2␣ phosphatases and compared the effect of halofuginone with that of salubrinal (results are available online at http://www.rhrc.re.kr/bbs/board.php?bo_table⫽b_02_05 &wr_id⫽95877&sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽ 0&page⫽0). Because tunicamycin, an inducer of endoplasmic reticulum stress, induced cell death when cells were cultured for more than 2 days, as was the case in our experiments, tunicamycin could not be used for comparison. Although halofuginone significantly reduced Th17 cell expression and enhanced Treg cell expression, treatment with salubrinal did not result in any difference in the frequency of Th17 or Treg cell expression. Regarding IDO expression, in contrast to the elevation of IDO expression upon halofuginone treatment, salubrinal reduced the expression of IDO. Salubrinal suppressed osteoclastogenesis, similar to halofuginone; however, the degree of suppression by halofuginone was far greater. This finding suggests that the inhibitory effect of halofuginone on osteoclastogenesis may be due in part to the starvation response. Collectively, the effect of halofuginone on Th17 cell regulation or IDO expression was not associated with the starvation response. However, the starvation response seems to play a role in the suppression of osteoclastogenesis by halofuginone. Inhibition of both the development of Th17 cells and osteoclast formation in human cells by halofuginone. We investigated whether halofuginone has inhibitory effects on the development of Th17 cells or the formation of osteoclasts in a human cell culture system in vitro. Halofuginone-treated T cells stimulated under

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Th17 cell–polarizing conditions showed the suppressed production of IL-17A, IL-17F, and IL-22 by Th17 cells, but there were no changes in the expression of FoxP3 (Figure 6A). Halofuginone treatment increased IDO expression by human non-T cells, as it did in murine splenocytes (results are available online at http://www. rhrc.re.kr/bbs/board.php?bo_table⫽b_02_05&wr_id⫽ 95877&sca⫽&sfl⫽&stx⫽&sst⫽&sod⫽&spt⫽0&page⫽0). In the presence of RANKL and M-CSF, the addition of halofuginone prevented the differentiation of osteoclasts in human monocytes and prevented bone resorption activity (Figure 6B). This treatment also reduced the expression of osteoclastogenic markers, such as TRAP, NF-ATc1, CTR, cathepsin K, MMP-9, and RANK (Figure 6C). Taken together, these results indicate that halofuginone inhibits Th17 differentiation as well as osteoclastogenesis in humans, consistent with its effects in mice. DISCUSSION Chronic inflammation can lead to bone destruction in diseases such as RA. In the present study, we showed that halofuginone not only inhibited Th17 differentiation, but also directly prevented osteoclast formation and suppressed RANKL-mediated osteoclastogenesis through the NF-ATc1 pathway, leading to amelioration of inflammatory autoimmune arthritis. It has been reported that halofuginone has antitumor activity in various cancer models (23–26) and antifibrotic properties in several organs (27). Recently, Sundrud and colleagues reported that halofuginone specifically inhibits the Th17 cell– associated autoimmune inflammatory disease EAE by activating the amino acid starvation response (16). We demonstrated the simultaneous presence of Th17 cells and FoxP3⫹ Treg cells in halofuginone-treated mice with CIA. These findings are important for understanding the pathology and treatment of autoimmune diseases because an imbalance between Th17 and Treg cell function and distribution ultimately leads to various inflammatory and autoimmune disorders. Recently, plasticity of Treg and Th17 cells has been suggested to play a role in modulating inflammatory disease (28). Induction of FoxP3 in Th17 cells by halofuginone may contribute to the conversion of Th17 cells to Treg cells, although the possibility of Th17 cells developing into FoxP3⫹ Treg cells has not been examined in detail. Further investigation of the molecular basis underlying the plasticity of Th17 is required. Our results showed that reciprocal FoxP3 induction occurs only under Th17polarizing conditions, which include TGF␤, suggesting

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that halofuginone treatment has an effect on ERK signaling. Cellular activation by TGF␤1 regulates both Smad-dependent and Smad-independent pathways, such as ERK, in T cells (29). Recently, ERK signaling was also reported to negatively regulate the development of Th17 cells (30). Those studies raise the possibility that the activation of ERK upon Th17 differentiation is involved in mediating reciprocal changes in IL-17 and FoxP3 expression by halofuginone. We demonstrated that halofuginone enhanced ERK phosphorylation and FoxP3 expression in Th17 cells. Consistent with this finding, previous studies have suggested that ERK activation involves the expression of FoxP3 induced by TGF␤ (31). Moreover, although high concentrations of halofuginone impaired TGF␤ signaling in fibroblasts, our data, along with the findings of other studies (16), showed that low doses of halofuginone that repress Th17 differentiation did not inhibit TGF␤-induced Smad phosphorylation. These observations suggest that TGF␤/Smad3-independent ERK activation may play an important role in the induction of FoxP3 by halofuginone in Th17 cell development, although several studies have explored the role of Smad molecules downstream in the development of FoxP3⫹ cells induced by TGF␤ (32,33). Our findings were consistent with those of other studies showing that FoxP3 expression was attenuated in TGF␤-primed CD4⫹ cells in both wild-type and ERK-knockout mice, suggesting that the ERK pathway in FoxP3 induction is independent of Smad3 (32,33). Depletion of amino acids and inhibition of glucose metabolism have been shown to enhance the generation of antigen-specific Treg cells (34,35). In addition, the depletion of extracellular amino acids, either by amino acid catabolic enzymes, such as IDO, arginase 1, and asparaginase, or by halofuginone, results in activation of the protein kinase general control nonrepressed 2 (GCN-2) in T cells (16,36–39). Consequently, Th17 cell differentiation is suppressed, whereas Treg cell development and T cell anergy are enhanced. These previous observations are consistent with our results showing an increase in the number of Treg cells and a decrease in IL-17 expression in the spleens of mice with CIA treated with halofuginone. Therefore, it is likely that halofuginone contributes to changes in metabolic reprogramming in T cell differentiation. However, the detailed metabolic profiles and signals of differentiated and memory T cells remain to be explored. IDO suppresses adaptive T cell–mediated immunity in inflammation, host immune defense, and maternal tolerance via tryptophan depletion (40). Specifically,

IDO activates regulatory T cells and blocks their conversion to Th17-like T cells (41). Surprisingly, we found that halofuginone induced IDO expression in CD11c DCs and, consequently, suppressed IL-17 production in Th17-polarized CD4⫹ T cells but did not alter FoxP3 expression (data not shown). These observations suggest that halofuginone-induced IDO enzymatic activity leading to tryptophan depletion is responsible for pathogenic inflammation in autoimmune arthritis mediated by IL-17. Osteoclasts are the only cell type capable of resorbing mineralized bone, and inflammation enhances its activity, leading to bone erosion. Although halofuginone was previously shown to prevent bone loss induced by estrogen deprivation, it failed to directly affect osteoclast differentiation (42). In that study, high doses of halofuginone were used in cultures of bone marrow cells in vitro, and low concentrations of halofuginone (0.1–1 ␮g/ml) had no effect on osteoclast formation. However, we found that halofuginone had an inhibitory effect on osteoclast differentiation and function in vivo as well as in vitro. Furthermore, the inhibitory effect of halofuginone on osteoclast formation was seen at concentrations ⬃5–50-fold lower than those inhibiting Th17 differentiation. The differences between our findings and those reported previously may have been due to the relative purity of halofuginone. The halofuginone used in our experiments was purified by high-performance liquid chromatography to ⬎99% purity, which may affect cell viability, because unintended contaminants may influence the inhibitory effect of halofuginone on osteoclast formation. This effect demonstrated in the present study was not due to a form of toxicity, such as osteoclast apoptosis (data not shown). Halofuginone inhibits osteoclast formation by targeting members of the AP-1 family, such as c-Fos, JunB, and JDP-2, and the NF-AT pathway. The essential roles of NF-ATc1 and AP-1 transcription factors in RANKL signaling are now well established. NF-ATc1 is up-regulated by RANKL and plays a crucial role in osteoclast differentiation and function (9). The NFATc1 promoter contains NF-AT binding sites, and NF-ATc1 specifically autoregulates its own promoter during osteoclastogenesis, thus enabling the robust induction of NF-ATc1 (43). The AP-1 transcription factors are also important in the process of osteoclastogenesis, and AP-1 proteins, including Fos and Jun, can bind to NF-AT to transactivate target genes (44). Previous studies of signaling pathways involved in osteoclast formation indicated that NF-ATc1 acts downstream of c-Fos in osteoclastogenesis regulated by RANKL (45).

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NF-ATc1 has also been shown to be involved in transcriptional regulation of IL-17 in both humans and mice (46,47). Consistent with these reports, we found that halofuginone reduced NF-ATc1 in Th17 cell differentiation as well as osteoclast maturation. Although the precise molecular mechanisms underlying the reduced activity of NF-ATc1 by halofuginone in different cell types are unclear, our data indicated a specific role of halofuginone in diminishing NF-ATc1 activity. We also demonstrated that RANKL-mediated JDP-2 induction was significantly suppressed by halofuginone treatment. JDP-2 is a member of the AP-1 family that can heterodimerize with c-Jun and other Jun proteins (48), and has been identified as an important signaling molecule for RANKL-induced osteoclast differentiation via activation of both TRAP and cathepsin K gene promoters (49). The relative degree of reduction in JDP-2 expression is markedly different from those of reduced c-Fos and JunB in the presence of halofuginone, suggesting that JDP-2 may be more important than other AP-1 family members in controlling osteoclast differentiation. These findings demonstrate a novel role of halofuginone in regulating the differentiation of osteoclasts through suppression of AP-1 and NF-AT transcription. In addition to inhibition of transcription factors, these effects are achieved by modulating, at least in part, the expression of cyclin D1, a key regulator of cell cycle progression and growth. M-CSF has been reported to promote proliferation of primary macrophages by activating D-type cyclins (50). In addition, RANKL downregulates the cyclin D–dependent kinase CDK-6, which is a negative regulator of the transition from the G1 phase to the differentiation stage (51). These observations suggested that halofuginone-induced cyclin D1 up-regulation may exert inhibitory effects on osteoclast differentiation through cell cycle regulation. This study revealed a critical role of halofuginone in the inhibition of inflammation as well as bone destruction in RA. Halofuginone can directly target both activated Th17 cells and osteoclast progenitor cells, which are important to exert effector responses in autoimmune arthritis. Understanding the mechanisms by which halofuginone regulates both T cell immune responses and bone systems have profound importance for potential therapeutic intervention in inflammatory disease– related bone loss. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved

the final version to be published. Drs. Cho and S.-H. Park had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. M.-K. Park, J.-S. Park, E.-M. Park, Lim, S.-M. Kim, D.-G. Lee, Baek, Yang, Woo, J. Lee, Kwok, H.-Y. Kim, Cho, S.-H. Park. Acquisition of data. M.-K. Park, J.-S. Park, E.-M. Park, Lim, S.-M. Kim, D.-G. Lee, Baek, Yang, Woo, J. Lee, Kwok, H.-Y. Kim, Cho, S.-H. Park. Analysis and interpretation of data. M.-K. Park, J.-S. Park, E.-M. Park, Lim, S.-M. Kim, D.-G. Lee, Baek, Yang, Woo, J. Lee, Kwok, H.-Y. Kim, Cho, S.-H. Park.

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Halofuginone ameliorates autoimmune arthritis in mice by regulating the balance between Th17 and Treg cells and inhibiting osteoclastogenesis.

The small molecule halofuginone has been shown to inhibit fibrosis, angiogenesis, and tumor progression. This study was undertaken to evaluate the eff...
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