Biomaterials 44 (2015) 45e54

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The antibody atliximab attenuates collagen-induced arthritis by neutralizing AIMP1, an inflammatory cytokine that enhances osteoclastogenesis Shin Hee Hong a, Jin Gu Cho a, b, Kang Jun Yoon c, Dae-Seog Lim d, Chul Hoon Kim e, Sang-Won Lee f, Sang Gyu Park a, * a

College of Pharmacy, Ajou University, Suwon, Gyunggido, Republic of Korea Department of Biomedical Science, CHA University, Sungnamsi, Gyunggido 463-836, Republic of Korea Department of Neurosurgery, St Peter's Kangnam Hospital, Seoul, Republic of Korea d Department of Applied Bioscience, CHA University, 222 Yatapdong, Bundanggu, Sungnamsi, Gyunggido 463836, Republic of Korea e Department of Pharmacology, Yonsei University College of Medicine, 134 Shinchon, Seodaemungu, Seoul 120-749, Republic of Korea f Department of Rheumatology, Yonsei University College of Medicine, 134 Shinchon, Seodaemungu, Seoul 120-749, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 September 2014 Accepted 16 December 2014 Available online 12 January 2015

ARS-interacting multifunctional protein 1 (AIMP1) induces production of inflammatory cytokines from immune cells. Since osteoclastogenesis is promoted by positive regulation of inflammatory cytokines, whether AIMP1 could promote osteoclastogenesis was investigated. AIMP1 induced osteoclastogenesis and acted synergistically with RANKL to promote osteoclastogenesis. Down-regulation of CD23, an AIMP1 receptor, abolished AIMP1-mediated osteoclastogenesis. Enzyme-linked immunosorbent assays showed that the AIMP1 level was significantly higher in the peripheral blood (PB) and synovial fluid of rheumatoid arthritis patients than in normal PB. A monoclonal antibody (clone 15B3AF) that blocked the cytokine activity of AIMP1 inhibited the AIMP1-mediated production of inflammatory cytokines. Clone 15B3AF inhibited the AIMP1-mediated osteoclastogenesis in vitro. We then cloned the complementary determining regions of clone 15B3AF and generated a chimeric antibody (atliximab). In a collageninduced arthritis mouse model (CIA), atliximab administration significantly attenuated disease severity and improved various histopathological parameters. Three-dimensional micro-computed tomography scanning confirmed that atliximab enhanced the joint structures in CIA mice. Furthermore, atliximab decreased the expression of inflammatory cytokines in the serum and inflamed joints of CIA mice. Taken together, our findings suggest that AIMP1 exacerbates RA by promoting inflammation and osteoclastogenesis and that atliximab could be developed as a therapeutic antibody to target inflammatory diseases, including RA. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Cytokine Osteoclastogenesis Arthritis AIMP1

1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in multiple joints [1]. A variety of autoantigens such as type II collagen, proteoglycans, and glycoprotein stimulate sequential immune reactions via activation of CD4þ T cells, which is observed in RA [1]. Activated CD4þ T cells

* Corresponding author. Laboratory for Tracing of Gene Function, College of Pharmacy, Ajou University, Suwon, Gyunggido, Republic of Korea. Tel.: þ82 10 2314 2130. E-mail address: [email protected] (S.G. Park). http://dx.doi.org/10.1016/j.biomaterials.2014.12.017 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

induce the differentiation of B cells into plasma cells that produce autoantibodies such as rheumatoid factor and anti-cyclic citrullinated peptide (CCP) [1]. In addition, activated CD4þ T cells produce interferon-g (IFN-g), which subsequently stimulates monocytes/ macrophages to produce the inflammatory cytokines interleukin1b (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor-a (TNFa) and promotes the expression of matrix metalloproteinase from synovial fibroblasts. These molecules mediate long-term cartilage degradation and bone erosion, resulting in chronic pain and joint dysfunction [2e4]. Bone destruction in RA is associated with osteoclastogenesis. Osteoclasts are formed by the differentiation and fusion of monocytes/macrophages, which serve as osteoclast precursor cells [5].

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Receptor activator of nuclear factor-kappa B (NFkB) ligand (RANKL), a member of the TNF family, is a critical osteoclastogenic cytokine [6]. The binding of RANKL to its receptor (RANK) recruits TNF receptor-associated factor 6, which activates downstream signaling molecules, including mitogen-activated protein kinases (MAPKs), NFkB, activator protein 1 (AP-1), and nuclear factor of activated T cells c1 (NFATc1), leading to the induction of osteoclast differentiation and the formation of multinucleated osteoclasts [7e10]. In addition, in response to RANKL, mature, multinucleated osteoclasts undergo internal structural changes, including the rearrangement of actin filaments and the formation of tight junctions between the bone surface and basal membrane to form sealed compartments called external vacuoles, which erodes the underlying bone [11,12]. Aminoacyl-tRNA synthetase (ARS)-interacting multifunctional protein 1 (AIMP1) is a cofactor of the mammalian ARS multicomplex in the cytoplasm [13]. AIMP1 is secreted in response to hypoxia, heat shock, and TNFa stimulation. It then functions as an inflammatory cytokine, targeting monocytes/macrophages, endothelial cells, neutrophils, dendritic cells (DCs), and T cells [14e22]. Secreted AIMP1 activates monocytes/macrophages via signaling cascades that include p38 MAPK, extracellular signal-regulated kinase (ERK), and NF-kB, leading to the secretion of proinflammatory cytokines such as TNFa, interleukin-8 (IL-8), and macrophage chemotactic protein-1 (MCP-1) [17,18,23]. In addition,

AIMP1 enhances the expression of intercellular adhesion molecule1 (ICAM-1) which plays a critical role in lymphocyte extravasation, inflammation and atherosclerosis [17,18,24]. Furthermore, AIMP1 stimulates the maturation of DCs, which induces the expression of IL-1b, IL-6, and IL-12, cytokines critical for sequential immune activation [16,25,26]. IL-1b and IL-6 are known to stimulate osteoclastogenesis [1]. In macrophages, AIMP1 forms a positive feedback loop with TNFa to amplify the inflammatory response [17,21]. On the basis of these studies, we hypothesized that AIMP1 is a causal cytokine in RA development associated with inflammation and osteoclastogenesis. In this study, we assessed the effects of AIMP1 on osteoclastogenesis and generated an AIMP1 neutralizing antibody that blocks cytokine activity, which was then used to develop a chimeric antibody, atliximab (AIMP1 targeting eli, -xi, mab). In vivo analysis using a collagen-induced arthritis (CIA) mouse model showed that neutralization of AIMP1 with atliximab reduced various pathological parameters. Therefore, we propose that atliximab could be developed into a therapeutic antibody to treat inflammatory diseases, including RA. 2. Materials and methods 2.1. Osteoclastogenesis assay Raw264.7 cells (2  103cells) were seeded onto 24-well plates in high glucose Dulbecco's modified Eagle's medium containing 10% FBS and 1% penicillin/streptomycin

Fig. 1. AIMP1 synergistically induces osteoclastogenesis of Raw264.7 cells with RANKL. (AeC) Osteoclastogenesis of Raw264.7 cells was induced by RANKL (50 ng/ml) in the presence or absence of AIMP1 for 3 days. Multinucleated osteoclasts (OC) were counted after TRAP staining. Bone resorption activity was evaluated by measuring the fluorescence intensity of the conditioned medium. Data represent the mean ± SEM of three independent experiments (*, vs. AIMP1-/RANKL). (DeE) Raw264.7 cells were treated with RANKL (50 ng/ml) in the presence or absence of AIMP1 (50 nM) for the indicated time. IkB and pERK were detected with specific antibodies (D), and the expression of NFATc1 was examined (E). (FeH) Raw 264.7 cells were transfected with 20 nM of control or CD23 si-RNA for 48 h and treated with RANKL (50 ng/ml) in the presence or absence of AIMP1 (50 nM) for 30 min. IkB, pERK, and CD23 were detected with their specific antibodies. The activation of NFkB was determined by IkB degradation. Tubulin was used as a loading control. In addition, osteoclastogenesis was induced for 3 days, and multinucleated osteoclasts were quantitated by TRAP staining (G and H). RANKL (50 ng/ml) and AIMP1 (50 nM) was treated for 1 day to examine the expression of NFATc1 and CD23 (H, inset). Data represent the mean ± SEM of three independent experiments (**, vs.AIMP1-/si-con; *, vs. AIMP1-/si-con; y, vs. AIMP1þ/si-con). *P < 0.05, **P < 0.01, and yP < 0.01.

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Fig. 2. Inflammatory cytokine levels in human subjects. (AeE) Cytokine levels were measured by ELISA in the PB of the normal cohort and in the PB and SF of RA patients (*, vs. normal PB; **, vs. normal PB; z, vs. RA PB; n ¼ 19e28 per group). (FeI) Correlation analysis of AIMP1 with TNFa, MCP-1, IL-6, and sIL-6R in the SF of RA Patients. *P < 0.01, **P < 0.05 and zP < 0.05. and were cultured for 24 h in humidified CO2 incubator. Osteoclastogenesis was induced by the addition of RANKL (50 ng/ml) in the presence or absence of AIMP1 in minimal essential medium supplemented with 10% FBS and 1% penicillin/streptomycin for 3 days. Osteoclast differentiation was examined by counting the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts using a TRAP assay kit

(SigmaeAldrich) according to the manufacturer's instructions [27]. For bone resorption assay, Raw264.7 cells were differentiated into osteoclasts on a fluoresceinated calcium phosphate-coated plate (Cosmo Bio Co., Japan). The culture medium was then transferred to a 96-well plate, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

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Fig. 3. AIMP1 neutralizing antibody inhibits AIMP1-enhanced secretion of inflammatory cytokines and osteoclastogenesis. (A) THP-1 cells, a human monocyte cell line, were treated with 10 nM of AIMP1 in the presence or absence of various AIMP1 monoclonal IgG antibodies (5 mg/ml) for 12 h. TNFa production was analyzed by ELISA. Data represent the

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3. Results 3.1. AIMP1 promotes osteoclastogenesis AIMP1 is secreted from macrophage in response to TNFa stimulation [21]. AIMP1 then induces the secretion of inflammatory cytokines from monocytes/macrophages, creating a positive feedback loop that amplifies the inflammatory response [17]. Because inflammatory cytokines such as TNFa, IL-1b, and IL-6 are osteoclastogenic, we examined whether AIMP1 could promote the osteoclastogenesis of macrophage in vitro. AIMP1 promoted osteoclastogenesis of Raw264.7 cells and human bone marrowderived macrophage (hBMM) in a dose-dependent manner, but not to level of RANKL (Supplementary Fig. 1AeF). Quantitative RTPCR analysis showed that AIMP1 increased expression of c-FOS, NFATc1 and TRAP (Supplementary Fig. 2A). Recently, CD23, a low affinity receptor for IgE, was isolated as a receptor for AIMP1 that induces the secretion of TNFa from monocytes and peripheral blood mononuclear cells [28]. CD23 regulates immune cell activation, and its expression is increased in RA [29,30]. A CD23 neutralizing antibody ameliorates CIA, and CD23-deficient mice show delayed onset and reduced severity of CIA, suggesting that CD23 is involved in RA development [31]. Therefore, to determine whether AIMP1 promoted osteoclastogenesis via CD23, we knocked down CD23 expression in Raw264.7 cells by using siRNA. Down-regulation of CD23 attenuated the AIMP1-mediated increased expression of c-FOS, NFATc1 and TRAP, leading to reduced osteoclastogenesis of Raw264.7 cells (Supplementary Fig. 2BeE). Next, we assessed whether AIMP1 could have synergistic activity with RANKL in osteoclastogenesis. When added with RANKL, AIMP1 promoted osteoclastogenesis of Raw264.7 cells and hBMM in a dose-dependent manner (Fig. 1AeC, Supplementary Fig. 3AeC). Because AIMP1 induces inflammatory cytokine secretion from monocytes/macrophages via ERK and NFkB signaling, and because RANKL stimulates osteoclastogenesis via the activation of ERK, NFkB, and AP-1 [8,9,19], we examined whether AIMP1 could enhance RANKL signaling. AIMP1 acted synergistically with RANKL to activate ERK and NFkB, leading to the increased expression of NFATc1 (Fig. 1DeE) [28e31]. As shown in Fig. 1FeH, downregulation of CD23 abolished the AIMP1-mediated amplification of NFkB, pERK, and NFATc1, leading to a significant reduction in osteoclastogenesis. These findings led us to investigate whether AIMP1 levels were elevated in RA patients. Information for human cohort is summarized in Supplementary Table 2. We assessed the levels of inflammatory cytokines, including AIMP1, in the peripheral blood (PB) and synovial fluid (SF) of RA patients. IL-6, MCP-1, soluble IL-6R (sIL-6R) and TNFa in the PB and SF of RA patients were significantly higher than the levels in normal PB (Fig. 2AeC), which is in agreement with previous reports [32e34]. Interestingly, AIMP1 levels in the PB and SF of RA patients were also higher than normal PB (normal PB, 4.48 ± 3.18 ng/ml; RA PB, 21.09 ± 23.06 ng/ ml; RA SF, 52.35 ± 55.10 ng/ml) (Fig. 2E). Correlation analysis of IL-6,

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MCP-1, sIL-6R and TNFa with AIMP1 in the SF of RA patients showed that the increases in inflammatory cytokines were significantly associated with the AIMP1 level (Fig. 2FeI). These results suggest that AIMP1 is correlates, at least in part, with RA development. 3.2. AIMP1 neutralizing antibody inhibits AIMP1 cytokine activity Next, we generated and screened monoclonal antibodies to neutralize the cytokine activity of AIMP1. Among them, clone 15B3AF inhibited AIMP1-mediated TNFa secretion in THP-1 monocyte (Fig. 3A). Immunoglobulin isotyping showed that clone 15B3AF is IgG1/kappa chain antibody (data not shown). In addition, western blotting showed that 15B3AF IgG recognized mouse AIMP1 as well as human AIMP1 (Fig. 3B). The epitope of clone 15B3AF was identified by western blotting with purified full-length (amino acids [aa] 1e312), N-terminal (aa 1e146), and C-terminal (aa 147e312) human AIMP proteins. 15B3AF IgG specifically recognized the N-terminus of AIMP1 (Fig. 3C). An enzyme-linked immunosorbent assay (ELISA) using purified protein also showed that 15B3AF IgG specifically recognized the N- terminal domain of AIMP1 (Supplementary Fig. 4). The finding that 15B3AF IgG blocked AIMP1 cytokine activity and interacted specifically with the Nterminal domain of AIMP1 suggested that the epitope for 15B3AF IgG resides between aa 101 and 146 in AIMP1. Therefore, we synthesized different AIMP1 peptides, designated P1 to P4 (Fig. 3D). P1 corresponds to the fibroblast proliferation-inducing domain of AIMP1; P2, P3, and P4 are cytokine domains, but P3 and P4 are confined to the C-terminal domain of AIMP1. Competitive ELISAs showed that the P2 peptide inhibited the binding of 15B3AF IgG to AIMP1 in a dose-dependent manner (Fig. 3E). This result suggests that the association of 15B3AF IgG with P2 region of AIMP1 is critical for blocking AIMP1-mediated TNFa secretion, in agreement with finding that the region between aa 101-192 in AIMP1 is critical for TNFa production [28]. Because AIMP1 induces the secretion of inflammatory cytokines via the activation of ERK and NFkB [19], we examined whether 15B3AF IgG could inhibit the AIMP1-mediated activation of ERK and NFkB in THP-1 monocytes. 15B3AF IgG efficiently inhibited the AIMP1-mediated activation of ERK and NFkB (data not shown). ELISAs using THP-1 monocytes and mouse DCs confirmed that 15B3AF IgG inhibited the AIMP1-mediated secretion of inflammatory cytokines in a dose-dependent manner (Fig. 3 F and G). Furthermore, 15B3AF IgG reduced the AIMP1-mediated increase in NFATc1 expression and osteoclastogenesis of Raw264.7 cells and hBMM in a dose-dependent manner (Fig. 3H and I, Supplementary Fig. 5AeC). To assess whether the complementary determining regions (CDRs) of 15B3AF IgG could work as immunogen, splenocytes were incubated with 15B3AF IgG, and an ELISPOT assay with an IFNg antibody was performed. However, 15B3AF IgG was not immunogenic (data not shown). In addition, dendritic cell-T cell proliferation assay using CD8þ T cell depleted PBMC showed that 15B3AF IgG was not immunogenic (data not shown).

mean ± SEM of three independent experiments (*, vs. vehicle/AIMP1). (B) Whole cell lysates (30 mg) from HEK293 and Raw264.7 cells were separated by 10% SDS-PAGE, and AIMP1 was blotted with 15B3AF IgG. (C) To determine the 15B3AF IgG epitope, purified full-length, N-terminal, and C-terminal AIMP1 proteins were resolved by 12% SDS-PAGE, and stained with Coomassie blue (5 mg/lane; left) and blotted with 15B3AF IgG (10 ng/lane; right). (D) The amino acid sequence alignment of human AIMP1 and mouse AIMP1 is shown. Synthesized peptides are indicated by red boxes. The cytokine domain is underlined. (E) To determine the minimal epitope of 15B3AF IgG, we performed competitive ELISAs using the peptides described above. A 96-well plate was coated with AIMP1 full-length protein (100 ng/well), and purified 15B3AF IgG (0.2 mg/ml) was added in the presence or absence of the competitive peptides as indicated. Data represent the mean ± SEM of three independent experiments (*, vs. vehicle). To examine whether 15B3AF IgG could inhibit the secretion of AIMP1-induced inflammatory cytokines, THP-1 monocytes (F) and mouse primary DCs (G) were treated with AIMP1 (10 nM) in the presence or absence of 15B3AF IgG as indicated, and the secretion of inflammatory cytokines was measured by ELISA. Data represent the mean ± SEM of three independent experiments (y and z, vs. AIMP1þ/15B3AF-). (H) Raw 264.7 cells were treated with RANKL (50 ng/ml) in the presence or absence of AIMP (10 nM) or an AIMP1 (10 nM)/15B3AF IgG mixture, as indicated, and differentiated into osteoclasts. Multinucleated osteoclasts were counted (I, top), and the expression of NFATc1 was examined by immunoblot (inset). Tubulin was used as a loading control. The bone absorption activity of osteoclasts was evaluated as described in the Materials and Methods section (I, bottom). Data represent the mean ± SEM of three independent experiments (*, vs. AIMP1-/15B3AF-; ** and y, vs. AIMP1þ/15B3AF-). *P < 0.001, **P < 0.001, yP < 0.05, and zP < 0.01.

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Fig. 4. Atliximab attenuates the severity of arthritis in a CIA mouse model. Atliximab was administered to CIA mice at the doses as indicated. The mean paw thickness (A) and mean arthritis score (B) of four legs were evaluated (* and **, vs. CIA/vehicle; y, vs. CIA/Atliximab 2 mg/kg; n ¼ 8e10 per group). (C) Histopathological analysis of atliximab-treated CIA mice. CIA mice showed severe inflammation (red arrow head), synovial hyperplasia (black arrow head), cartilage destruction (a), and bone erosion (b) in the joint sections. In contrast, the extent of arthritis and bone destruction was significantly reduced in the joints of mice treated with 2 mg/kg or 5 mg/kg atliximab (original magnification x100, hematoxylin and eosin staining). Immunohistochemical (IHC) analysis of joint tissues from vehicle-treated CIA mice showed markedly positive staining for AIMP1, TNFa, IL-1b, IL-6

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3.3. A chimeric antibody against AIMP1 reduces the severity of arthritis in a CIA mouse model We cloned the CDRs from the variable light chain and heavy chain of 15B3AF IgG and grafted them into human IgG as shown in Supplementary Fig. 6A to generate a construct encoding the chimeric antibody atliximab (AIMP1 targeting eli, -xi, mab). We transiently transfected the construct into a Chinese hamster ovary (CHO) cell line and purified atliximab using protein A Sepharose (Supplementary Fig. 6B). 15B3AF IgG and atliximab showed similar activities in ELISAs and TNFa secretion assays with AIMP1 (Supplementary Fig. 6C and D). Binding affinity determination using surface plasmon resonance between human recombinant AIMP1 and atliximab showed that KD value is 2.33  1012 M (±0.16). Next, we used a CIA mouse model to examine the therapeutic efficacy of atliximab. Macroscopic evidence of arthritis, such as erythema or swelling, was markedly observed in vehicle treated CIA mice (Supplementary Fig. 7A). Interestingly, intraperitoneal injection of atliximab significantly attenuated arthritis severity, including paw thickness and mean arthritis score, in a dosedependent manner (Fig. 4A and B, Supplementary Fig. 7A). In addition, area under the curve analysis showed that atliximab significantly reduced paw thickness and the mean arthritis score (Supplementary Fig. 7BeC). Histopathological evaluation and immunohistochemical analysis of paw and knee joint sections from vehicle-treated CIA mice showed inflammatory cell infiltration, synovial hyperplasia and partial bone destruction (Fig. 4C hematoxylin and eosin staining). The sections exhibited extensive staining for AIMP1, TNFa, IL-1b, IL-6, and MCP1, which were localized in inflamed cells around the joints (Fig. 4C). Atliximab administration reduced not only the extent of inflammatory cell infiltration and bone destruction but also the expression of AIMP1, TNFa, IL-1b, IL-6, and MCP1 in a dose-dependent manner (Fig. 4CeD). Analysis of histopathological parameters confirmed that atliximab reduced inflammation, cartilage destruction and bone destruction in a dose dependent manner (Fig. 4E). Next, we used three-dimensional micro-computed tomography (CT) imaging analysis to quantify the level of bone alterations. Severe bone destruction was observed in vehicle-treated CIA mice. However, atliximab administration preserved bone integrity in a dose-dependent manner (Fig. 5A). In particular, the bone structure in CIA mice treated with 5 mg/kg atliximab was comparable to that in normal mice without arthritis (Fig. 5A). In addition, six parameters were analyzed: bone volume (BV), bone volume/tissue volume (BV/TV), bone surface area/bone volume (BS/BV), bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular separation (Tb.Se). The BV and BV/TV measurements enabled us to compare bone samples of different sizes when assessing the extent of bone preservation. The BS/BV parameter reflects the loss of bone surface due to erosion. Tb.Th inversely correlates with periarticular osteopenia induced by joint inflammation. BMD correlates with bone erosion [35]. When CIA mice were administered with atliximab, BV, BV/TV, BMD, and Tb.Th increased, and BS/BV and Tb.Se decreased in a dose-dependent manner (Fig. 5B). To validate the significance of these changes, we evaluated the correlation coefficients. Tb.Th correlated with BV/TV and BS/BV (r2 ¼ 0.8496 and r2 ¼ 0.7673, respectively, p < 0.001), and BV/TV correlated with BMD and BS/BV (r2 ¼ 0.9383 and r2 ¼ 0.8707, respectively, p < 0.0001) (Fig. 5C).

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CD4þCD25þFoxp3þ (forkhead box p3) regulatory T cells (Tregs) are a distinct population of suppressor T cells that maintain peripheral immune tolerance. Treatment with a TNFa inhibitor increases the CD4þCD25þFoxp3þ Treg population [36e39]. Atliximab administration increased the CD4þCD25þFoxp3þ Treg population in the spleen of CIA mice in a dose-dependent manner (Supplementary Fig. 8A). In addition, treatment of mice with atliximab significantly reduced the IFNgþCD4þ population and CD4þIL-17Aþpopulation (Supplementary Fig. 8BeC). To investigate whether Atliximab could decrease serum levels of inflammatory cytokines, we examined the concentration of IFNg, TNFa, IL-1b, IL-6, MCP-1, and AIMP1 in the serum of CIA mice. Atliximab significantly reduced serum concentrations of inflammatory cytokines including AIMP1 in a dose dependent manner (Supplementary Fig. 8DeI). In contrast, atliximab treatment increased the level of IL-4, an immuno suppressive cytokine secreted by Th2 cells (Supplementary Fig. 8K). Thus, in the CIA mouse model, atliximab decreased the levels of inflammatory cytokines in the serum and inflamed joints and reduced various rheumatoid pathological parameters. 4. Discussion RA is a chronic inflammatory and destructive arthropathy that is difficult to cure. RA is initiated by the infiltration of inflammatory cells, primarily CD4þ T cells, which are the main orchestrators of the immune responses. Activated CD4þ T cells stimulate monocytes/macrophage and synovial fibroblasts to produce cytokines such as IL-1b, IL-6, and TNFa [1]. DCs migrate into the joint in response to inflammatory cytokines and chemokines, localize in the rheumatoid synovial tissue perivascular region [40,41], and activate CD4þ T cells to produce IFNg, thus creating a positive feedback loop between monocytes/macrophages, DCs, and CD4þ T cells. RANKL induces the differentiation of osteoclasts from monocytes/macrophages via TNFa, and IL-1b [42,43]. AIMP1 secreted from macrophages in response to TNFa stimulation directly activates monocytes/macrophages to produce IL-1b, IL-8, and TNFa and stimulates DCs to produce IL-1b, IL-6, IL-12, and TNFa [16,17,21]. Therefore we investigated whether AIMP1 could enhance osteoclastogenesis and found that AIMP1 acted synergistically with RANKL to stimulate osteoclastogenesis (Fig. 1AeE). In addition, we assessed whether RANKL, a TNFa family member, could also enhance AIMP1 expression in Raw264.7 cells. AIMP1 expression was increased about 2 folds by RANKL treatment (data not shown), suggesting that the increased AIMP1 in synovial fluid, at least, derives from monocytes/macrophages stimulated with TNFa and RANKL. The knock-down of AIMP1 using si-RNA reduced RANKL-mediated osteoclastogenesis of macrophage (Supplementary Fig. 9AeC), indicating that AIMP1 is critical for osteoclastogenesis. Interestingly, AIMP1 increased expression of RANKL in osteoblast (Supplementary Fig. 10A), suggesting that AIMP1 and RANKL form positive feed-back loop to promote osteoclastogensis. Monocytes/macrophages express high levels of CD23 [44]. Activation of CD23 promotes the release of inflammatory cytokines, including TNFa, IL-1b, and IL-6 [45], and AIMP1 induces the secretion of TNFa via CD23 [28]. Thus, AIMP1 could be involved in RA development. CD23 regulates immune cell activation and its expression is increased in RA by NFkB activation [29,30,46], suggesting that CD23 plays a critical role in the amplification of

and MCP-1, which localized primarily in the inflamed cells around joints. In contrast, 2 mg/kg or 5 mg/kg atliximab reduced the staining for AIMP1, TNFa, IL-1b, IL-6 and MCP-1, when compared to treatment with vehicle or 1 mg/kg atliximab (original magnification x100, IHC). (D) AIMP1, TNFa, IL-1b, IL-6, and MCP-1 expression was semiquantitatively analyzed by densitometric scanning. Treatment with 2 mg/kg, 5 mg/kg of atliximab significantly reduced inflammation in the joints (**, vs. atliximab; n ¼ 8e10 per group). (E) Histopathological parameters, including inflammation and cartilage and bone destruction, were analyzed by two independent pathologists (*, vs. CIA/vehicle; n ¼ 8e10 per group). *P < 0.05, **P < 0.01, and yP < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Micro-computerized tomography scanning confirms the efficacy of atliximab in CIA mice. (A) Severe bone erosions were observed in both the foreleg and hind leg of CIA mice. In contrast, treatment of CIA mice with 2 or 5 mg/kg atliximab reduced joint destruction. (B) Bone volume (BV), trabecular thickness (Tb. Th), bone volume/tissue volume (BV/ TV), and bone mineral density (BMD) increased significantly in CIA mice treated with 2 or 5 mg/kg atliximab, whereas bone surface area/bone volume (BS/BV) and trabecular separation (Tb. Se) decreased significantly (*, vs. CIA/vehicle; n ¼ 8e10 per group). (C) Significant positive correlations were observed between Tb.Th. and BV/TV and between BV/TV and BMD. Significant negative correlations were observed between Tb.Th and BS/BV and between Bv/Tv and BS/BV. *P < 0.05.

inflammation during RA development. We also found that AIMP1 increased the expression of CD23 (Fig. 1H). Suppression of NFkB activation with N-acetyl-leucinyl-leucinyl-norleucinal, an inhibitor of IkB proteolysis, abrogated the AIMP1-mediated increase in CD23 (data not shown). A CD23 blocking antibody ameliorates CIA, and genetic depletion of CD23 in mice delays the onset and reduces the

severity of CIA [31]. A chimeric antibody, lumiliximab, that targets CD23 was recently developed. Treatment of antigen-presenting cells with lumiliximab reduced the secretion of inflammatory cytokines such as IL-1b, TNFa [47], suggesting that AIMP1-CD23 signaling is a critical axis for the development of inflammatory disease such as RA.

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Various biologic therapies have been developed to suppress inflammation and bone destruction [48,49]. The major targets of the biologics are the immune effector cells including T lymphocytes, B lymphocytes, and macrophages, responsible for inflammation. The first drugs approved for the treatment of RA were TNFa inhibitors. However, TNFa-targeting agents are not effective in all RA patients. About 30% of patients treated with TNFa inhibitors fail to achieve a 20% improvement in symptoms according to the American College of Rheumatology criteria (ACR20; primary failure or inefficacy) [50e52], and some patients acquired drug resistance during therapy (secondary failure) [53]. Therefore, we hypothesized that neutralization of AIMP1, an inducer of osteoclastogenesis as well as inflammatory cytokines, could break the positive feedback loop that promotes inflammation, leading to the amelioration of RA. 15B3AF IgG efficiently bound to the cytokine domain of AIMP1, resulting in a reduction in the AIMP1-mediated secretion of inflammatory cytokines. We also assessed whether 15B3AF IgG could specifically bind to AIMP1 because non-specific binding of 15B3AF IgG could induce side effect after in vivo administration. A ProtoArray analysis using a protein microarray chip embedded with about 9000 human proteins was performed. 15B3AF IgG primarily bound to SCYE1, another name of AIMP1, confirming that 15B3AF IgG bound specifically to AIMP1. 15B3AF IgG also associated with guanine nucleotide binding protein G subunit gamma-T1 (GNGT1), a photoreceptor that is mainly expressed in the eye (Supplementary Table 3). Sequence alignment using ClustalW2 showed that aa 114134 of human AIMP1 shared about 40% similarity with GNGT1, in agreement with the epitope mapping data in Fig. 3D and E. Further studies are needed to determine whether the binding affinity between 15B3AF IgG and GNGT1 is meaningful. To further develop the AIMP1 neutralizing antibody, 15B3AF IgG CDRs were grafted into human IgG1 to generate a chimeric antibody, Atliximab. Atliximab and 15B3AF IgG showed similar binding affinities for AIMP1 and efficiently inhibited AIMP1-mediated TNFa production in human monocytes (Supplementary Fig. 6C and D). There were no differences in the white blood cell count, and hemoglobin. Liver and kidney toxicities were not observed in mice treated with 10 or 20 mg/kg Atliximab (Supplementary Table 4). In addition, there were no differences in body weight change (data not shown). 5. Conclusions AIMP1 cytokine synergistically increases osteoclastogenesis of macrophages with RANKL. Development of atliximab, an AIMP1 neutralizing humanized chimeric antibody, inhibited AIMP1mediated osteoclastogenesis in vitro. In addition, application of atlixiamb into CIA mouse model siginificantly attenuated arthritis. Therefore, further development of atliximab into a fully humanized antibody may produce an effective drug for the treatment of inflammatory diseases, including RA. Acknowledgement This study was supported by a grant from the Korea Health Care Technology R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea (A092039), and by the Bio and Medical Technology Devlopment Program of the National Research Foundation (NRF), funded by Koean Government Grant NRF2014M3A9D9069584. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.12.017.

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