ARTHRITIS & RHEUMATOLOGY Vol. 67, No. 4, April 2015, pp 893–902 DOI 10.1002/art.39007 © 2015, American College of Rheumatology
Tofacitinib Facilitates the Expansion of Myeloid-Derived Suppressor Cells and Ameliorates Arthritis in SKG Mice Keisuke Nishimura, Jun Saegusa, Fumichika Matsuki, Kengo Akashi, Goichi Kageyama, and Akio Morinobu In vitro, tofacitinib facilitated the differentiation of BM cells to MDSCs, and inhibited their differentiation to dendritic cells. Conclusion. Tofacitinib facilitates the expansion of MDSCs and ameliorates arthritis in SKG mice.
Objective. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that have the ability to suppress T cell responses. The aim of this study was to evaluate the effects of the JAK inhibitor tofacitinib on MDSCs in a mouse model of rheumatoid arthritis. Methods. Arthritis was induced in SKG mice by zymosan A (ZyA) injection. MDSCs isolated from the bone marrow (BM) of donor SKG mice with arthritis were adoptively transferred to recipient mice with arthritis. In a separate experiment, tofacitinib was administered to arthritic SKG mice subcutaneously via osmotic pump, in some cases followed by injection of an anti–Gr-1 monoclonal antibody (mAb). BM cells from untreated mice were cultured for 5 days with granulocyte–macrophage colony-stimulating factor, with or without tofacitinib, and then analyzed by flow cytometry. Results. The numbers of MDSCs and polymorphonuclear MDSCs (PMN-MDSCs) were significantly increased in the spleens of SKG mice following ZyA injection. Adoptive transfer of MDSCs to recipient arthritic mice reduced the severity of arthritis compared to that in untreated control mice. Treatment with tofacitinib also ameliorated the progression of arthritis in SKG mice and induced significantly higher numbers of MDSCs and PMN-MDSCs in the BM of these animals. Furthermore, administration of an anti–Gr-1 mAb reduced the antiarthritic effect of tofacitinib in SKG mice.
Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage and bone destruction. Dendritic cells (DCs), monocytes, T cells, B cells, and neutrophils infiltrate the synovium and interact with each other and synovial fibroblasts to induce chronic synovitis (1,2). Cytokine production by various cells in the joint is central to the pathogenesis of RA. Antibodies targeting tumor necrosis factor ␣ and interleukin-6 (IL-6) are current therapeutic strategies for RA. The JAK kinases are tyrosine kinases that mediate the signaling pathways activated by various cytokines and growth factors. The JAK family consists of JAK-1, JAK-2, JAK-3, and tyrosine kinase 2, and different cytokines activate different sets of JAK family members. Tofacitinib is a small-molecule JAK inhibitor that inhibits JAK-1 and JAK-3, and suppresses inflammatory signaling downstream of ␥c-chain cytokines (IL-2, IL-4, IL-7, and IL-15), as well as IL-6 and interferon-␥ (3). Since multiple cytokines have been implicated in the pathogenesis of RA, tofacitinib currently represents a novel therapy for RA (4,5). However, the antirheumatic effects of tofacitinib, especially its influence on myeloid cell differentiation, are not fully understood. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are differentiated from immature myeloid precursor cells and characterized by the coexpression of Gr-1 and CD11b in mice. MDSCs have immunosuppressive properties; they suppress T cell responses by producing arginase 1 (Arg-1) (6–8), inducible nitric oxide synthase (iNOS) (9), and reactive oxygen species (ROS) (9,10). Phenotypically,
Supported in part by Grant-in-Aid for Scientific Research 24659473 from the Japan Society for Promotion of Science. Keisuke Nishimura, MD, Jun Saegusa, MD, PhD, Fumichika Matsuki, PhD, Kengo Akashi, MD, Goichi Kageyama, MD, PhD, Akio Morinobu, MD, PhD: Kobe University Graduate School of Medicine, Kobe, Japan. Address correspondence to Akio Morinobu, MD, PhD, Department of Rheumatology and Clinical Immunology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected]. Submitted for publication March 26, 2014; accepted in revised form December 19, 2014. 893
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MDSCs are divided into 2 subsets, polymorphonuclear MDSCs (PMN-MDSCs; defined as CD11b⫹Ly-6G⫹Ly6Clow cells), and monocytic MDSCs (M-MDSCs; defined as CD11b⫹Ly-6G⫺Ly-6Chigh cells) (11–13). PMNMDSCs express high levels of ROS and low levels of NO, whereas M-MDSCs express low levels of ROS and high levels of NO, and both subsets express Arg-1 (11). MDSCs are increased in tumor-bearing mice and in patients with cancer, and contribute to tumor survival by inhibiting tumor-specific T cell responses. Thus, MDSCs are potent immunosuppressive cells and potential target cells for cancer treatment. MDSC expansion is also associated with infection and autoimmunity (14– 20), and the spleens of mice with collagen-induced arthritis (CIA) have increased numbers of MDSCs (20). Immature myeloid precursor cells in the bone marrow (BM) are thought to differentiate into MDSCs in response to inflammatory stimuli, and then exit the BM and enter the circulatory system. However, the differentiation of MDSCs and their role in suppressing inflammation remain unclear. In the present study, we investigated the role of MDSCs and the effect of tofacitinib on MDSCs in the SKG mouse model of arthritis, a well-established model that exhibits many features of RA, including chronic destructive arthritis with a Th17 phenotype (21). We show that MDSCs possess antirheumatic properties, and that tofacitinib facilitates their expansion. Furthermore, the antiarthritic effect of tofacitinib was abrogated when MDSCs were depleted by the administration of an anti– Gr-1 monoclonal antibody (mAb), suggesting that the effect of tofacitinib is mediated, at least in part, by MDSCs. MATERIALS AND METHODS Animals. Female SKG mice and female BALB/c mice were obtained from CLEA Japan, Inc. Mice were housed in the Kobe University animal facility at a constant temperature, with provision of laboratory chow and water ad libitum. All procedures were carried out in accordance with the recommendations of the Institutional Animal Care Committee of Kobe University. Reagents and antibodies. Zymosan A (ZyA), DMSO, G6, PF-956980 hydrate, and 2-mercaptoethanol (2-ME) were purchased from Sigma-Aldrich. Tofacitinib (Selleck Chemicals), GLPG0634 (AdooQ Bioscience), a carboxyfluorescein diacetate succinimidyl ester (CFSE-DA) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS) (MP Biomedicals), 1% penicillin– streptomycin (Lonza Walkersville), and recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) (PeproTech) were also used. Fluorescein isothiocyanate (FITC)–conjugated anti–Gr-1, FITC-conjugated anti–Ly-6G, phycoerythrin-conjugated anti-CD11b, allophycocyanin (APC)-
conjugated anti–Ly-6C, and APC-conjugated anti-F4/80 were from BD PharMingen. Arthritis induction. Eight-week-old SKG mice were treated with 2 mg ZyA, as previously described (21). Briefly, ZyA suspended in saline was intraperitoneally injected on day 0. Arthritis developed between 14 and 21 days after injection. Evaluation of arthritis. The development and severity of arthritis were assessed using a previously described system for scoring clinical arthritis (22,23), as follows: 0 ⫽ no joint swelling, 0.1 ⫽ swelling of one digit joint, 0.5 ⫽ mild swelling of the wrist or ankle, and 1.0 ⫽ severe swelling of the wrist or ankle. Arthritis scores were totaled for each mouse. The maximum possible clinical arthritis score was 5.8. Histology. The hind paws of the mice were removed, fixed in 4% paraformaldehyde, decalcified in EDTA, embedded in paraffin, and sectioned. The samples were then stained with hematoxylin and eosin. Histologic evaluation was performed using a previously described scoring system (23), in which 0 ⫽ no inflammation, 1 ⫽ slight thickening of the synovial cell layer and/or some inflammatory cells in the sublining, 2 ⫽ thickening of the synovial lining, infiltration of the sublining, and localized cartilage erosions, and 3 ⫽ infiltration in the synovial space, pannus formation, cartilage destruction, and bone erosion. Isolation of MDSCs and CD4ⴙ T cells. MDSCs were isolated from a single-cell suspension prepared from the BM of arthritic SKG mice, using a biotinylated mAb against Gr-1, streptavidin-coated magnetic beads, and the AutoMACS system (all from Miltenyi Biotec), according to the manufacturer’s protocol. CD4⫹ T cells were purified from single-cell suspensions prepared from the spleens of normal mice, using negative selection with a biotinylated antibody mixture and antibiotin-coated magnetic beads (all from Miltenyi Biotec), following the manufacturer’s protocol. Flow cytometry. Cells were washed with Flow Cytometry Staining Buffer (eBioscience), and were stained with anti– Gr-1, anti–Ly-6G, anti–Ly-6C, and anti-CD11b mAb for 30 minutes at 4°C. The cells were acquired on a FACSCalibur (BD Bioscience) and analyzed using FlowJo software (Tree Star). Western blot analysis. BM cells from SKG mice treated with or without ZyA were lysed with radioimmunoprecipitation assay buffer (Thermo Scientific) containing a protease inhibitor cocktail (Roche Diagnostics) at 4°C for 30 minutes. Protein samples (30 g) were boiled for 5 minutes in sodium dodecyl sulfate sample buffer (Wako Pure Chemical Industries). The expression of iNOS and Arg1 was determined by immunoblot analysis using purified anti-mouse iNOS and Arg1 antibodies (Cell Signaling Technology). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) was used as a secondary antibody. The protein bands were visualized using an enhanced chemiluminescence kit (Thermo Scientific). The membranes were stripped and reprobed with an HRP-conjugated antibody to -actin (Sigma-Aldrich) to confirm equal sample loading. Adoptive transfer experiments. Gr-1⫹CD11b⫹ MDSCs were isolated from a single-cell suspension prepared from the BM of arthritic SKG mice. These cells (2 ⫻ 106/ mouse) were transferred to SKG mice through the tail vein on days 7, 17, and 27 after ZyA injection. Phosphate buffered saline (PBS) was injected as a control. Treatment of SKG mice with tofacitinib. Tofacitinib was dissolved in DMSO. Two weeks after ZyA injection, Alzet
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osmotic mini pumps (Durect) were implanted subcutaneously into the back of each mouse to deliver tofacitinib at a dosage of 15 mg/kg/day, or DMSO as a control. In vivo depletion of MDSCs. To deplete MDSCs in vivo, 0.2 mg of anti–Gr-1 mAb (RB6-8C5) or rat IgG2b isotype control (both from Bio X Cell) was intraperitoneally injected into tofacitinib-treated SKG mice every 3 days, from day 16 through day 41. Generation of adherent cells from BM progenitors. BM cells (1 ⫻ 106) from SKG mice were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin– streptomycin, 10 ng/ml GM-CSF, and 50 M 2-ME, with or without 1.0 M GLPG0634, 0.3 M G6, 0.3 M PF-956980, or 0.5 M tofacitinib. The cultures were maintained at 37°C in a 5% CO2–humidified atmosphere in 24-well plates. On day 3 of culture, floating cells were gently removed and the medium was replaced with fresh medium containing a cytokine and tofacitinib. Cells were collected on day 5 and analyzed by flow cytometry. Proliferation assay. Isolated CD4⫹ T cells were incubated with 10 M CFSE-DA, according to the manufacturer’s protocol. The cells were then suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin.
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CFSE-DA–labeled CD4⫹ T cells (1 ⫻ 105) in 200 l of medium were cultured in a 96-well flat-bottomed plate with BM cells from control or tofacitinib-treated mice, and stimulated with 10 g/ml anti-CD3 antibody and 5 g/ml anti-CD28 antibody for 2 days. CD4⫹ T cell proliferation was determined by measuring CFSE-DA fluorescence using flow cytometry. Statistical analysis. Results are expressed as the mean ⫾ SEM. Statistical comparisons were performed using Student’s t-test. P values less than 0.05 were considered statistically significant.
RESULTS Accumulation of MDSCs in both the BM and spleens of arthritic mice. To study the role of MDSCs in inflammatory arthritis, we first examined these cells in the BM and spleens of arthritic SKG mice. The clinical arthritis score in ZyA-treated SKG mice was a mean ⫾ SEM 5.3 ⫾ 0.1, whereas no arthritis was detected in the untreated control SKG mice. Both the BM and spleens of ZyA-treated mice contained an increased percentage of CD11b⫹Gr-1⫹ MDSCs (Figures 1A and B). How-
Figure 1. Myeloid-derived suppressor cells (MDSCs) accumulate in the bone marrow (BM) and spleens of arthritic SKG mice. A, BM cells from zymosan A (ZyA)–treated or untreated control SKG mice were stained with anti–Gr-1, anti-CD11b, anti–Ly-6C, and anti–Ly-6G antibodies. Representative flow cytometry plots show the distribution of MDSCs (Gr-1⫹CD11b⫹), polymorphonuclear MDSCs (PMN-MDSCs; CD11b⫹Ly6G⫹Ly-6Clow), and monocytic MDSCs (M-MDSCs; CD11b⫹Ly-6G⫺Ly-6Chigh) in each group. B, Frequencies of MDSCs, PMN-MDSCs, and M-MDSCs in the BM and spleens of control and ZyA-treated SKG mice were analyzed by flow cytometry. Symbols represent individual mice; bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍ 0.001. C, Expression of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in BM cells from ZyA-treated and control SKG mice was assessed by immunoblotting, with -actin serving as a positive control.
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ever, the percentage of PMN-MDSCs (CD11b⫹Ly6G⫹Ly-6Clow) was significantly increased only in the spleens of ZyA-treated mice (Figure 1B). The percentage of M-MDSCs (CD11b⫹Ly-6G⫺Ly-6Chigh) was significantly increased in the BM of ZyA-treated mice, although these cells represented only a small proportion of the total population of BM cells. Compared to untreated control SKG mice, the BM cells from ZyAtreated SKG mice also expressed higher levels of iNOS and Arg1 (Figure 1C). We next investigated the production of cytokines by MDSCs. The production of IL-10 and transforming growth factor  in BM cells from ZyA-treated SKG mice was not increased compared to that in untreated SKG mice (results not shown). These results suggest that arthritic SKG mice produce higher numbers of BM-MDSCs, characterized by increased production of iNOS and Arg1. Amelioration of arthritis following adoptive transfer of MDSCs. The adoptive transfer of MDSCs is reported to inhibit the development of CIA and antigen-
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induced arthritis (AIA) in murine models (20,24). To examine the effect of MDSCs on arthritis in SKG mice, we isolated MDSCs from the BM of these mice and adoptively transferred them into recipient mice with arthritis on days 7, 17, and 27 after ZyA injection. The purity of the isolated MDSCs was ⬎99% (see Supplementary Figure 1A, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/ 10.1002/art.39007/abstract). Isolated MDSCs consisted mainly of PMN-MDSCs (see Supplementary Figure 1B, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.39007/ abstract). The adoptive transfer of these MDSCs significantly decreased the severity of arthritis in SKG mice compared to that in the PBS-treated control group (Figure 2A). We next examined the histologic differences between the MDSC-treated and PBS-treated SKG mice. The mice in each group were killed 32 days after the induction of arthritis, and histologic features were com-
Figure 2. Adoptive transfer of MDSCs ameliorates arthritis in SKG mice. A, Clinical arthritis scores were determined in ZyA-injected SKG mice that were treated with isolated MDSCs (2 ⫻ 106) or phosphate buffered saline (PBS) (as control) on days 7, 17, and 27 after ZyA injection. B, The hind paws of MDSC- or PBS-treated SKG mice were assessed for histopathologic changes on day 32 after ZyA injection. Hematoxylin and eosin stained; original magnification ⫻ 40. C, Histologic arthritis scores were determined bilaterally in the hind paws of MDSC- and PBS-treated SKG mice. Results in A and C are the mean ⫾ SEM of 5 mice per group. ⴱ ⫽ P ⬍ 0.05 versus PBS controls. See Figure 1 for other definitions.
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pared. Adoptive transfer of MDSCs significantly reduced the severity of synovial hyperplasia and extent of bone erosion (Figure 2B), and the histologic scores of arthritis in the MDSC-treated mice were significantly lower than those in the PBS-treated mice (Figure 2C). These results, combined with previous findings, indicate that MDSC transfer ameliorates arthritis in various mouse models of RA, and suggest that MDSCs are normally produced during the course of the disease to control excessive inflammation. Increased accumulation of MDSCs and PMNMDSCs in the BM of tofacitinib-treated SKG mice. We next examined the effects of tofacitinib on arthritis in SKG mice. Similar to the findings in a previous study (25), continuous administration of tofacitinib significantly ameliorated the progression of arthritis, as measured by the clinical arthritis scores, in SKG mice (Figure 3A). To investigate the effect of tofacitinib on MDSCs, we examined their numbers in the BM of tofacitinib-treated SKG mice. Administration of tofacitinib significantly increased the percentage of MDSCs in the BM of both arthritic mice and nonarthritic mice
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(Figure 3B), indicating that tofacitinib alone can induce the production of MDSCs. Furthermore, the percentage of PMN-MDSCs in the BM was increased in tofacitinibtreated arthritic mice (Figure 3B). The percentage of M-MDSCs in the BM was not significantly different between tofacitinib-treated mice and DMSO-treated control mice, either with or without ZyA injection. We did not find significant differences in the frequency of MDSCs in the spleens between tofacitinib-treated mice and DMSO-treated control mice (results not shown). These results indicate that tofacitinib induces the expansion of MDSCs in the BM of SKG mice. Abrogation of the antiarthritic effect of tofacitinib following depletion of MDSCs by anti–Gr-1 mAb. To investigate the role of MDSCs in tofacitinib-treated SKG mice, we depleted Gr-1⫹ MDSCs in the BM of tofacitinib-treated arthritic mice by administering an anti–Gr-1 mAb every 3 days. First, to examine the effect of the anti–Gr-1 mAb, we administered the anti–Gr-1 mAb both to SKG mice injected with ZyA and to SKG mice that had not received ZyA injection. A single injection of anti–Gr-1 mAb significantly decreased the
Figure 3. Tofacitinib treatment leads to expansion of MDSCs in the BM and amelioration of arthritis in SKG mice. Tofacitinib (Tofa) (15 mg/kg/day) and/or DMSO was continuously administered to ZyA-treated or untreated SKG mice (ZyA ⫹ tofacitinib n ⫽ 8; ZyA ⫹ DMSO n ⫽ 10; tofacitinib alone n ⫽ 7; DMSO alone n ⫽ 8). A, Clinical arthritis scores were determined up to 42 days after ZyA injection. B, Frequencies of MDSCs, PMN-MDSCs, and M-MDSCs were determined in the BM of mice in each treatment group. Symbols in B represent individual mice; bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus ZyA ⫹ DMSO or as indicated. See Figure 1 for other definitions.
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Figure 4. Abrogation of the antiarthritic effect of tofacitinib (Tofa) by treatment with an anti–Gr-1 antibody to deplete MDSCs. A, A neutralizing monoclonal antibody (mAb) against Gr-1 or an isotype control was injected into tofacitinib- or DMSO-treated arthritic SKG mice, and clinical arthritis scores were determined up to 41 days after ZyA injection. B, The hind paws of SKG mice in each treatment group were evaluated for histopathologic changes on day 41 after ZyA injection. Hematoxylin and eosin stained; original magnification ⫻ 40. C, Histologic scores were determined in the left hind paws of SKG mice in each treatment group. Results in A and C are the mean ⫾ SEM (tofacitinib ⫹ anti–Gr-1 n ⫽ 5; tofacitinib ⫹ isotype n ⫽ 4; DMSO ⫹ isotype n ⫽ 3). ⴱ ⫽ P ⬍ 0.05 versus tofacitinib ⫹ isotype. See Figure 1 for other definitions.
numbers of BM-MDSCs in SKG mice (see Supplementary Figure 2A, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/ art.39007/abstract). The anti–Gr-1 mAb worsened the severity of arthritis in the tofacitinib-treated SKG mice. The clinical arthritis score in tofacitinib-treated mice treated with anti–Gr-1 mAb was comparable to that in the untreated control arthritic mice (Figure 4A). We next examined the histologic differences among these SKG mice. The administration of the anti–Gr-1 mAb also reduced the antiarthritic effect of tofacitinib, as shown by the results of histologic assessments (Figures 4B and C). These results suggest that tofacitinib ameliorates arthritis in SKG mice by increasing the number of Gr-1⫹ MDSCs. The percentage of MDSCs in mice injected with anti–Gr-1 mAb was comparable to that in control mice at the time of termination (day 41) (see Supplementary Figure 2B, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/
art.39007/abstract). The effect of the anti–Gr-1 mAb on MDSC depletion in vivo seemed to be transient, as has been described previously in another study (26). However, repeated infusion of the anti–Gr-1 mAb was enough to suppress the function of MDSCs, leading to worsening of arthritis. Facilitation of MDSC differentiation by tofacitinib in vitro. We next examined the effects of tofacitinib on the differentiation of MDSCs in vitro. BM cells were cultured with GM-CSF in the presence or absence of tofacitinib. When cultured with GM-CSF alone, most cells were Gr-1 negative. However, tofacitinib treatment increased the number of Gr-1–positive cells. In contrast, tofacitinib inhibited the development of CD11b⫹CD11c⫹ DCs (Figure 5A). We also examined the ability of BM cells from tofacitinib-treated mice to suppress T cell activation. CFSE-DA–labeled CD4⫹ T cells were cultured with mock-treated control BM cells or tofacitinib-treated BM cells, and then stimulated with anti-CD3 and anti-CD28
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Figure 5. Tofacitinib (Tofa) facilitates the in vitro differentiation of MDSCs. A, BM cells from SKG mice were cultured in the presence of granulocyte–macrophage colony-stimulating factor and treated with 0.5 M tofacitinib or DMSO for 5 days. The cells were then stained with anti–Gr-1, anti-CD11b, anti-CD11c, and anti-F4/80 antibodies and analyzed by flow cytometry. B, Carboxyfluorescein diacetate succinimidyl ester (CFSE-DA)–labeled naive CD4⫹ T cells were cultured alone or in a 1:1 ratio with vehicle (DMSO)–treated or tofacitinib-treated SKG mouse BM cells, and then stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After 2 days of culture, CD4⫹ T cell proliferation was examined by measuring CFSE-DA fluorescence with flow cytometry. See Figure 1 for other definitions.
mAb. In contrast to the mock-treated BM cells, tofacitinib-treated BM cells were incapable of enhancing T cell proliferation (Figure 5B). To further clarify the effect of the inhibition of JAK on the differentiation of BM cells to MDSCs, we then cultured BM cells with GM-CSF in the presence of selective JAK inhibitors or tofacitinib. The number of MDSCs was increased by the addition of GLPG0634, a selective JAK-1 inhibitor, or PF-956980, a selective JAK-3 inhibitor. In contrast, a selective JAK-2 inhibitor, G6, did not affect the number of MDSCs (Figure 6). Furthermore, the number of CDllb⫹CDllc⫹DCs was decreased by the addition of PF-956980. These results indicate that tofacitinib facilitates the differentiation of MDSCs through the inhibition of JAK-1 and/or JAK-3.
Figure 6. Inhibition of JAK-1 or JAK-3 facilitates the differentiation of MDSCs in vitro. BM cells from SKG mice were cultured in the presence of granulocyte–macrophage colony-stimulating factor with or without 1.0 M GLPG0634 (GLPG), 0.3 M G6, 0.3 M PF-956980 (PF), or 0.5 M tofacitinib (Tofa) for 5 days (DMSO served as vehicle control). The percentages of MDSCs and CD11b⫹CD11c⫹ dendritic cells were analyzed by flow cytometry. Results are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
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DISCUSSION MDSCs are a heterogeneous cell population with potent immunosuppressive activities. Their role is well established in cancer but not in inflammatory diseases. In this study, we have shown that MDSCs ameliorate disease activity in an animal model of RA, and that MDSCs play an important role in the tofacitinibmediated suppression of disease activity. We also have shown, for the first time, that tofacitinib facilitates the expansion of MDSCs in arthritis. Previous studies have shown that MDSCs are increased in the spleens of mice with CIA or AIA, and that transfer of MDSCs ameliorates arthritis in both models (20,24). In addition, depletion of MDSCs by an anti–Gr-1 mAb prevents mice with CIA from undergoing spontaneous recovery. In the present study, we have shown an important role for MDSCs in the SKG mouse model. SKG mice exhibit a more aggressive and longer lasting arthritis than that in mice with CIA. Our findings are especially relevant because the numbers of MDSCs are increased in the peripheral blood of patients with RA (27), a chronic arthritis that rarely spontaneously resolves. We also found that MDSCs primarily accumulated in the BM, and that adoptive transfer of mouse BM-MDSCs reduced the clinical arthritis scores in recipient SKG mice. The immunomodulatory functions of MDSCs are well established in cancer but not in autoimmune diseases. The suppression of CD8⫹ T cells in cancer is predominantly mediated by the enzymatic activities of Arg-1 and iNOS and by ROS. Arg1 and iNOS have been detected in the synovial fluid and spleens of mice with proteoglycan-induced arthritis, but not in the spleens of nonarthritic mice (28). MDSCs isolated from the spleens of mice with CIA inhibit CD4⫹ T cell proliferation through Arg1 activity in vitro (20). Our study also demonstrated the expression of Arg1 and iNOS in the BM of arthritic SKG mice, suggesting that both molecules may play a role in suppressing T cell responses. Notably, in experimental autoimmune encephalomyelitis, an increased expression of programmed death ligand 1 was shown to mediate the protective effects of MDSCs, whereas neither NO nor Arg1 was detected in the PMN-MDSCs (19). Thus, MDSCs may exhibit an array of protective mechanisms in autoimmune diseases, the details of which require further clarification. Various factors may induce the proliferation of MDSCs, including cyclooxygenase 2, prostaglandins (8,29,30), stem cell factor (29), M-CSF, IL-6 (31), GMCSF (30), vascular endothelial growth factor (30), and IL-10 (32). We examined the effect of tofacitinib on the
production of IL-10 and IL-6 from BM cells, but found that tofacitinib treatment did not up-regulate the production of IL-10 and IL-6 in BM cells (results not shown). It is still unknown whether tofacitinib influences other factors, such as those described above, in the pathways leading to MDSC expansion. Since so many factors seem to be involved in the expansion of MDSCs, the exact mechanism by which tofacitinib augments MDSC production requires further clarification. It has been reported that JAK-3⫺/⫺ mice show increased numbers of Fc␥ receptor (Fc␥R)/Mac-1 and Fc␥R/Gr-1 double-positive cells in the BM and spleen (33), compatible with the phenotype of MDSCs, prompting us to examine the effects of tofacitinib on MDSCs. A recent study showed that polyunsaturated fatty acids enhance the accumulation of MDSCs (34), and that JSI-124, a selective JAK-2/STAT-3 inhibitor, abrogates this effect. In that study, JAK-2/STAT-3 inhibition reduced the number of MDSCs, while in our study, JAK-1/JAK-3 inhibition enhanced the number of MDSCs. In addition, GM-CSF, an upstream activator of JAK-2, is reported to be critical for MDSC proliferation in cancer (35). Thus, we speculate that activation of JAK-2 and inhibition of JAK-1 and JAK-3 play critical roles in the expansion of MDSCs. We also found that the addition of tofacitinib to GM-CSF–treated BM cells led to increased numbers of MDSCs and reduced numbers of DCs in vitro. GM-CSF is critical for the generation of both DCs and MDSCs. The GM-CSF receptor interacts with two JAK-2 molecules but does not associate with JAK-1 or JAK-3. Thus, the expansion of MDSCs and inhibition of DCs by tofacitinib may involve the inhibition of JAK-1 and/or JAK-3 downstream of other cytokines produced by the BM cells themselves, rather than suppression of the GM-CSF signal. Kubo et al found that tofacitinib suppressed the expression of CD80/CD86 and the T cell stimulatory capability in lipopolysaccharide-stimulated human monocyte–derived DCs (36). They also showed that induction of CD80/CD86 expression was suppressed by a JAK-3 inhibitor, but not by a JAK-2 inhibitor. In comparison, we have shown that a JAK-3 inhibitor suppressed the differentiation of mouse BM cells into CD11b⫹CD11c⫹ DCs. These results indicate that JAK-3 inhibition suppresses the function and differentiation of DCs, and then reduces the T cell stimulatory capacity. A previous report indicated that CD11b⫺/low high Ly-6C cells, which are phenotypically similar to the CD11b⫹Ly-6G⫺Ly-6Chigh M-MDSC population, are osteoclast precursors (37). Thus, we examined the effect of tofacitinib on CD11b⫺/lowLy-6Chigh cells, but did
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not find a significant difference in the frequency of CD11b⫺/lowLy-6Chigh cells in the BM between tofacitiniband vehicle-treated SKG mice (results not shown). This result suggests that tofacitinib does not affect the number of osteoclast precursors in arthritic mice. In summary, MDSCs play crucial roles in the regulation of SKG mouse arthritis, and a JAK inhibitor, tofacitinib, enhances their expansion. This report describes a novel mode of antiarthritic action for tofacitinib, and the results suggest that JAKs play a critical role in the differentiation of MDSCs. ACKNOWLEDGMENT The authors thank Shino Tanaka for providing technical assistance.
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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. Dr. Morinobu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Nishimura, Saegusa, Kageyama, Morinobu. Acquisition of data. Nishimura, Matsuki, Akashi. Analysis and interpretation of data. Nishimura, Saegusa, Kageyama, Morinobu.
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Tofacitinib Facilitates the Expansion of Myeloid-Derived Suppressor Cells and Ameliorates Arthritis in SKG Mice Keisuke Nishimura, Jun Saegusa, Fumichika Matsuki, Kengo Akashi, Goichi Kageyama, and Akio Morinobu In vitro, tofacitinib facilitated the differentiation of BM cells to MDSCs, and inhibited their differentiation to dendritic cells. Conclusion. Tofacitinib facilitates the expansion of MDSCs and ameliorates arthritis in SKG mice.
Objective. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that have the ability to suppress T cell responses. The aim of this study was to evaluate the effects of the JAK inhibitor tofacitinib on MDSCs in a mouse model of rheumatoid arthritis. Methods. Arthritis was induced in SKG mice by zymosan A (ZyA) injection. MDSCs isolated from the bone marrow (BM) of donor SKG mice with arthritis were adoptively transferred to recipient mice with arthritis. In a separate experiment, tofacitinib was administered to arthritic SKG mice subcutaneously via osmotic pump, in some cases followed by injection of an anti–Gr-1 monoclonal antibody (mAb). BM cells from untreated mice were cultured for 5 days with granulocyte–macrophage colony-stimulating factor, with or without tofacitinib, and then analyzed by flow cytometry. Results. The numbers of MDSCs and polymorphonuclear MDSCs (PMN-MDSCs) were significantly increased in the spleens of SKG mice following ZyA injection. Adoptive transfer of MDSCs to recipient arthritic mice reduced the severity of arthritis compared to that in untreated control mice. Treatment with tofacitinib also ameliorated the progression of arthritis in SKG mice and induced significantly higher numbers of MDSCs and PMN-MDSCs in the BM of these animals. Furthermore, administration of an anti–Gr-1 mAb reduced the antiarthritic effect of tofacitinib in SKG mice.
Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage and bone destruction. Dendritic cells (DCs), monocytes, T cells, B cells, and neutrophils infiltrate the synovium and interact with each other and synovial fibroblasts to induce chronic synovitis (1,2). Cytokine production by various cells in the joint is central to the pathogenesis of RA. Antibodies targeting tumor necrosis factor ␣ and interleukin-6 (IL-6) are current therapeutic strategies for RA. The JAK kinases are tyrosine kinases that mediate the signaling pathways activated by various cytokines and growth factors. The JAK family consists of JAK-1, JAK-2, JAK-3, and tyrosine kinase 2, and different cytokines activate different sets of JAK family members. Tofacitinib is a small-molecule JAK inhibitor that inhibits JAK-1 and JAK-3, and suppresses inflammatory signaling downstream of ␥c-chain cytokines (IL-2, IL-4, IL-7, and IL-15), as well as IL-6 and interferon-␥ (3). Since multiple cytokines have been implicated in the pathogenesis of RA, tofacitinib currently represents a novel therapy for RA (4,5). However, the antirheumatic effects of tofacitinib, especially its influence on myeloid cell differentiation, are not fully understood. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are differentiated from immature myeloid precursor cells and characterized by the coexpression of Gr-1 and CD11b in mice. MDSCs have immunosuppressive properties; they suppress T cell responses by producing arginase 1 (Arg-1) (6–8), inducible nitric oxide synthase (iNOS) (9), and reactive oxygen species (ROS) (9,10). Phenotypically,
Supported in part by Grant-in-Aid for Scientific Research 24659473 from the Japan Society for Promotion of Science. Keisuke Nishimura, MD, Jun Saegusa, MD, PhD, Fumichika Matsuki, PhD, Kengo Akashi, MD, Goichi Kageyama, MD, PhD, Akio Morinobu, MD, PhD: Kobe University Graduate School of Medicine, Kobe, Japan. Address correspondence to Akio Morinobu, MD, PhD, Department of Rheumatology and Clinical Immunology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected]. Submitted for publication March 26, 2014; accepted in revised form December 19, 2014. 893
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MDSCs are divided into 2 subsets, polymorphonuclear MDSCs (PMN-MDSCs; defined as CD11b⫹Ly-6G⫹Ly6Clow cells), and monocytic MDSCs (M-MDSCs; defined as CD11b⫹Ly-6G⫺Ly-6Chigh cells) (11–13). PMNMDSCs express high levels of ROS and low levels of NO, whereas M-MDSCs express low levels of ROS and high levels of NO, and both subsets express Arg-1 (11). MDSCs are increased in tumor-bearing mice and in patients with cancer, and contribute to tumor survival by inhibiting tumor-specific T cell responses. Thus, MDSCs are potent immunosuppressive cells and potential target cells for cancer treatment. MDSC expansion is also associated with infection and autoimmunity (14– 20), and the spleens of mice with collagen-induced arthritis (CIA) have increased numbers of MDSCs (20). Immature myeloid precursor cells in the bone marrow (BM) are thought to differentiate into MDSCs in response to inflammatory stimuli, and then exit the BM and enter the circulatory system. However, the differentiation of MDSCs and their role in suppressing inflammation remain unclear. In the present study, we investigated the role of MDSCs and the effect of tofacitinib on MDSCs in the SKG mouse model of arthritis, a well-established model that exhibits many features of RA, including chronic destructive arthritis with a Th17 phenotype (21). We show that MDSCs possess antirheumatic properties, and that tofacitinib facilitates their expansion. Furthermore, the antiarthritic effect of tofacitinib was abrogated when MDSCs were depleted by the administration of an anti– Gr-1 monoclonal antibody (mAb), suggesting that the effect of tofacitinib is mediated, at least in part, by MDSCs. MATERIALS AND METHODS Animals. Female SKG mice and female BALB/c mice were obtained from CLEA Japan, Inc. Mice were housed in the Kobe University animal facility at a constant temperature, with provision of laboratory chow and water ad libitum. All procedures were carried out in accordance with the recommendations of the Institutional Animal Care Committee of Kobe University. Reagents and antibodies. Zymosan A (ZyA), DMSO, G6, PF-956980 hydrate, and 2-mercaptoethanol (2-ME) were purchased from Sigma-Aldrich. Tofacitinib (Selleck Chemicals), GLPG0634 (AdooQ Bioscience), a carboxyfluorescein diacetate succinimidyl ester (CFSE-DA) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS) (MP Biomedicals), 1% penicillin– streptomycin (Lonza Walkersville), and recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) (PeproTech) were also used. Fluorescein isothiocyanate (FITC)–conjugated anti–Gr-1, FITC-conjugated anti–Ly-6G, phycoerythrin-conjugated anti-CD11b, allophycocyanin (APC)-
conjugated anti–Ly-6C, and APC-conjugated anti-F4/80 were from BD PharMingen. Arthritis induction. Eight-week-old SKG mice were treated with 2 mg ZyA, as previously described (21). Briefly, ZyA suspended in saline was intraperitoneally injected on day 0. Arthritis developed between 14 and 21 days after injection. Evaluation of arthritis. The development and severity of arthritis were assessed using a previously described system for scoring clinical arthritis (22,23), as follows: 0 ⫽ no joint swelling, 0.1 ⫽ swelling of one digit joint, 0.5 ⫽ mild swelling of the wrist or ankle, and 1.0 ⫽ severe swelling of the wrist or ankle. Arthritis scores were totaled for each mouse. The maximum possible clinical arthritis score was 5.8. Histology. The hind paws of the mice were removed, fixed in 4% paraformaldehyde, decalcified in EDTA, embedded in paraffin, and sectioned. The samples were then stained with hematoxylin and eosin. Histologic evaluation was performed using a previously described scoring system (23), in which 0 ⫽ no inflammation, 1 ⫽ slight thickening of the synovial cell layer and/or some inflammatory cells in the sublining, 2 ⫽ thickening of the synovial lining, infiltration of the sublining, and localized cartilage erosions, and 3 ⫽ infiltration in the synovial space, pannus formation, cartilage destruction, and bone erosion. Isolation of MDSCs and CD4ⴙ T cells. MDSCs were isolated from a single-cell suspension prepared from the BM of arthritic SKG mice, using a biotinylated mAb against Gr-1, streptavidin-coated magnetic beads, and the AutoMACS system (all from Miltenyi Biotec), according to the manufacturer’s protocol. CD4⫹ T cells were purified from single-cell suspensions prepared from the spleens of normal mice, using negative selection with a biotinylated antibody mixture and antibiotin-coated magnetic beads (all from Miltenyi Biotec), following the manufacturer’s protocol. Flow cytometry. Cells were washed with Flow Cytometry Staining Buffer (eBioscience), and were stained with anti– Gr-1, anti–Ly-6G, anti–Ly-6C, and anti-CD11b mAb for 30 minutes at 4°C. The cells were acquired on a FACSCalibur (BD Bioscience) and analyzed using FlowJo software (Tree Star). Western blot analysis. BM cells from SKG mice treated with or without ZyA were lysed with radioimmunoprecipitation assay buffer (Thermo Scientific) containing a protease inhibitor cocktail (Roche Diagnostics) at 4°C for 30 minutes. Protein samples (30 g) were boiled for 5 minutes in sodium dodecyl sulfate sample buffer (Wako Pure Chemical Industries). The expression of iNOS and Arg1 was determined by immunoblot analysis using purified anti-mouse iNOS and Arg1 antibodies (Cell Signaling Technology). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) was used as a secondary antibody. The protein bands were visualized using an enhanced chemiluminescence kit (Thermo Scientific). The membranes were stripped and reprobed with an HRP-conjugated antibody to -actin (Sigma-Aldrich) to confirm equal sample loading. Adoptive transfer experiments. Gr-1⫹CD11b⫹ MDSCs were isolated from a single-cell suspension prepared from the BM of arthritic SKG mice. These cells (2 ⫻ 106/ mouse) were transferred to SKG mice through the tail vein on days 7, 17, and 27 after ZyA injection. Phosphate buffered saline (PBS) was injected as a control. Treatment of SKG mice with tofacitinib. Tofacitinib was dissolved in DMSO. Two weeks after ZyA injection, Alzet
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osmotic mini pumps (Durect) were implanted subcutaneously into the back of each mouse to deliver tofacitinib at a dosage of 15 mg/kg/day, or DMSO as a control. In vivo depletion of MDSCs. To deplete MDSCs in vivo, 0.2 mg of anti–Gr-1 mAb (RB6-8C5) or rat IgG2b isotype control (both from Bio X Cell) was intraperitoneally injected into tofacitinib-treated SKG mice every 3 days, from day 16 through day 41. Generation of adherent cells from BM progenitors. BM cells (1 ⫻ 106) from SKG mice were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin– streptomycin, 10 ng/ml GM-CSF, and 50 M 2-ME, with or without 1.0 M GLPG0634, 0.3 M G6, 0.3 M PF-956980, or 0.5 M tofacitinib. The cultures were maintained at 37°C in a 5% CO2–humidified atmosphere in 24-well plates. On day 3 of culture, floating cells were gently removed and the medium was replaced with fresh medium containing a cytokine and tofacitinib. Cells were collected on day 5 and analyzed by flow cytometry. Proliferation assay. Isolated CD4⫹ T cells were incubated with 10 M CFSE-DA, according to the manufacturer’s protocol. The cells were then suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin.
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CFSE-DA–labeled CD4⫹ T cells (1 ⫻ 105) in 200 l of medium were cultured in a 96-well flat-bottomed plate with BM cells from control or tofacitinib-treated mice, and stimulated with 10 g/ml anti-CD3 antibody and 5 g/ml anti-CD28 antibody for 2 days. CD4⫹ T cell proliferation was determined by measuring CFSE-DA fluorescence using flow cytometry. Statistical analysis. Results are expressed as the mean ⫾ SEM. Statistical comparisons were performed using Student’s t-test. P values less than 0.05 were considered statistically significant.
RESULTS Accumulation of MDSCs in both the BM and spleens of arthritic mice. To study the role of MDSCs in inflammatory arthritis, we first examined these cells in the BM and spleens of arthritic SKG mice. The clinical arthritis score in ZyA-treated SKG mice was a mean ⫾ SEM 5.3 ⫾ 0.1, whereas no arthritis was detected in the untreated control SKG mice. Both the BM and spleens of ZyA-treated mice contained an increased percentage of CD11b⫹Gr-1⫹ MDSCs (Figures 1A and B). How-
Figure 1. Myeloid-derived suppressor cells (MDSCs) accumulate in the bone marrow (BM) and spleens of arthritic SKG mice. A, BM cells from zymosan A (ZyA)–treated or untreated control SKG mice were stained with anti–Gr-1, anti-CD11b, anti–Ly-6C, and anti–Ly-6G antibodies. Representative flow cytometry plots show the distribution of MDSCs (Gr-1⫹CD11b⫹), polymorphonuclear MDSCs (PMN-MDSCs; CD11b⫹Ly6G⫹Ly-6Clow), and monocytic MDSCs (M-MDSCs; CD11b⫹Ly-6G⫺Ly-6Chigh) in each group. B, Frequencies of MDSCs, PMN-MDSCs, and M-MDSCs in the BM and spleens of control and ZyA-treated SKG mice were analyzed by flow cytometry. Symbols represent individual mice; bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍ 0.001. C, Expression of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in BM cells from ZyA-treated and control SKG mice was assessed by immunoblotting, with -actin serving as a positive control.
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ever, the percentage of PMN-MDSCs (CD11b⫹Ly6G⫹Ly-6Clow) was significantly increased only in the spleens of ZyA-treated mice (Figure 1B). The percentage of M-MDSCs (CD11b⫹Ly-6G⫺Ly-6Chigh) was significantly increased in the BM of ZyA-treated mice, although these cells represented only a small proportion of the total population of BM cells. Compared to untreated control SKG mice, the BM cells from ZyAtreated SKG mice also expressed higher levels of iNOS and Arg1 (Figure 1C). We next investigated the production of cytokines by MDSCs. The production of IL-10 and transforming growth factor  in BM cells from ZyA-treated SKG mice was not increased compared to that in untreated SKG mice (results not shown). These results suggest that arthritic SKG mice produce higher numbers of BM-MDSCs, characterized by increased production of iNOS and Arg1. Amelioration of arthritis following adoptive transfer of MDSCs. The adoptive transfer of MDSCs is reported to inhibit the development of CIA and antigen-
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induced arthritis (AIA) in murine models (20,24). To examine the effect of MDSCs on arthritis in SKG mice, we isolated MDSCs from the BM of these mice and adoptively transferred them into recipient mice with arthritis on days 7, 17, and 27 after ZyA injection. The purity of the isolated MDSCs was ⬎99% (see Supplementary Figure 1A, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/ 10.1002/art.39007/abstract). Isolated MDSCs consisted mainly of PMN-MDSCs (see Supplementary Figure 1B, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.39007/ abstract). The adoptive transfer of these MDSCs significantly decreased the severity of arthritis in SKG mice compared to that in the PBS-treated control group (Figure 2A). We next examined the histologic differences between the MDSC-treated and PBS-treated SKG mice. The mice in each group were killed 32 days after the induction of arthritis, and histologic features were com-
Figure 2. Adoptive transfer of MDSCs ameliorates arthritis in SKG mice. A, Clinical arthritis scores were determined in ZyA-injected SKG mice that were treated with isolated MDSCs (2 ⫻ 106) or phosphate buffered saline (PBS) (as control) on days 7, 17, and 27 after ZyA injection. B, The hind paws of MDSC- or PBS-treated SKG mice were assessed for histopathologic changes on day 32 after ZyA injection. Hematoxylin and eosin stained; original magnification ⫻ 40. C, Histologic arthritis scores were determined bilaterally in the hind paws of MDSC- and PBS-treated SKG mice. Results in A and C are the mean ⫾ SEM of 5 mice per group. ⴱ ⫽ P ⬍ 0.05 versus PBS controls. See Figure 1 for other definitions.
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pared. Adoptive transfer of MDSCs significantly reduced the severity of synovial hyperplasia and extent of bone erosion (Figure 2B), and the histologic scores of arthritis in the MDSC-treated mice were significantly lower than those in the PBS-treated mice (Figure 2C). These results, combined with previous findings, indicate that MDSC transfer ameliorates arthritis in various mouse models of RA, and suggest that MDSCs are normally produced during the course of the disease to control excessive inflammation. Increased accumulation of MDSCs and PMNMDSCs in the BM of tofacitinib-treated SKG mice. We next examined the effects of tofacitinib on arthritis in SKG mice. Similar to the findings in a previous study (25), continuous administration of tofacitinib significantly ameliorated the progression of arthritis, as measured by the clinical arthritis scores, in SKG mice (Figure 3A). To investigate the effect of tofacitinib on MDSCs, we examined their numbers in the BM of tofacitinib-treated SKG mice. Administration of tofacitinib significantly increased the percentage of MDSCs in the BM of both arthritic mice and nonarthritic mice
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(Figure 3B), indicating that tofacitinib alone can induce the production of MDSCs. Furthermore, the percentage of PMN-MDSCs in the BM was increased in tofacitinibtreated arthritic mice (Figure 3B). The percentage of M-MDSCs in the BM was not significantly different between tofacitinib-treated mice and DMSO-treated control mice, either with or without ZyA injection. We did not find significant differences in the frequency of MDSCs in the spleens between tofacitinib-treated mice and DMSO-treated control mice (results not shown). These results indicate that tofacitinib induces the expansion of MDSCs in the BM of SKG mice. Abrogation of the antiarthritic effect of tofacitinib following depletion of MDSCs by anti–Gr-1 mAb. To investigate the role of MDSCs in tofacitinib-treated SKG mice, we depleted Gr-1⫹ MDSCs in the BM of tofacitinib-treated arthritic mice by administering an anti–Gr-1 mAb every 3 days. First, to examine the effect of the anti–Gr-1 mAb, we administered the anti–Gr-1 mAb both to SKG mice injected with ZyA and to SKG mice that had not received ZyA injection. A single injection of anti–Gr-1 mAb significantly decreased the
Figure 3. Tofacitinib treatment leads to expansion of MDSCs in the BM and amelioration of arthritis in SKG mice. Tofacitinib (Tofa) (15 mg/kg/day) and/or DMSO was continuously administered to ZyA-treated or untreated SKG mice (ZyA ⫹ tofacitinib n ⫽ 8; ZyA ⫹ DMSO n ⫽ 10; tofacitinib alone n ⫽ 7; DMSO alone n ⫽ 8). A, Clinical arthritis scores were determined up to 42 days after ZyA injection. B, Frequencies of MDSCs, PMN-MDSCs, and M-MDSCs were determined in the BM of mice in each treatment group. Symbols in B represent individual mice; bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus ZyA ⫹ DMSO or as indicated. See Figure 1 for other definitions.
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Figure 4. Abrogation of the antiarthritic effect of tofacitinib (Tofa) by treatment with an anti–Gr-1 antibody to deplete MDSCs. A, A neutralizing monoclonal antibody (mAb) against Gr-1 or an isotype control was injected into tofacitinib- or DMSO-treated arthritic SKG mice, and clinical arthritis scores were determined up to 41 days after ZyA injection. B, The hind paws of SKG mice in each treatment group were evaluated for histopathologic changes on day 41 after ZyA injection. Hematoxylin and eosin stained; original magnification ⫻ 40. C, Histologic scores were determined in the left hind paws of SKG mice in each treatment group. Results in A and C are the mean ⫾ SEM (tofacitinib ⫹ anti–Gr-1 n ⫽ 5; tofacitinib ⫹ isotype n ⫽ 4; DMSO ⫹ isotype n ⫽ 3). ⴱ ⫽ P ⬍ 0.05 versus tofacitinib ⫹ isotype. See Figure 1 for other definitions.
numbers of BM-MDSCs in SKG mice (see Supplementary Figure 2A, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/ art.39007/abstract). The anti–Gr-1 mAb worsened the severity of arthritis in the tofacitinib-treated SKG mice. The clinical arthritis score in tofacitinib-treated mice treated with anti–Gr-1 mAb was comparable to that in the untreated control arthritic mice (Figure 4A). We next examined the histologic differences among these SKG mice. The administration of the anti–Gr-1 mAb also reduced the antiarthritic effect of tofacitinib, as shown by the results of histologic assessments (Figures 4B and C). These results suggest that tofacitinib ameliorates arthritis in SKG mice by increasing the number of Gr-1⫹ MDSCs. The percentage of MDSCs in mice injected with anti–Gr-1 mAb was comparable to that in control mice at the time of termination (day 41) (see Supplementary Figure 2B, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/
art.39007/abstract). The effect of the anti–Gr-1 mAb on MDSC depletion in vivo seemed to be transient, as has been described previously in another study (26). However, repeated infusion of the anti–Gr-1 mAb was enough to suppress the function of MDSCs, leading to worsening of arthritis. Facilitation of MDSC differentiation by tofacitinib in vitro. We next examined the effects of tofacitinib on the differentiation of MDSCs in vitro. BM cells were cultured with GM-CSF in the presence or absence of tofacitinib. When cultured with GM-CSF alone, most cells were Gr-1 negative. However, tofacitinib treatment increased the number of Gr-1–positive cells. In contrast, tofacitinib inhibited the development of CD11b⫹CD11c⫹ DCs (Figure 5A). We also examined the ability of BM cells from tofacitinib-treated mice to suppress T cell activation. CFSE-DA–labeled CD4⫹ T cells were cultured with mock-treated control BM cells or tofacitinib-treated BM cells, and then stimulated with anti-CD3 and anti-CD28
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Figure 5. Tofacitinib (Tofa) facilitates the in vitro differentiation of MDSCs. A, BM cells from SKG mice were cultured in the presence of granulocyte–macrophage colony-stimulating factor and treated with 0.5 M tofacitinib or DMSO for 5 days. The cells were then stained with anti–Gr-1, anti-CD11b, anti-CD11c, and anti-F4/80 antibodies and analyzed by flow cytometry. B, Carboxyfluorescein diacetate succinimidyl ester (CFSE-DA)–labeled naive CD4⫹ T cells were cultured alone or in a 1:1 ratio with vehicle (DMSO)–treated or tofacitinib-treated SKG mouse BM cells, and then stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After 2 days of culture, CD4⫹ T cell proliferation was examined by measuring CFSE-DA fluorescence with flow cytometry. See Figure 1 for other definitions.
mAb. In contrast to the mock-treated BM cells, tofacitinib-treated BM cells were incapable of enhancing T cell proliferation (Figure 5B). To further clarify the effect of the inhibition of JAK on the differentiation of BM cells to MDSCs, we then cultured BM cells with GM-CSF in the presence of selective JAK inhibitors or tofacitinib. The number of MDSCs was increased by the addition of GLPG0634, a selective JAK-1 inhibitor, or PF-956980, a selective JAK-3 inhibitor. In contrast, a selective JAK-2 inhibitor, G6, did not affect the number of MDSCs (Figure 6). Furthermore, the number of CDllb⫹CDllc⫹DCs was decreased by the addition of PF-956980. These results indicate that tofacitinib facilitates the differentiation of MDSCs through the inhibition of JAK-1 and/or JAK-3.
Figure 6. Inhibition of JAK-1 or JAK-3 facilitates the differentiation of MDSCs in vitro. BM cells from SKG mice were cultured in the presence of granulocyte–macrophage colony-stimulating factor with or without 1.0 M GLPG0634 (GLPG), 0.3 M G6, 0.3 M PF-956980 (PF), or 0.5 M tofacitinib (Tofa) for 5 days (DMSO served as vehicle control). The percentages of MDSCs and CD11b⫹CD11c⫹ dendritic cells were analyzed by flow cytometry. Results are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
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DISCUSSION MDSCs are a heterogeneous cell population with potent immunosuppressive activities. Their role is well established in cancer but not in inflammatory diseases. In this study, we have shown that MDSCs ameliorate disease activity in an animal model of RA, and that MDSCs play an important role in the tofacitinibmediated suppression of disease activity. We also have shown, for the first time, that tofacitinib facilitates the expansion of MDSCs in arthritis. Previous studies have shown that MDSCs are increased in the spleens of mice with CIA or AIA, and that transfer of MDSCs ameliorates arthritis in both models (20,24). In addition, depletion of MDSCs by an anti–Gr-1 mAb prevents mice with CIA from undergoing spontaneous recovery. In the present study, we have shown an important role for MDSCs in the SKG mouse model. SKG mice exhibit a more aggressive and longer lasting arthritis than that in mice with CIA. Our findings are especially relevant because the numbers of MDSCs are increased in the peripheral blood of patients with RA (27), a chronic arthritis that rarely spontaneously resolves. We also found that MDSCs primarily accumulated in the BM, and that adoptive transfer of mouse BM-MDSCs reduced the clinical arthritis scores in recipient SKG mice. The immunomodulatory functions of MDSCs are well established in cancer but not in autoimmune diseases. The suppression of CD8⫹ T cells in cancer is predominantly mediated by the enzymatic activities of Arg-1 and iNOS and by ROS. Arg1 and iNOS have been detected in the synovial fluid and spleens of mice with proteoglycan-induced arthritis, but not in the spleens of nonarthritic mice (28). MDSCs isolated from the spleens of mice with CIA inhibit CD4⫹ T cell proliferation through Arg1 activity in vitro (20). Our study also demonstrated the expression of Arg1 and iNOS in the BM of arthritic SKG mice, suggesting that both molecules may play a role in suppressing T cell responses. Notably, in experimental autoimmune encephalomyelitis, an increased expression of programmed death ligand 1 was shown to mediate the protective effects of MDSCs, whereas neither NO nor Arg1 was detected in the PMN-MDSCs (19). Thus, MDSCs may exhibit an array of protective mechanisms in autoimmune diseases, the details of which require further clarification. Various factors may induce the proliferation of MDSCs, including cyclooxygenase 2, prostaglandins (8,29,30), stem cell factor (29), M-CSF, IL-6 (31), GMCSF (30), vascular endothelial growth factor (30), and IL-10 (32). We examined the effect of tofacitinib on the
production of IL-10 and IL-6 from BM cells, but found that tofacitinib treatment did not up-regulate the production of IL-10 and IL-6 in BM cells (results not shown). It is still unknown whether tofacitinib influences other factors, such as those described above, in the pathways leading to MDSC expansion. Since so many factors seem to be involved in the expansion of MDSCs, the exact mechanism by which tofacitinib augments MDSC production requires further clarification. It has been reported that JAK-3⫺/⫺ mice show increased numbers of Fc␥ receptor (Fc␥R)/Mac-1 and Fc␥R/Gr-1 double-positive cells in the BM and spleen (33), compatible with the phenotype of MDSCs, prompting us to examine the effects of tofacitinib on MDSCs. A recent study showed that polyunsaturated fatty acids enhance the accumulation of MDSCs (34), and that JSI-124, a selective JAK-2/STAT-3 inhibitor, abrogates this effect. In that study, JAK-2/STAT-3 inhibition reduced the number of MDSCs, while in our study, JAK-1/JAK-3 inhibition enhanced the number of MDSCs. In addition, GM-CSF, an upstream activator of JAK-2, is reported to be critical for MDSC proliferation in cancer (35). Thus, we speculate that activation of JAK-2 and inhibition of JAK-1 and JAK-3 play critical roles in the expansion of MDSCs. We also found that the addition of tofacitinib to GM-CSF–treated BM cells led to increased numbers of MDSCs and reduced numbers of DCs in vitro. GM-CSF is critical for the generation of both DCs and MDSCs. The GM-CSF receptor interacts with two JAK-2 molecules but does not associate with JAK-1 or JAK-3. Thus, the expansion of MDSCs and inhibition of DCs by tofacitinib may involve the inhibition of JAK-1 and/or JAK-3 downstream of other cytokines produced by the BM cells themselves, rather than suppression of the GM-CSF signal. Kubo et al found that tofacitinib suppressed the expression of CD80/CD86 and the T cell stimulatory capability in lipopolysaccharide-stimulated human monocyte–derived DCs (36). They also showed that induction of CD80/CD86 expression was suppressed by a JAK-3 inhibitor, but not by a JAK-2 inhibitor. In comparison, we have shown that a JAK-3 inhibitor suppressed the differentiation of mouse BM cells into CD11b⫹CD11c⫹ DCs. These results indicate that JAK-3 inhibition suppresses the function and differentiation of DCs, and then reduces the T cell stimulatory capacity. A previous report indicated that CD11b⫺/low high Ly-6C cells, which are phenotypically similar to the CD11b⫹Ly-6G⫺Ly-6Chigh M-MDSC population, are osteoclast precursors (37). Thus, we examined the effect of tofacitinib on CD11b⫺/lowLy-6Chigh cells, but did
EFFECTS OF TOFACITINIB ON MDSCs AND MURINE ARTHRITIS
not find a significant difference in the frequency of CD11b⫺/lowLy-6Chigh cells in the BM between tofacitiniband vehicle-treated SKG mice (results not shown). This result suggests that tofacitinib does not affect the number of osteoclast precursors in arthritic mice. In summary, MDSCs play crucial roles in the regulation of SKG mouse arthritis, and a JAK inhibitor, tofacitinib, enhances their expansion. This report describes a novel mode of antiarthritic action for tofacitinib, and the results suggest that JAKs play a critical role in the differentiation of MDSCs. ACKNOWLEDGMENT The authors thank Shino Tanaka for providing technical assistance.
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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. Dr. Morinobu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Nishimura, Saegusa, Kageyama, Morinobu. Acquisition of data. Nishimura, Matsuki, Akashi. Analysis and interpretation of data. Nishimura, Saegusa, Kageyama, Morinobu.
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