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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To investigate the impact of STA-21, a promising STAT-3 inhibitor, on the development and progression of inflammatory arthritis and to determine the possible mechanisms by which STA-21 has antiarthritic effects in interleukin-1 receptor antagonist–knockout (IL-1Ra–KO) mice, an animal model of rheumatoid arthritis (RA).

Methods

IL-1Ra–KO mice were treated with intraperitoneal injections of STA-21 (0.5 mg/kg) or vehicle 3 times per week for 3 weeks. The mouse joints were assessed for clinical and histologic features of inflammatory arthritis. CD4+CD25+FoxP3+ Treg cells and CD4+IL-17+ cells were defined. Human peripheral blood mononuclear cell–derived monocytes or mouse bone marrow–derived monocyte/macrophage (BMM) cells were cultured in the presence of macrophage colony-stimulating factor alone or together with RANKL and various concentrations of STA-21, followed by staining of the cells for tartrate-resistant acid phosphatase activity to determine osteoclast formation.

Results

STA-21 suppressed inflammatory arthritis in IL-1Ra–KO mice. The proportion of Th17 cells was decreased and the proportion of Treg cells expressing FoxP3 was markedly increased in the spleens of STA-21–treated mice. Adoptive transfer of CD4+CD25+ T cells obtained from STA-21–treated IL-1Ra–KO mice markedly suppressed inflammatory arthritis. In vitro treatment with STA-21 induced the expression of FoxP3 and repressed IL-17 expression in both mouse and human CD4+ T cells. Moreover, STA-21 prevented both mouse BMM cells and human monocytes from differentiating into osteoclasts in vitro.

Conclusion

STA-21 improved the clinical course of arthritis in IL-1Ra–KO mice. It increased not only the number of Treg cells but also the function of the Treg cells. It also suppressed Th17 cells and osteoclast formation. These data suggest that STA-21 might be an effective treatment for patients with RA.

Rheumatoid arthritis (RA) is a multisystem autoimmune disease of unknown etiology that is characterized by a hyperplastic synovial membrane, also called pannus, capable of destroying adjacent articular cartilage and bone ([1, 2]). Among the various pathologic events occurring in the affected joints, bone destruction is of utmost importance clinically because it is related to functional impairment and the progression of joint damage among patients with RA whose disease is in prolonged remission ([3]). Osteoclasts, which are specialized bone-resorbing cells regulated by RANKL and macrophage colony-stimulating factor (M-CSF), are mainly implicated in the development of bony erosion in RA ([4, 5]).

Among the various immune cells, CD4+ T cells are critically implicated in the pathogenesis of RA. CD4+ T cells can differentiate into various subsets. It has been observed that CD4+ T cells differentiate into 2 T helper cell subsets with reciprocal functions and patterns of cytokine secretion, termed Th1 and Th2 ([6]). Previously, RA was considered to be a purely Th1-mediated disease. This classic paradigm was maintained until 2005, when a distinct lineage of proinflammatory T helper cells, termed Th17 cells, was identified ([7, 8]). Th17 cells are characterized by the production of a proinflammatory cytokine, interleukin-17 (IL-17; also known as IL-17A). Various cytokines, such as IL-6, transforming growth factor β (TGFβ), IL-23, and IL-1β, contribute to the differentiation and/or amplification of Th17 cells ([9, 10]). Currently, it is certain that Th17 cells and IL-17 significantly contribute to the development of RA ([10, 11]).

STAT-3 is a transcription factor encoded by the STAT-3 gene ([12]). The STAT-3 protein exists in a latent form in the cytoplasm. STAT-3 becomes phosphorylated on tyrosine residues upon receptor activation by cytokines, such as IL-6, and forms homo- or heterodimers that translocate to the cell nucleus, where they act as transcription activators. STAT-3 is the major transcription factor in the differentiation of Th17 cells ([13]), and it was reported that STAT-3 is activated in inflamed synovium, as demonstrated in an animal model of RA ([14, 15]). There have been several previous reports indicating that STAT-3 plays essential roles in inflammatory arthritis ([16-19]).

STA-21 is a small molecule with potent STAT-3–inhibiting activity. It impedes STAT-3 DNA binding activity, STAT-3 dimerization, and STAT-3–dependent luciferase activity. It also inhibits breast cancer cells that express constitutive active STAT-3 ([20]). However, the effect of STA-21 in RA has not yet been determined.

Therefore, we hypothesized that STA-21, a potent STAT-3 inhibitor, would suppress arthritis in an animal model of RA, utilizing IL-1 receptor antagonist–knockout (IL-1Ra–KO) mice. To clarify the mechanisms by which STA-21 exerts a therapeutic effect, we demonstrated that STA-21 decreased the proportion of Th17 cells (a proinflammatory T helper cell subset that is deeply implicated in the development of RA [11]) and increased the proportion of FoxP3-expressing Treg cells (which suppress autoimmune processes and maintain peripheral tolerance [21]), both in vitro and in vivo. We also performed adoptive transfer of CD4+CD25+ T cells, which had been induced by STA-21, into recipient IL-1Ra–KO mice, to verify that Treg cells induced by STA-21 also work in vivo. In addition, we investigated the impact of STA-21 on osteoclasts, which are critical cells that have been deeply implicated in bone destruction in RA, in vitro and in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Animals

Male C57BL/6 and DBA/1J mice, all ages 4–6 weeks, were purchased from Orient Bio. IL-1Ra–KO mice were kindly obtained from Prof. Yoichiro Iwakura (University of Tokyo, Japan). The mice were maintained as described previously ([22]). All experimental procedures were examined and approved by the Animal Research Ethics Committee of the Catholic University of Korea, which conforms to the guidelines of the National Institutes of Health.

Induction of type II collagen–induced arthritis (CIA) and treatment with STA-21.

Induction of CIA was conducted as described previously ([23]). Seven days after the first immunization, mice were given 9 consecutive intraperitoneal (IP) injections of 0.5 mg/kg STA-21 (Santa Cruz Biotechnology) in saline or saline alone (as vehicle control), 3 times per week for 3 weeks. The score of arthritis severity in the joints of these mice was determined twice weekly; the arthritis score was expressed as the sum of the scores for 4 limbs. To assess the influence of STA-21 on symptom severity in the IL-1Ra–KO mouse model, 13–16-week-old male IL-1Ra–KO mice were treated with 0.5 mg/kg STA-21 in saline or with vehicle alone, via IP injections 3 times per week for 3 weeks. On day 40 after the first STA-21 injection, CD4+CD25+ T cells were sorted, using a MoFlo flow cytometer, from the spleens of STA-21–treated mice or vehicle-treated mice. Thereafter, 5 × 105 cells were adoptively transferred into IL-1Ra–KO mice.

Histology

Mouse joint tissue was fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier, and embedded in paraffin. Tissue sections (7 μm) were prepared and stained with hematoxylin and eosin, Safranin O, and toluidine blue, to detect proteoglycans. Confocal microscopy was used to detect immunostaining for Th17 and Treg cells, as previously described ([22]).

Differentiation of effector T cells

Isolation of mouse and human CD4+ T cells and differentiation of effector T cells was performed as described previously ([22]). Cells were pretreated with STA-21 for 2 hours, and then stimulated under polarizing conditions.

Evaluation of in vitro osteoclastogenesis in mouse cells

Isolation of mouse bone marrow–derived monocyte/macrophage (BMM) cells, differentiation of osteoclast precursor cells (preosteoclasts), tartrate-resistant acid phosphatase (TRAP) staining, and bone resorption analysis were performed as described previously ([24]).

Transfection of STAT-3 small interfering RNA (siRNA).

RAW 264.7 cells were transfected with 100 nM STAT-3 siRNA or control siRNA (both from Dharmacon) using FuGene HD (Roche), in accordance with the manufacturer's instructions. One day later, these cells were stimulated with RANKL (50 ng/ml). After 3 days, the cells were harvested and the expression of TRAP was evaluated by real-time polymerase chain reaction (PCR).

Evaluation of in vitro osteoclastogenesis in human cells

The generation of human osteoclasts, TRAP staining, and bone resorption analysis were performed as described previously ([24]). All subjects gave their informed consent to participate before the study was initiated. The study received the approval of the Institutional Review Board of Seoul St. Mary's Hospital, South Korea.

Gene expression analysis using real-time PCR

A LightCycler 2.0 instrument (software version 4.0; Roche Diagnostics) was used for PCR amplification. All reactions were performed with LightCycler FastStart DNA Master SYBR Green I (Takara) according to the manufacturer's instructions (a list of the primers used is available from the corresponding author upon request). The messenger RNA (mRNA) expression levels were normalized to those of β-actin mRNA.

Enzyme-linked immunosorbent assay (ELISA).

The amounts of IL-17 in culture supernatants derived from mouse or human sera were measured by sandwich ELISA (R&D Systems). Horseradish peroxidase–avidin (R&D Systems) was used for color development. Absorbance was measured at 405 nm on an ELISA microplate reader (Molecular Devices).

Measurement of IgG2a

Blood was obtained from the orbital sinus of mice, and the serum was stored at −20°C until used. Anti–type II collagen IgG2a antibodies were measured using the mouse IgG2a ELISA quantitation kit (Bethyl Laboratories).

Flow cytometry

Expression of cytokines and transcription factors was assessed by intracellular staining with the following antibodies: in mice, fluorescein isothiocyanate (FITC)–conjugated anti–IL-17, FITC-conjugated anti-FoxP3, phycoerythrin (PE)–conjugated anti-FoxP3, FITC-conjugated anti–inducible costimulator (anti-ICOS), FITC-conjugated anti–programmed death protein 1 (anti–PD-1), and FITC-conjugated anti–intercellular adhesion molecule 1 (anti–ICAM-1) (all from eBioscience); in humans, PerCP-conjugated anti-CD4, anti-CD25, FITC-conjugated anti-FoxP3, and PE-conjugated anti–IL-17 (all from eBioscience). Cells were stimulated for 4 hours with phorbol myristate acetate (PMA) and ionomycin, with the addition of GolgiStop (BD Biosciences). The cultured cells were surface labeled for 30 minutes and then permeabilized with Cytofix/Cytoperm solution (BD PharMingen). Thereafter, the cells were intracellularly stained with fluorescent antibodies before being analyzed on a FACSCalibur flow cytometer. For flow cytometry analysis of phospho–STAT-3 (using PE-conjugated anti-Tyr705) and phospho–STAT-5 (using PE-conjugated anti-Tyr694), murine CD4+ T cells were cultured with IL-6 for 30 minutes and the level of STAT-3 phosphorylation was measured according to the manufacturer's instructions. Events were collected and analyzed with FlowJo software (Tree Star).

Isolation of cells from the mouse paw joints

Paws from the mice were digested in 0.125% Dispase II (Roche), 0.2% type II collagenase, and 0.2% type IV collagenase (Sigma-Aldrich) in Hanks' balanced salt solution (Gibco) for 75 minutes at 37°C, and then passed through a cell strainer. Cells were stimulated with 25 ng/ml PMA and 250 ng/ml ionomycin for 4 hours in the presence of GolgiStop. Intracellular staining of Th17 cells was performed as described above.

Statistical analysis

Statistical analyses were performed using SAS software (version 9.2; SAS Institute). All experimental values are presented as the mean ± SD. Comparisons of numerical data between 2 groups were performed using Student's t-tests or the Mann-Whitney U test. Differences in the mean values between the various groups were analyzed using analysis of variance with a post hoc test. P values less than 0.05 (2-tailed) were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Suppression of inflammatory arthritis in STA-21–treated mice

We investigated whether treatment with STA-21 would suppress the rheumatoid inflammation and joint destruction in IL-1Ra–KO mice. The results showed that IP injections of STA-21 (0.5 mg/kg) reduced the arthritis score and the incidence of arthritis compared to that in mice receiving IP injections of vehicle (Figure 1A). Histologic examination of the joints revealed that the paws and ankles of STA-21–treated mice exhibited a lower degree of inflammation and attenuated cartilage damage, as determined on day 40 after the first STA-21 injection, when compared to the paws and ankles of vehicle-treated mice (Figure 1B). In addition, the serum levels of IgG2a were significantly lower in mice treated with STA-21 (Figure 1C).

image

Figure 1. Treatment with STA-21 suppresses inflammatory arthritis in mice. A, Arthritis score and incidence of arthritis in interleukin-1 receptor antagonist–knockout (IL-1Ra–KO) mice following treatment with STA-21 or vehicle. B, Histologic examination of joint tissue sections from IL-1Ra–KO mice in each group, using staining with hematoxylin and eosin (H&E), Safranin O, or toluidine blue. C, Levels of circulating IgG2a in the serum of IL-1Ra–KO mice in each group. D, Expression of IL-17 and FoxP3 mRNA, as determined by real-time polymerase chain reaction, in splenocytes from IL-1Ra–KO mice in each group. E, Intracellular flow cytometry analysis of the expression of IL-17 and FoxP3 in splenocytes from IL-1Ra–KO mice in each group. Cells were cultured with phorbol myristate acetate (25 ng/ml) and ionomycin (250 ng/ml) for 4 hours, and then stained with antibodies against IL-17 and FoxP3. Values are the percentage of positive cells. F, Arthritis score in mice with type II collagen–induced arthritis (CIA) following treatment with STA-21 or vehicle. G, Histologic examination of the joints from mice with CIA in each group. H, Flow cytometry analysis of the synovial CD4+IL-17+ T cell subset in mice with CIA in each group. Values are the percentage of positive cells. Results in A (left), C, D, and F are the mean ± SD of 3 mice per group. Results in A (right) are the percentage with 95% confidence intervals. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus vehicle.

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We also evaluated the expression of IL-17, which is the main cytokine that characterizes Th17 cells ([7]), and the expression of FoxP3, the master regulator in the development and function of Treg cells ([25]), in the splenocytes of IL-1Ra–KO mice, using real-time PCR and flow cytometry. As shown in Figures 1D and E, the mice treated with STA-21 showed a decreased expression of IL-17 and an increased expression of FoxP3.

In addition, we investigated whether treatment with STA-21 would suppress the development and progression of inflammatory arthritis in mice with CIA. As shown in Figure 1F, IP injections of STA-21 in mice with CIA reduced the arthritis score compared to that in mice with CIA receiving IP injections of vehicle. Histologic examination of the joints indicated that the paws and ankles of STA-21–treated mice exhibited a lower degree of inflammation and attenuated cartilage damage as compared to the paws and ankles of vehicle-treated mice (Figure 1G). On day 62 after the first immunization, mixed joint cells were separated. We then examined the synovial subset of CD4+IL-17+ T cells (Th17 cells) using flow cytometry. The results showed that mice with CIA treated with STA-21, as compared to mice treated with vehicle, had profoundly decreased numbers of synovial Th17 cells (Figure 1H).

Decrease in Th17 cells and increase in FoxP3-expressing Treg cell populations following treatment with STA-21 in IL-1Ra–KO mice

We next investigated whether STA-21 would affect the population of new T helper cell subsets, that is, Treg cells and Th17 cells. To achieve this, we examined the numbers of CD4+IL-17+ T cells (Th17 cells) and CD4+CD25+FoxP3+ Treg cells in the mouse spleens, by performing confocal staining of the spleen tissue. The results indicated that the spleen tissue samples from mice treated with STA-21 showed decreased numbers of Th17 cells and increased numbers of FoxP3+ Treg cells, when compared to the spleen tissue samples from mice treated with vehicle (Figure 2A).

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Figure 2. Treatment with STA-21 decreases the number of Th17 cells and increases the number of FoxP3+ Treg cells in interleukin-1 (IL-1) receptor antagonist–knockout mice. Samples of spleen tissue from mice treated with STA-21 or vehicle were stained for A, CD4+IL-17+ T cells and CD4+CD25+FoxP3+ T cells using antibodies specific for CD4, IL-17, CD25, and FoxP3, B, CD4+STAT-3+IL-17+ T cells and CD4+pSTAT-3(Y705)+IL-17+ T cells using antibodies specific for CD4, STAT-3, pSTAT-3 (Y705), and IL-17, and C, CD4+STAT-5+ T cells and CD4+pSTAT-5+ T cells using antibodies specific for CD4, STAT-5, and pSTAT-5. In the left panels, confocal microscopy was used to visually enumerate, at higher magnification, each of the cell subsets (performed by 4 investigators). In the right panels, the distribution of the cell subsets in each group is presented. Results are the mean ± SD of 3 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01.

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STAT-3 is the major transcription factor involved in the differentiation of Th17 cells ([13]), whereas STAT-5 is required for the development of FoxP3+ Treg cells ([26]). Therefore, we determined the number of CD4+STAT-3+IL-17+ T cells and CD4+STAT-5+ T cells in the mouse spleen tissue. As expected, in comparison with the vehicle control group, the spleen tissue from mice treated with STA-21 showed decreased numbers of CD4+STAT-3+IL-17+ T cells and CD4+pSTAT-3(Y705)+IL-17+ T cells (Figure 2B) and increased numbers of CD4+STAT-5+ T cells and CD4+pSTAT-5+ T cells (Figure 2C). In addition, we found that STAT-5 colocalized with FoxP3 in the mouse CD4+ T cells (results not shown).

Repression of IL-17 and induction of FoxP3 in CD4+ T cells, under conditions of Th17 cell differentiation, following treatment with STA-21 in mice

We examined the effect of STA-21 on Th17 cell differentiation in vitro. CD4+ T cells from mice were cultured in the presence of anti-CD3, TGFβ, and IL-6, with or without STA-21, for 72 hours. After stimulation under conditions favoring the development of Th17 cells, we found that in vitro treatment with STA-21 substantially decreased the proportion of IL-17–expressing CD4+ T cells and increased the proportion of CD4+ T cells expressing FoxP3, as determined by flow cytometry (Figure 3A). There was no evidence of increased cell death in the STA-21–treated group (results not shown).

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Figure 3. STA-21 represses the expression of interleukin-17 (IL-17) and induces the expression of FoxP3 in mouse CD4+ T cells. A–D, CD4+ T cells from the spleens of 6-week-old DBA/1J mice were cultured under Th17 cell–inducing conditions with or without various concentrations of STA-21. A, After 72 hours of culture, the cells were stained with antibodies against CD4, CD25, IL-17, and FoxP3 for intracellular flow cytometry analysis. Values are the percentage of positive cells. Representative results are shown. B, Expression of IL-17 and FoxP3 mRNA in the cells was determined by real-time polymerase chain reaction (PCR). C, The concentration of IL-17 in culture supernatants of mouse serum was measured by enzyme-linked immunosorbent assay. D, The mRNA expression of molecules that are associated with Th17 cell differentiation, including retinoic acid receptor–related orphan nuclear receptor γt, aryl hydrocarbon receptor, interferon regulatory factor 4, and CCL20, was determined by real-time PCR. E, CD4+ T cells isolated from the spleens of 6-week-old DBA/1J mice were cultured under Th17 cell–inducing conditions with or without retinoic acid or various concentrations of STA-21. After 72 hours of culture, the expression of FoxP3 and the Treg cell–associated molecules inducible costimulator (ICOS), programmed death protein 1 (PD-1), and intercellular adhesion molecule 1 (ICAM-1) was analyzed by flow cytometry. Values are the percentage of positive cells. Representative results are shown. Results are the mean ± SD of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus the other treatment conditions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38305/abstract.

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We also checked the mRNA levels of IL-17 and FoxP3. As shown in Figure 3B, in vitro treatment with STA-21 decreased the expression of IL-17 and increased the expression of FoxP3 in mouse CD4+ T cells. STA-21 also decreased the levels of IL-17 in the culture supernatants of mouse serum (Figure 3C).

In addition, treatment with STA-21 decreased the mRNA levels of retinoic acid receptor–related orphan nuclear receptor γt (RORγt), aryl hydrocarbon receptor, interferon regulatory factor 4, and CCL20 (Figure 3D), all of which are factors implicated in Th17 cell differentiation. Flow cytometry analysis showed that in vitro treatment with STA-21 not only up-regulated FoxP3, but also up-regulated the levels of ICOS, PD-1, and ICAM-1 (Figure 3E), all of which are implicated in the differentiation of Treg cells, to a level similar to that of retinoic acid, which is known to induce Treg cells ([27]).

Marked attenuation of inflammatory arthritis following adoptive transfer of CD4+CD25+ T cells obtained from STA-21–treated IL-1Ra–KO mice

In an attempt to clarify whether Treg cells induced by STA-21 really work in vivo, we transferred CD4+CD25+ T cells (5 × 105 cells) isolated from the spleens of STA-21–treated or vehicle-treated IL-1Ra–KO mice into IL-1Ra–KO recipient mice. As shown in Figure 4A, adoptive transfer of CD4+CD25+ T cells from STA-21–treated IL-1Ra–KO mice markedly decreased the arthritis severity score in IL-1Ra–KO recipient mice compared to that in vehicle-treated IL-1Ra–KO mice. These findings verify that Treg cells induced by STA-21 have immunoregulatory functions in vivo.

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Figure 4. Adoptive transfer of CD4+CD25+ T cells isolated from STA-21–treated interleukin-1 receptor antagonist–knockout (IL-1Ra–KO) mice suppresses the development of inflammatory arthritis. CD4+CD25+ T cells (5 × 105 cells) isolated on day 40 postimmunization from the spleens of STA-21– or vehicle-treated IL-1Ra–KO mice were adoptively transferred into IL-1Ra–KO recipient mice. A, The arthritis severity score was evaluated in each group for 10 weeks after adoptive transfer. B, Levels of IgG2a were determined in serum samples from each group by enzyme-linked immunosorbent assay (ELISA) on day 70 after adoptive transfer. C, Splenocytes from mice in each group were cultured in medium alone for 3 days, and the concentrations of IL-17 and IL-10 in the culture supernatants were determined by ELISA. D, Joint tissue sections from the mice in each group were assessed for histologic features, using staining with hematoxylin and eosin (H&E), Safranin O, and toluidine blue (representative results shown) (left), and histologic scores of inflammation and cartilage damage were determined (right). E, Samples of spleen tissue from the mice in each group were stained for CD4+IL-17+ T cells and CD4+CD25+FoxP3+ T cells, using antibodies specific for CD4, IL-17, CD25, and FoxP3. Both cell populations were analyzed using laser confocal microscopy (left), and the distribution of the 2 cell populations in each group is shown (right). Results are the mean ± SD of 5 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus vehicle.

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We next examined the effect of the transferred CD4+CD25+ T cells on the antigen-specific humoral immune response. We measured IgG2a levels in the sera of recipient mice. The results showed that the serum levels of IgG2a were significantly lower in recipient mice that had received CD4+CD25+ T cells from STA-21–treated IL-1Ra–KO mice (Figure 4B).

Furthermore, spontaneous production of IL-17 and IL-10 by cultured splenocytes from the mice was measured by ELISA. As shown in Figure 4C, levels of IL-17 were lower and levels of IL-10, the prototype antiinflammatory cytokine produced by Treg cells, were higher in the recipient mice that received CD4+CD25+ T cells from STA-21–treated IL-1Ra–KO mice. Histologic examination showed that the paws and ankles from the recipient mice that received CD4+CD25+ T cells from STA-21–treated IL-1Ra–KO mice exhibited a lower degree of inflammation and attenuated cartilage damage as compared to the paws and ankles from vehicle-treated IL-1Ra–KO mice (Figure 4D).

We also examined the numbers of CD4+IL-17+ T cells (Th17 cells) and CD4+CD25+FoxP3+ Treg cells in the spleens of the mice. Results of confocal staining of the spleen tissue showed that the recipient mice that received CD4+CD25+ T cells from STA-21–treated IL-1Ra–KO mice showed decreased numbers of Th17 cells and increased numbers of FoxP3+ Treg cells (Figure 4E).

Inhibition of osteoclastogenesis by STA-21 in mice

Osteoclasts are primarily involved in the bone destruction of RA. RANKL is the key osteoclastogenic molecule expressed by osteoclastogenesis-supporting cells such as activated CD4+ T cells, osteoblasts, and synoviocytes ([28, 29]), and it binds to its single receptor, RANK, and thereby mediates osteoclastogenesis that leads to bone destruction. To examine the effect of STA-21 on osteoclast formation, we performed TRAP staining on tissue sections from the joints of IL-1Ra–KO mice treated with STA-21 or vehicle. As shown in Figure 5A, the joint tissue of STA-21–treated mice showed a markedly reduced formation of osteoclasts compared to the joint tissue of vehicle-treated mice.

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Figure 5. STA-21 inhibits osteoclastogenesis in mice. A, Synovial tissue sections were obtained from the joints of interleukin-1 receptor antagonist–knockout mice that were left untreated (wild-type [WT]) or treated with STA-21 or vehicle. Tissue samples were stained with tartrate-resistant acid phosphatase (TRAP) antibodies (brown staining) (left), and the numbers of TRAP+ cells (osteoclasts) per ankle joint were determined (right). B, Osteoclast precursor cells derived from bone marrow–derived monocyte/macrophage (BMM) cells of normal mice were further cultured in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml) alone or together with RANKL (50 ng/ml) and various concentrations of STA-21. After 4 days, the cells were stained for TRAP (representative images shown), and the bone resorption activity of the BMM cells was examined by placing the cells on dentin slices and culturing as described above; after 21 days, the slices were stained with hematoxylin. C and D, BMM cells were cultured with M-CSF (M) and RANKL (R) with or without various concentrations of STA-21. C, The number of multinucleated TRAP+ cells and percentage of pit area were determined. D, The mRNA expression of various osteoclastogenic markers was analyzed using real-time polymerase chain reaction (PCR). E, RAW 264.7 cells were transfected with STAT-3 small interfering RNA (siRNA) or control siRNA, and then cultured with RANKL (50 ng/ml) for 72 hours. Levels of STAT-3 and TRAP mRNA were determined using real-time PCR. Results are the mean ± SD of 3 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus the other treatment conditions.

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Our next experiment was to examine whether STA-21 inhibits osteoclast formation in vitro. To induce osteoclastogenesis, BMM cells were prepared from normal mice and then stimulated with M-CSF alone or together with RANKL. The addition of STA-21 to the cell cultures markedly inhibited osteoclastogenesis. These findings were evaluated by counting the number of TRAP-positive cells per well (Figures 5B and C). Bone resorption analysis showed similar results (Figures 5B and C). In addition, the mRNA levels of various osteoclastogenic markers, such as β3 integrin, cathepsin K, and matrix metalloproteinase 9 (MMP-9), were also remarkably decreased by the addition of STA-21 (Figure 5D).

RAW 264.7 cells were transfected with STAT-3 siRNA or control siRNA, and then cultured in the presence of RANKL (50 ng/ml) for 72 hours. Messenger RNA levels of TRAP, a marker of osteoclastogenesis, were determined by real-time PCR. As shown in Figure 5E, knockdown of STAT-3 in RAW 264.7 cells significantly decreased the mRNA levels of both TRAP and STAT-3.

Inhibition of Th17 cell differentiation and promotion of FoxP3 expression by STA-21 in human CD4+ T cells

To determine the effect of STA-21 on the differentiation of human Th17 cells in vitro, CD4+ T cells obtained from healthy human volunteers were cultured in the presence of anti-CD3, IL-1β, and IL-6, with or without STA-21, for 72 hours. After stimulation under conditions favoring the development of Th17 cells, we found that in vitro treatment with STA-21 substantially decreased the proportion of IL-17–expressing CD4+ T cells and increased the proportion of CD4+ T cells expressing FoxP3, as determined by flow cytometry (Figure 6A). There was no evidence of increased cell death in the STA-21–treated group (results not shown).

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Figure 6. STA-21 inhibits both Th17 cell differentiation and osteoclastogenesis in humans. CD4+ T cells isolated from healthy human volunteers were cultured under Th17 cell–inducing conditions in the presence or absence of various concentrations of STA-21. A, After 72 hours in culture, the cells were stained with antibodies against CD4, CD25, interleukin-17 (IL-17), and FoxP3 for intracellular flow cytometry. Values are the percentage of positive cells. Representative results are shown. B, Expression of IL-17 mRNA in the cells was determined by real-time polymerase chain reaction (PCR) (left), and the concentration of IL-17 in culture supernatants was measured by enzyme-linked immunosorbent assay (right). C and D, Human monocytes obtained from healthy volunteers were cultured in the presence of macrophage colony-stimulating factor (M-CSF) (M) (25 ng/ml) alone or together with RANKL (R) (30 ng/ml) and various concentrations of STA-21. C, After 9 days, the cells were stained for tartrate-resistant acid phosphatase (TRAP), and the bone resorption activity of the cells was examined by placing the cells on dentin slices and culturing as described above; after 21 days, the slices were stained with hematoxylin (top). In addition, the number of multinucleated TRAP+ cells and percentage of pit area were determined (bottom). D, Levels of matrix metalloproteinase 9, RANK, and cathepsin K mRNA were quantified by real-time PCR. Results are the mean ± SD of 3 individuals per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus the other treatment conditions.

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We also checked the mRNA levels of IL-17. As shown in Figure 6B, in vitro treatment with STA-21 decreased the expression of IL-17 in human CD4+ T cells. STA-21 also decreased the levels of IL-17 in the culture supernatants.

Inhibition of osteoclastogenesis by STA-21 in humans

We also investigated the effect of STA-21 on osteoclastogenesis in humans. Monocytes were prepared from healthy human volunteers and then stimulated with M-CSF alone or together with RANKL, to induce osteoclastogenesis. The addition of STA-21 to the cell cultures prevented human monocytes from differentiating into mature osteoclasts, as determined by TRAP staining (Figure 6C). Bone resorption analysis demonstrated similar results (Figure 6C). Treatment with STA-21 also reduced the mRNA expression of osteoclastogenic markers, such as MMP-9, RANK, and cathepsin K (Figure 6D). These findings suggest that STA-21 inhibits osteoclastogenesis in humans.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The present study was designed to examine whether STA-21, a potent STAT-3 inhibitor, would suppress inflammatory arthritis. In this study, we have demonstrated that treatment with STA-21 reduced the clinical and histologic scores in IL-1Ra–KO mice, a spontaneous animal model of RA. We presented evidence showing that there are 2 probable mechanisms underlying the antiarthritic effect of STA-21. One is via its regulatory effects on Th17 cells and Treg cells, and the other is through the inhibition of osteoclast formation.

More than 20 years ago, 2 main subsets of CD4+ T cells with different functions and patterns of cytokine secretion were identified, known as Th1 and Th2 cells ([6]). The Th1/Th2 paradigm was maintained until distinct T helper cell subsets, such as Th17 cells ([7, 8]), Treg cells ([30]), and follicular T helper cells, were identified. Treg cells, in which the expression of transcription factor FoxP3 occurs, play a major role in the maintenance of immunologic self tolerance and immune homeostasis ([30, 31]). In contrast, Th17 cells are a unique CD4+ T cell subset, and these cells are characterized by the production of IL-17. Th17 cells are now considered crucial in the pathogenesis of various autoimmune diseases and chronic inflammatory disorders, including RA ([32, 33]). Therefore, therapeutic approaches that increase the number of Treg cells and decrease the number of Th17 cells simultaneously are likely to be the most effective strategies to treat patients with RA.

STAT-3 is the major transcription factor that is critical for Th17 cell differentiation ([13]). STAT-3 is activated and phosphorylated by JAK. Activated STAT-3 translocates into the nucleus and then promotes the transcription of RORγt, the essential transcription factor of Th17 cells. It also regulates the expression of IL-17, IL-21, and IL-23R, all of which are of the utmost importance in the effector function of Th17 cells. Thus, mice that lack STAT-3 in CD4+ T cells are unable to generate Th17 cells and are resistant to animal models of autoimmunity ([34, 35]). Therefore, one can speculate that STA-21, a potent STAT-3 inhibitor, would inhibit Th17 cell differentiation and thereby suppress rheumatoid inflammation. As expected, our results showed that in vitro treatment with STA-21 inhibits Th17 cell differentiation under Th17-polarizing conditions, both in mice and in humans (Figures 3A and 6A). In addition, STA-21 decreased the number of Th17 cells, and thereby suppressed inflammatory arthritis in IL-1Ra–KO mice (Figures 2A and B).

Recently, Yang and colleagues proposed the competition model, in which the relative activation of STAT-3 and STAT-5 directly dictates the outcome of IL-17 production in CD4+ T cells ([36]). They demonstrated that STAT-3 and STAT-5 bind to multiple common sites across the locus encoding IL-17, and that the induction of STAT-5 binding by IL-2 was associated with less binding of STAT-3 at these sites. They also found that efficient Th17 cell differentiation occurred in the presence of very low doses of IL-6, as long as STAT-5 activation was antagonized, verifying that the opposing effects of STAT-3 and STAT-5 act on the same locus encoding IL-17 ([36]). Therefore, it could be expected that therapeutic agents inhibiting STAT-3 could up-regulate STAT-5 at the same time. Accordingly, our results showed that in vitro treatment with STA-21 up-regulated FoxP3 expression in CD4+ T cells, even in conditions of Th17 cell differentiation, both in mice and in humans (Figures 3A and 6A). Furthermore, STA-21 induced greater numbers of Treg cells expressing FoxP3 or STAT-5, both of which are critical transcription factors for Treg cells, in IL-1Ra–KO mice (Figures 2A and C).

Of note, it is not the number of Treg cells, but the function of Treg cells that is important in the highly inflammatory microenvironment of RA, because the function of Treg cells might be abnormal in patients with RA ([37]). Thus, we performed adoptive transfer of CD4+CD25+ T cells induced by STA-21 into IL-1Ra–KO mice, and the results confirmed that Treg cells induced by STA-21 have immunoregulatory effects in vivo (Figure 4A). Further investigations using molecular-based analysis will be needed to understand the exact mechanism of reciprocal regulation of STAT-3 and STAT-5 by STA-21.

It is apparent that various proinflammatory cytokines play a crucial role in the pathogenesis of RA, based on the clinical efficacy of anticytokine therapies. For such cytokines to cause inflammation and thereby contribute to the development of RA, the appropriate intracellular signaling should be activated. Members of the JAK family of kinases play an essential role in the signaling pathway of diverse cytokines that are critically implicated in the pathogenesis of RA. Therefore, small-molecule inhibitors targeting intracellular signaling pathways have been developed for the treatment of RA ([38]). Among them, tofacitinib, which is an orally available JAK inhibitor, was recently approved by the US Food and Drug Administration for the treatment of RA ([39]). In vitro experiments indicated that treatment with tofacitinib inhibited the production of both interferon-γ and IL-17 in human CD4+ T cells, presumably suppressing Th1 and Th17 cells ([40]). STAT-3 is a downstream signal of JAK and specifically implicated in the differentiation of proinflammatory Th17 cells, the latter of which are deeply involved in the pathogenesis of RA. Thus, STA-21, a promising STAT-3 inhibitor, could be a good therapeutic strategy for the treatment of RA.

Bone destruction is critically important for the prognosis in RA patients, because it is closely associated with functional impairment. In spite of continuous treatment with conventional disease-modifying antirheumatic drugs (DMARDs), disease progression does not stop ([41, 42]) and some patients still have to undergo joint replacement surgery because of progressive bone destruction ([43]). Introduction of biologic agents, such as the tumor necrosis factor α (TNFα) inhibitors, has caused a profound paradigm shift in the treatment of RA, since the combinations of methotrexate, the prototype of conventional DMARDs, with TNFα inhibitors, such as infliximab, etanercept, and adalimumab, have proven to be significantly superior to methotrexate alone for improving the signs and symptoms of disease, but also for inhibiting its radiographic progression ([44-46]). As a result, almost all clinical trials of newly developed drugs targeting RA have focused on the degree of radiographic progression as well as the severity of disease activity as the primary outcome measurements. However, in some populations of RA patients, the disease remains refractory to biologic agents such as TNF inhibitors, and disease progression does not stop. Therefore, the development of new therapeutic strategies targeting refractory bone destruction will be mandatory.

STAT-3 is critical in the growth, differentiation, and survival of cells, and it was reported that STAT-3 activation in stromal/osteoblastic cells is required for the induction of RANKL and osteoclast formation ([47]). Protein inhibitor of activated STAT-3, a specific inhibitor of STAT-3 ([48]), attenuates osteoclastogenesis by down-regulating NF-ATc1 and osteoclast-associated receptor ([49]). Based on the results of previous investigations ([47-49]), we could expect that STA-21, a potent STAT-3 inhibitor, inhibits osteoclastogenesis. In the current study, we demonstrated that in vitro treatment with STA-21 inhibited osteoclastogenesis in both mice and humans (Figures 5B and 6C). Additionally, we confirmed that STA-21 inhibited osteoclastogenesis in vivo in IL-1Ra–KO mice, an animal model of RA (Figure 5A). Therefore, STA-21, which was proven to have antiosteoclastogenic potential in this study, could be a good therapeutic strategy for the treatment of patients with RA, especially in the prevention of bone destruction.

STAT-3 is also an essential transcription factor for cell survival. Therefore, in order to develop promising therapeutic agents targeting STAT-3, it would be of great importance to maintain the balance between the degree of pathologic STAT-3 and STAT-3 as a survival factor. STA-21 inhibits pathogenic Th17 cell differentiation and osteoclast formation. Nevertheless, it also expands the number and function of Treg cells. Thus, STA-21 might be a good therapeutic candidate to overcome the obstacles encountered in developing STAT-3–inhibiting agents.

In conclusion, we have demonstrated that STA-21, a promising STAT-3 inhibitor, improved the clinical course of inflammatory arthritis in IL-1Ra–KO mice. STA-21 suppressed Th17 cells and induced the expansion of Treg cells both in vitro and in vivo. Moreover, Treg cells induced by STA-21 had immunoregulatory effects in vivo. STA-21 also inhibited osteoclast formation in vivo during the development of inflammatory arthritis, as well as in vitro. These findings suggest that use of STA-21 could be a good therapeutic strategy to treat patients with RA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

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. Cho 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. J.-S. Park, Kwok, Cho.

Acquisition of data. J.-S. Park, Lim, E.-K. Kim, Ryu, S.-M. Kim, Oh.

Analysis and interpretation of data. J.-S. Park, Kwok, Lim, E.-K. Kim, Ju, S.-H. Park, H.-Y. Kim, Cho.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES