A bispecific soluble receptor fusion protein that targets TNF-α and IL-21 for synergistic therapy in inflammatory arthritis.

This study was aimed at investigating the therapeutic effects of BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, on the development of autoimmune arthritis in humans and mice. To verify the effects of BITRAP in human, peripheral blood mononuclear cells were cultured with BITRAP under IL-17-producing T (Th17) cell-polarizing conditions or osteoclast differentiation conditions. BITRAP treatment inhibited the production of IL-17 and vascular endothelial growth factor but increased the production of IL-10 in CD4+ T cells, as well as directly suppressed osteoclastogenesis. Collagen-induced arthritis (CIA) and IL-1R antagonist (IL-1Ra) knockout mice were treated with BITRAP. Following injection in CIA mice, BITRAP rapidly migrated into the inflamed joints and remained there for 72 hours. Application of BITRAP attenuated the severity of autoimmune arthritis in CIA and IL-1Ra knockout mice by reducing the numbers of inflammatory cytokine-expressing cells and Th17 cells and antibody secretion. Finally, BITRAP suppressed STAT3 phosphorylation, as well as production of IL-17 and TNF-α, in murine splenic CD4+ T cells. These findings suggest that BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, may be an effective treatment to overcome the limitations of anti-TNF therapy for patients with rheumatoid arthritis.

TWEAK promotes osteoclastogenesis in rheumatoid arthritis.

Bone destruction is critical in the functional disability of patients with rheumatoid arthritis (RA). Osteoclasts, specialized bone-resorbing cells regulated by cytokines, such as receptor activator of NF-κB ligand (RANKL), are primarily implicated in bone destruction in RA. The aim of the study was to examine whether tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, has osteoclastogenic activity in patients with RA and in animal models, including mice with collagen-induced arthritis (CIA) and IL-1 receptor antagonist knockout (IL-1RaKO) mice. TWEAK was increased in the synovium, synovial fluid, and serum of patients with RA and in the synovium of CIA mice and IL-1RaKO mice. TWEAK induced RANKL expression in mixed joint cells and splenocytes from CIA mice, IL-1RaKO mice, and fibroblast-like synoviocytes from patients with RA. Both osteoclast precursor cells and osteoclasts express TWEAK receptor fibroblast growth factor-inducible 14. In addition, TWEAK enhanced in vitro osteoclastogenesis without the presence of RANKL-providing cells and by inducing RANKL expression in fibroblast-like synoviocytes. Moreover, treatment with fibroblast growth factor-inducible 14-Fc inhibited RANKL-induced osteoclastogenesis, indicating that endogenous TWEAK also has osteoclastogenic activity. Our data demonstrated that TWEAK promotes osteoclastogenesis in RA, suggesting that therapeutic strategies targeting TWEAK could be effective for treatment of patients with RA, especially in preventing bone destruction.

Interleukin-21 promotes osteoclastogenesis in humans with rheumatoid arthritis and in mice with collagen-induced arthritis.

OBJECTIVE: Bone destruction is a critical pathology involved in the functional disability caused by rheumatoid arthritis (RA). Osteoclasts, which are specialized bone-resorbing cells regulated by cytokines such as RANKL, are implicated in bone destruction in RA. The aim of this study was to determine whether interleukin-21 (IL-21), a potent immunomodulatory 4-α-helical bundle type 1 cytokine, has osteoclastogenic activity in patients with RA and in mice with collagen-induced arthritis (CIA). METHODS: The expression of IL-21 in synovial tissue was examined using immunohistochemistry. The concentrations of IL-21 in serum and synovial fluid were determined by enzyme-linked immunosorbent assay. The levels of RANKL and osteoclastogenic markers were measured using real-time polymerase chain reaction. CD14+ monocytes from patients with RA or mouse bone marrow cells were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA or CD4+ T cells from mice with CIA in the presence of IL-21 and subsequently stained for tartrate-resistant acid phosphatase activity to determine osteoclast formation. RESULTS: IL-21 was up-regulated in the synovium, synovial fluid, and serum of patients with RA and in the synovium and serum of mice with CIA. IL-21 induced RANKL expression in mixed joint cells and CD4+ T cells from mice with CIA and in CD4+ T cells and FLS from patients with RA. Moreover, IL-21 enhanced in vitro osteoclastogenesis without the presence of RANKL-providing cells and by inducing RANKL expression in CD4+ T cells and FLS. CONCLUSION: Our data suggest that IL-21 promotes osteoclastogenesis in RA. We believe that therapeutic strategies targeting IL-21 might be effective for the treatment of patients with RA, especially in preventing bone destruction.

Foxp3+ regulatory T cells control humoral autoimmunity by suppressing the development of long-lived plasma cells.

Foxp3(+) regulatory T cells (Tregs) are crucial for maintaining T cell tolerance, but their role in humoral autoimmunity remains unclear. To address this, we combined a model of autoantibody-dependent arthritis (K/BxN) with Foxp3 mutant scurfy mice to generate Treg-deficient K/BxN mice, referred to as K/BxNsf mice. The disease symptoms of K/BxNsf mice were exacerbated, and this coincided with increases in extrafollicular Th cells, follicular Th cells, and germinal centers. Surprisingly, the K/BxNsf mice exhibited an abnormal accumulation of mature plasma cells in their spleens and a corresponding loss of bone marrow plasma cells. The plasma cells were unresponsive to the bone marrow homing chemokine CXCL12, despite normal expression of the chemokine receptor CXCR4. Importantly, they were long-lived and less susceptible to the cytotoxic action of cyclophosphamide. They also expressed less FcγRIIb and were less apoptotic in response to autoantigen-autoantibody immune complexes. This suggests that Tregs control plasma cell susceptibility to cell death induced by engagement of FcγRIIb with immune complexes. Direct cytotoxic effects of Tregs also contribute to the death of plasma cells. Thus, our results reveal that Tregs suppress the emergence of long-lived splenic plasma cells by affecting plasma cell-autonomous mechanisms as well as T cell help, thereby avoiding the persistence of humoral autoimmunity.

Interleukin-27 suppresses osteoclastogenesis via induction of interferon-γ.

Interleukin (IL)-27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL-27 on arthritis and bone erosion in the murine collagen-induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL-27 on osteoclastogenesis is associated with interferon-γ (IFN-γ) production by using an IFN-γ knockout mouse model. The IL-27-Fc was injected into both CIA and IFN-γ-deficient mice. The effects of IL-27-Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL-27-Fc-injected mice showed significantly lower arthritis indices and fewer tartrate-resistant acid-phosphatase-positive osteoclasts in their joint tissues than untreated mice. Interleukin-27 inhibited osteoclastogenesis from bone marrow-derived mononuclear cells in vitro, which was counteracted by the addition of anti-IFN-γ antibody. The IL-27-Fc did not affect arthritis in IFN-γ knockout mice. Interleukin-27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN-γ on priming osteoclasts. These results imply that IL-27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN-γ.

Transforming growth factor ß-transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor ß (TGFß)-transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen-induced arthritis (CIA). METHODS: DBA/1J mice with CIA were treated with syngeneic TGFß-induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFß-transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFß-transduced MSCs on osteoclast formation were analyzed in vitro and in vivo. RESULTS: Systemic infusion of syngeneic TGFß-transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFß-transduced MSCs potently suppressed type II collagen-specific T cell proliferation and down-regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen-specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFß-transduced MSCs inhibited osteoclast differentiation. CONCLUSION: TGFß-transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell-mediated immunity using gene-modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model.

IL-23, a clinically novel cytokine, targets CD4(+) T cells. Recent IL-1Ra(-/-) mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4(+) T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-kappaB ligand expression by CD4(+) T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-kappaB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra(-/-) mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4(+) T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.

Oral administration of type-II collagen suppresses IL-17-associated RANKL expression of CD4+ T cells in collagen-induced arthritis.

The receptor activator of nuclear factor kappaB ligand (RANKL) is an osteoclastogenic mediator, which is mainly expressed by stromal cells and osteoblast. However, T cells can also be an important provider for RANKL in special condition such as autoimmune arthritis. We examined the RANKL expression of hyporesponsive CD4+ T cells induced by oral feeding with type II collagen in collagen-induced arthritis (CIA) mice. The potential of RANKL expression in CD4+ T cells was downregulated in tolerance, as compared with CIA. One of possible explanations for this phenomenon is that CII-specific T cell activation was intrinsically impaired in oral tolerance, which caused suppression of RANKL expression of CD4+ T cells. We also investigated the extrinsic role of cytokine in this process. IL-17, well-known pro-inflammatory cytokine was upregulated in CIA and downregulated in tolerance. IL-17 had a potential to stimulate T cells to express RANKL in dose-dependent manner. IL-17-associated RANKL expression of CD4+ T cells was downregulated in oral tolerance, suggesting that the induction of tolerance ameliorates IL-17-induced RANKL expression of T cells in murine CIA. We also discovered that CIA - T cells could enhance osteoclastogenesis but not oral tolerance - T cells. Oral tolerance might be promising therapeutic option in viewpoints of modulating autoreactivity of CII which can induce not only IL-17 production but also RANKL expression in CD4+ T cells.

IL-17 induces the production of IL-16 in rheumatoid arthritis.

The purpose of this study was to investigate the expression of IL-16 in the rheumatoid synovium and the role of inflammatory cytokines and Toll-like receptor (TLR) ligands in IL-16 production by fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients. Immunohistochemical staining was performed with a monoclonal antibody to IL-16 in synovial tissues from patients with RA and likewise in patients with osteoarthritis (OA). FLS were isolated from RA synovial tissues and stimulated with IL-15, IL-1beta, IFN-gamma, and IL-17. The IL-16 mRNA level was assessed by semiquantitative RT-PCR and real time (RT) PCR and a comparison was made between IL-16 mRNA levels produced by RA-FLS and OA-FLS. Production of IL-16 was identified by a western blot assay, and IL-16 production after stimulation by specific ligands of TLR2 and TLR4 was assessed by RT-PCR. While immunohistochemical staining demonstrated strong expression of IL-16 mRNA in synovial tissues from patients with RA, similar findings were not present in the OA group. Moreover, mRNA expression of IL-16 by RA-FLS increased after treatment with IL-17 but not with IL-15, IL-1beta, and IFN-gamma. Specifically, IL-17 increased IL-16 mRNA level by RA-FLS and peripheral blood mononuclear cells in a dose-dependent manner. However, IL-17 did not stimulate IL-16 production in OA-FLS. Peptidoglycan, a selective TLR2 ligand, also increased production of IL-16 by RA-FLS dose- dependently, whereas LPS, a selective TLR4 ligand, had no such stimulatory effect. The results from our data demonstrate that IL-17 and TLR2 ligands stimulate the production of IL-16 by RA-FLS.

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-kappaB activation.

N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-kappaB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-kappaB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-alpha, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-gamma and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-kappaB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-kappaB activation and thus expression of inflammatory and osteoclastogenic genes.

Toll-like receptor 2 and 4 combination engagement upregulate IL-15 synergistically in human rheumatoid synovial fibroblasts.

Toll-like receptors (TLRs) are pattern-recognition receptors that connect innate and adaptive immunity. Interleukin-15 (IL-15) is a proinflammatory, innate response cytokine that mediates pleiotropic effector functions in inflammatory synovitis of rheumatoid arthritis (RA). The aim of this study was to clarify whether stimulation of TLR2 and TLR4 by their specific ligands induces the production of IL-15 in fibroblast-like synoviocytes (FLS) from RA patients. FLS were isolated from RA synovial tissues and stimulated with the TLR2 ligand bacterial peptidoglycan (PGN) and the TLR4 ligand lipopolysaccharide (LPS). IL-15 in the culture supernatants was measured by ELISA, and mRNA levels were assessed by RT-PCR and real time PCR. The expression of TLR2, TLR4, and IL-15 in the RA synovium was quantified by immunohistochemistry and compared with values obtained in osteoarthritis synovium. IL-15 production increased in culture supernatants of RA FLS stimulated with PGN or PGN plus LPS, and this was upregulated at the transcriptional level. In contrast, LPS did not increase the level of IL-15 although it augmented the stimulatory effect of PGN on IL-15 production. Inhibition of nuclear factor (NF)-kappaB with a specific inhibitor abrogated the stimulatory effect of PGN or PGN plus LPS on IL-15. Neutralization of TLR2 with a blocking monoclonal antibody significantly reduced IL-15 production (P<0.05), reflecting the functional relevance of TLR2 activation in the induction of IL-15 production. These data suggest that TLR2 activation in RA FLS by microbial constituents is involved in the induction of IL-15 and that TLR2 promotes inflammation through NF-kappaB. TLR4 augmented the stimulatory effect of TLR2 on IL-15, possibly contributing to the maintenance of synovitis in patients with RA.

Toll-like receptor 2 ligand mediates the upregulation of angiogenic factor, vascular endothelial growth factor and interleukin-8/CXCL8 in human rheumatoid synovial fibroblasts.

Rheumatoid arthritis (RA) is characterized by infiltrations of inflammatory cells accompanied by neovascularization in the joint. We hypothesized that cell activation via the toll-like receptor (TLR) may be involved in the induction of angiogenic molecules, which are relevant to the pathogenesis of RA. RA fibroblast like synoviocytes (FLS) were stimulated with TLR-2 ligand bacterial peptidoglycan (PGN), TLR-4 ligand lipopolysaccharide (LPS) and various cytokines. Vascular endothelial growth factor (VEGF) and IL-8 were measured by ELISA in culture supernatants; mRNA levels were assessed by RT-PCR and real time PCR. The levels of TLR-2, VEGF and IL-8 were analyzed by dual immunohistochemistry in RA synovium and compared with osteoarthritis (OA). Regulation of MyD88, IRAK4, IRAK1, IRAK-M and TRAF-6 mRNA expression levels by PGN were analyzed by RT-PCR. Phosphorylation of I kappa B alpha was evaluated by western blotting. Levels of VEGF and IL-8 were upregulated in culture supernatants of RA FLS stimulated with PGN, similar to the levels of IL-1beta and IL-17 stimulation. Neutralization of TLR-2 with a blocking monoclonal antibody significantly reduced both VEGF and IL-8 levels (P<0.05), which reflected the functional relevance of TLR-2 activation to the induction of VEGF and IL-8 production. Downstream intracellular signaling following TLR-2 stimulation involved MyD88-IRAK-4-TRAF-6 pathways, resulting in NF-kappaB activation. Thus, TLR-2 activation in RA FLS by microbial constituents could be involved in the induction of VEGF and IL-8 and thereby promote inflammation either directly or via angiogenesis. This possibly contributes to the perpetuation of synovitis in patients with RA.

Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR-2 and TLR-4 activation.

The interplay between the innate immune system and inflammatory bone destruction in the joints of individuals with rheumatoid arthritis (RA) remains unclear. This study was undertaken to explore the effect of toll-like receptor (TLR) signaling in fibroblast-like synoviocytes (FLS) on the expression of RANKL and induction of osteoclastogenic activity. The levels of RANKL mRNA and protein were measured using RT-PCR, real-time PCR, and immunostaining. Monocytes were cocultured with RA -FLS that had been stimulated with TLR ligands in fresh media and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. Osteoclast molecule markers were measured using real-time PCR. Expression of TLR-2 and TLR-4 was higher in RA-FLS than in OA-FLS and normal skin fibroblasts. TLR-2 and TLR-4 ligands induced RANKL expression in RA-FLS. TLR stimulation of RA-FLS also induced the production of IL-1beta and TNF-alpha to a lesser extent; however, it had no effect on IL-17 production. Inhibition of TLR induced IL-1beta production, which partially reversed the upregulation of RANKL induced by TLR ligands. RA-FLS stimulated by TLR-2 and TLR-4 ligands and cocultured with human monocytes induced high levels of expression of TRAP, RANK, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9, suggesting that RA-FLS promote osteoclast differentiation. Our results suggest that the TLR signaling pathway, through TLR-2 and TLR-4, induces RANKL expression in RA-FLS and the expression of RANKL promotes the differentiation of osteoclasts in RA synovium. Targeting specific TLRs may be a promising approach to prevent inflammatory bone destruction in the pathogenesis of RA.

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis.

To investigate the role of CD8alpha(+) DCs in the development of collagen-induced arthritis (CIA). The immunogenic properties of CD8alpha(+) and CD8alpha(-) DC subsets were investigated by mixed-lymphocyte reaction and cytokine enzyme-linked immunoassay. CII-pulsed CD8alpha(+) DCs or CD8alpha(-) DCs with CD4(+) T cells from CIA mice were adoptively transferred onto the hind footpad of DBA mice. The onset of arthritis and the arthritis index were examined for 14 weeks after adoptive transfer. Expression of MHC-II and CD80 but not CD86 and CD40 was higher in CD8alpha(+) DCs than in CD8alpha(-) DCs from the spleens of CIA mice. Culturing CD8alpha(+) DCs with CD4(+) T cells significantly increased the proliferative response of CD4(+) T cells in the presence of CII. The production of interleukin (IL)-12p70, IL-17, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was slightly increased in CD8alpha(+) DCs than in CD8alpha(-) DCs. DBA/1 mice that were adoptively transferred with CII-pulsed CD8alpha(+) DCs and CD4(+) T cells into the footpads showed accelerated onset of CIA compared to control group. By contrast, CD8alpha(-) DCs showed a partial inhibitory effect on CIA. These findings show that CD8alpha(+) DCs accelerated the onset of CIA when aoptively transferred with CD4(+) T cells and that CD8alpha(+) DCs provoke the development of CIA probably by stimulating the immune responses of CII-reactive CD4(+) T cells and by increasing the production of inflammatory cytokines.

Epigallocatechin-3-gallate ameliorates both obesity and autoinflammatory arthritis aggravated by obesity by altering the balance among CD4+ T-cell subsets.

Epigallocatechin-3-gallate (EGCG) is the most biologically active catechin in green tea. EGCG has been shown to have therapeutic effects in autoinflammatory diseases and obesity. Obesity is currently regarded-partly-as an inflammatory condition because of the inflammatory cytokines and higher Th1 cell differentiation detected in obese animal models and human cohort studies. In this work, the effects of EGCG on diet-induced obesity (DIO) mice and obese collagen-induced arthritis (CIA) mice were investigated. EGCG reduced the body weight and fat infiltration in liver tissue while improving serum lipid profiles in DIO mice. EGCG also induced a higher Treg/Th17 cell ratio in CD4(+) T-cell differentiation by decreasing the ratio of STAT3/STAT5 expression in DIO mice. EGCG was also effective in obese CIA mice. Reducing Th17 cells and increasing regulatory T (Treg) cells by affecting the STAT protein ratio were important effects of EGCG that might result in improved arthritic scores and levels of several inflammatory indicators. Thus, EGCG has an anti-inflammatory effect by suppressing STAT3 proteins and Th17-cell differentiation. EGCG thus shows promise for treating autoimmune conditions related to STAT3 or Th17 cells, such as metabolic syndrome, inflammatory arthritis, and some neoplastic diseases.

STA-21, a promising STAT-3 inhibitor that reciprocally regulates Th17 and Treg cells, inhibits osteoclastogenesis in mice and humans and alleviates autoimmune inflammation in an experimental model of rheumatoid arthritis.

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.

Phospholipase D1 has a pivotal role in interleukin-1ß-driven chronic autoimmune arthritis through regulation of NF-κB, hypoxia-inducible factor 1α, and FoxO3a.

Interleukin-1ß (IL-1ß) is a potent proinflammatory and immunoregulatory cytokine playing an important role in the progression of rheumatoid arthritis (RA). However, the signaling network of IL-1ß in synoviocytes from RA patients is still poorly understood. Here, we show for the first time that phospholipase D1 (PLD1), but not PLD2, is selectively upregulated in IL-1ß-stimulated synoviocytes, as well as synovium, from RA patients. IL-1ß enhanced the binding of NF-κB and ATF-2 to the PLD1 promoter, thereby enhancing PLD1 expression. PLD1 inhibition abolished the IL-1ß-induced expression of proinflammatory mediators and angiogenic factors by suppressing the binding of NF-κB or hypoxia-inducible factor 1α to the promoter of its target genes, as well as IL-1ß-induced proliferation or migration. However, suppression of PLD1 activity promoted cell cycle arrest via transactivation of FoxO3a. Furthermore, PLD1 inhibitor significantly suppressed joint inflammation and destruction in IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice, a model of spontaneous arthritis. Taken together, these results suggest that the abnormal upregulation of PLD1 may contribute to the pathogenesis of IL-1ß-induced chronic arthritis and that a selective PLD1 inhibitor might provide a potential therapeutic molecule for the treatment of chronic inflammatory autoimmune disorders.

IL-17-deficient allogeneic bone marrow transplantation prevents the induction of collagen-induced arthritis in DBA/1J mice.

IL-17-producing CD4+ T cells (Th17) play important functions in autoimmune diseases and allograft rejection of solid organs. We examined the effects of IL 17 and its mechanism of action on arthritis in a murine collagen-induced arthritis (CIA) model using bone marrow transplantation (BMT) system. DBA/1J mice were administered a lethal radiation dose and then rescued with bone marrow derived from either wild-type (WT) or IL-17-/- mice on C57BL/6 background mice. CIA was induced after the bone marrow transplant, and disease progression was characterized. DBA/1J mice with CIA that received IL-17-/- donor bone marrow showed potently inhibited development and severity of clinical arthritis as compared with CIA mice that received WT bone marrow. Reduced secretion of the pro-inflammatory cytokines tumor necrosis factor-α, IL-1ß, and IL-6, and collagen-specific T cell responses were observed in mice that received IL-17-/- bone marrow. IL-17 blockade also inhibited effector T cell proliferation by reciprocally regulating the Treg/Th17 ratio. IL-17 blockade prevented joint destruction in mice with CIA. These findings suggest that CIA with BMT is a viable method of immunological manipulation and that IL-17 deficiency suppresses severe joint destruction and inflammation in CIA mice. There may be clinical benefits in blocking IL-17 and BMT in the treatment of rheumatoid arthritis.

Grape-seed proanthocyanidin extract as suppressors of bone destruction in inflammatory autoimmune arthritis.

Chronic autoimmune inflammation, which is commonly observed in rheumatoid arthritis (RA), disrupts the delicate balance between bone resorption and formation causing thedestruction of the bone and joints. We undertook this study to verify the effects of natural grape-seed proanthocyanidin extract (GSPE), an antioxidant, on chronic inflammation and bone destruction. GSPE administration ameliorated the arthritic symptoms of collagen-induced arthritis (CIA), which are representative of cartilage and bone destruction. GSPE treatment reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and osteoclast activity and increased differentiation of mature osteoblasts. Receptor activator of NFκB ligand expression in fibroblasts from RA patients was abrogated with GSPE treatment. GSPE blocked human peripheral blood mononuclear cell-derived osteoclastogenesis and acted as an antioxidant. GSPE improved the arthritic manifestations of CIA mice by simultaneously suppressing osteoclast differentiation and promoting osteoblast differentiation. Our results suggest that GSPE may be beneficial for the treatment of inflammation-associated bone destruction.

Obesity aggravates the joint inflammation in a collagen-induced arthritis model through deviation to Th17 differentiation.

White fat cells secrete adipokines that induce inflammation and obesity has been reported to be characterized by high serum levels of inflammatory cytokines such as IL-6 and TNF-α. Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis, but the relationship between RA and obesity is controversial. We made an obese inflammatory arthritis model: obese collagen-induced arthritis (CIA). C57BL/6 mice were fed a 60-kcal high fat diet (HFD) from the age of 4 weeks and they were immunized twice with type II collagen (CII). After immunization, the obese CIA mice showed higher arthritis index scores and histology scores and a more increased incidence of developing arthritis than did the lean CIA mice. After treatment with CII, mixed lymphocyte reaction also showed CII-specific response more intensely in the obese CIA mice than lean CIA. The anti-CII IgG and anti-CII IgG2a levels in the sera of the obese CIA mice were higher than those of the lean CIA mice. The number of Th17 cells was higher and the IL-17 mRNA expression of the splenocytes in the obese CIA mice was higher than that of the lean CIA mice. Obese CIA mice also showed high IL-17 expression on synovium in immunohistochemistry. Although obesity may not play a pathogenic role in initiating arthritis, it could play an important role in amplifying the inflammation of arthritis through the Th1/Th17 response. The obese CIA murine model will be an important tool when we investigate the effect of several therapeutic target molecules to treat RA.