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.

Lactobacillus sakei suppresses collagen-induced arthritis and modulates the differentiation of T helper 17 cells and regulatory B cells.

BACKGROUND: To evaluate the immunomodulatory effect of Lactobacillus sakei in a mouse model of rheumatoid arthritis (RA) and in human immune cells. METHODS: We evaluated whether L. sakei reduced the severity of collagen-induced arthritis (CIA) and modulated interleukin (IL)-17 and IL-10 levels, as well as whether it affected the differentiation of CD4+ T cells and regulatory B cells. We evaluated osteoclastogenesis after culturing bone marrow-derived mononuclear cells with L. sakei. RESULTS: The differentiation of T helper 17 cells and the serum level of IL-17 were suppressed by L. sakei in both human peripheral blood mononuclear cells and mouse splenocytes. The serum level of IL-10 was significantly increased in the L. sakei-treated group, whereas the regulatory T cell population was unchanged. The population of regulatory B cells significantly increased the in L. sakei-treated group. Oral administration of L. sakei reduced the arthritis incidence and score in mice with CIA. Finally, osteoclastogenesis and the mRNA levels of osteoclast-related genes were suppressed in the L. sakei-treated group. CONCLUSION: L. sakei exerted an anti-inflammatory effect in an animal model of RA, regulated Th17 and regulatory B cell differentiation, and suppressed osteoclastogenesis. Our findings suggest that L. sakei has therapeutic potential for RA.

Protein inhibitor of activated STAT3 reduces peripheral arthritis and gut inflammation and regulates the Th17/Treg cell imbalance via STAT3 signaling in a mouse model of spondyloarthritis.

BACKGROUND: Spondyloarthritis (SpA) is chronic inflammatory arthritis, and interleukin (IL)-17 is crucial in SpA pathogenesis. Type 17 helper T (Th17) cells are one of major IL-17-secreting cells. Signal transducer and activator of transcription (STAT)-3 signaling induces Th17 differentiation. This study investigated the effects of protein inhibitor of activated STAT3 (PIAS3) on SpA pathogenesis. Curdlan was injected into SKG ZAP-70W163C mice for SpA induction. METHODS: The PIAS3 or Mock vector was inserted into mice for 10 weeks. Clinical and histologic scores of the paw, spine, and gut were evaluated. The expression of IL-17, tumor necrosis factor-α (TNF-α), STAT3, and bone morphogenic protein (BMP) was measured. Confocal microscopy and flow cytometry were used to assess Th cell differentiation. RESULTS: PIAS3 significantly diminished the histologic scores of the paw and gut. PIAS3-treated mice displayed decreased expression of IL-17, TNF-α, and STAT3 in the paw, spine, and gut. BMP-2/4 expression was lower in the spines of PIAS3-treated mice. Th cell differentiation was polarized toward the upregulation of regulatory T cells (Tregs) and the downregulation of Th17 in PIAS3-treated mice. CONCLUSION: PIAS3 had beneficial effects in mice with SpA by reducing peripheral arthritis and gut inflammation. Pro-inflammatory cytokines and Th17/Treg differentiation were controlled by PIAS3. In addition, BMPs were decreased in the spines of PIAS3-treated mice. These findings suggest that PIAS3 could have therapeutic benefits in patients with SpA.

JAK2-STAT3 blockade by AG490 suppresses autoimmune arthritis in mice via reciprocal regulation of regulatory T Cells and Th17 cells.

IL-6-mediated STAT3 signaling is essential for Th17 differentiation and plays a central role in the pathogenesis of rheumatoid arthritis. To investigate the molecular mechanism underlying the antirheumatic effects and T cell regulatory effects of STAT3 inhibition, we studied the effects of the JAK 2 inhibitor AG490 on Th17 cell/regulatory T cell (Treg) balance and osteoclastogenesis. AG490 was administered to mice with collagen-induced arthritis (CIA) via i.p. injection, and its in vivo effects were determined. Differential expression of proinflammatory cytokines, including IL-17A, IL-1ß, and IL-6, was analyzed by immunohistochemistry. Levels of phosphorylated STAT3 and STAT5 and differentiation of Th17 cells and Tregs after AG490 treatment in our CIA model were analyzed by immunostaining. In vitro development of Th17 cells and Tregs was analyzed by flow cytometry and real-time PCR. AG490 ameliorated the arthritic phenotype in CIA and increased the proportion of Foxp3(+) Tregs. In contrast, the proportion of IL-17A-producing T cells and levels of inflammatory markers were reduced in AG490-treated mice. Numbers of p-STAT3(+) CD4(+) T cells and p-STAT5(+) CD4(+) T cells were reduced and elevated, respectively, after treatment with AG490. Furthermore, AG490 markedly increased the expression of molecules associated with Treg development (ICOS, programmed cell death protein 1, ICAM-1, and CD103). The development and function of osteoclasts were suppressed by AG490 treatment. Our results suggest that AG490, specifically regulating the JAK2/STAT3 pathway, may be a promising treatment for rheumatoid arthritis.

p53 controls autoimmune arthritis via STAT-mediated regulation of the Th17 cell/Treg cell balance in mice.

OBJECTIVE: To investigate the connection between p53 and interleukin-17-producing Th17 cell/Treg cell balance in rheumatoid arthritis (RA). METHODS: Th17 cell and Treg cell frequencies were analyzed by flow cytometry, and cytokine levels in the supernatant were determined using enzyme-linked immunosorbent assays. The expression of transcription factors was analyzed by immunostaining and Western blotting, and the interactions between p53 and STAT-3 or STAT-5 were determined by immunoprecipitation-Western blot analysis. A p53 agonist was administered in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. RESULTS: CD4+ T cells from p53-/- mice decreased the activity of STAT-5, lowered the level of phosphorylated STAT-5, and compromised Treg cell differentiation. The protein p53 bound STAT-5 directly, and this interaction was enhanced with increasing p53 activity. Under inflammatory conditions, p53 suppressed Th17 cell differentiation and skewed T cells toward Treg cell differentiation through the activation of STAT-5 signaling cascades. In mice with CIA, injection of a p53 overexpression vector or an antagonist of Mdm2 had the effect of controlling arthritis development in vivo. The regulatory effect of p53 was recapitulated in the cells of RA patients, with more pronounced suppression due to the repressed status of p53 in RA. CONCLUSION: We demonstrated a link between p53-mediated and STAT-mediated regulation of Th17 cells/Treg cells in RA. Our results suggest that factors involved in this pathway might constitute novel therapeutic targets for the treatment of RA.

Interleukin-2/anti-interleukin-2 monoclonal antibody immune complex suppresses collagen-induced arthritis in mice by fortifying interleukin-2/STAT5 signalling pathways.

In this study, we investigated the effects of administration of interleukin-2 (IL-2)/JES6-1 (anti-IL-2 monoclonal antibody) immune complexes on the expansion and activation of regulatory T (Treg) cells, the down-regulation of T helper type 17 (Th17) cells, and the control of the severity of collagen-induced arthritis (CIA). Wild-type and CIA-induced wild-type mice were injected intraperitoneally (i.p.) with IL-2 or IL-2/JES6-1 complex three times at 2-day intervals. Treg cell surface markers were analysed by flow cytometry. After injecting IL-2 or IL-2/JES6-1, the time kinetics of IL-2 signalling molecules was examined by FACS and Western blotting. Concentrations of IL-17 and IL-10 were measured by ELISA. Injection of IL-2/JES6-1 increased the proportion of Foxp3+ Treg cells among splenic CD4+ T cells, which reached the highest level on day 4 after injection. Up-regulation of CTLA4, GITR and glycoprotein-A repetitions predominant (GARP) was observed. Activation of p-signal transducer and activator of transcription 5 (STAT5) was apparent within 3 hr after injection of IL-2/JES6-1 complexes. Expression of IL-2 signalling molecules, including p-AKT and p-p38/mitogen-activated protein kinase, was also higher in splenocytes treated with IL-2/JES6-1 complexes. Injection of IL-2/JES6-1 complexes suppressed the induction of CIA and the production of IL-17 and inflammatory responses while increasing the level of IL-10 in the spleen. The expansion of Treg cells (via STAT5) and the concomitant increase in IL-2 signalling pathways by IL-2/JES6-1 complexes suggests their potential use as a novel therapeutic agent for the treatment of autoimmune arthritis.

EC-18 prevents autoimmune arthritis by suppressing inflammatory cytokines and osteoclastogenesis.

BACKGROUND: EC-18, a synthetic monoacetyldiaglyceride, exhibits protective effects against lung inflammation, allergic asthma, and abdominal sepsis. However, there have been no investigations to determine whether EC-18 has preventive potential in autoimmune diseases, especially rheumatoid arthritis (RA). METHODS: To investigate the efficacy of EC-18 on the development of RA, EC-18 was administered in a collagen-induced arthritis (CIA) murine model and disease severity and the level of inflammatory cytokines in the joint were investigated. The effect of EC-18 on the inflammation-related factors was investigated by flow cytometry, ELISA, western blot, and real-time PCR in splenocytes from mice and in peripheral blood mononuclear cells from healthy and patients with RA. The effect of EC-18 on osteoclastogenesis was investigated. RESULTS: EC-18 effectively reduced the clinical and histological severity of arthritis, similar to Janus kinase inhibitors include tofacitinib and baricitinib, in CIA. Furthermore, EC-18 exhibited a synergistic effect with methotrexate in preventing CIA. Treatment with EC-18 effectively reduced the production of inflammatory cytokines in immune cells and osteoclast differentiation in mice and patients with RA. CONCLUSION: These results suggest that EC-18 may be an effective strategy for RA.

A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells.

OBJECTIVE: The interleukin (IL)-12 cytokine family is closely related to the development of T helper cells, which are responsible for autoimmune disease enhancement or suppression. IL-12 family members are generally heterodimers and share three α-subunits (p35, p19, and p28) and two ß-subunits (p40 and EBI3). However, a ß-sheet p40 homodimer has been shown to exist and antagonize IL-12 and IL-23 signaling 1. Therefore, we assumed the existence of a p40-EBI3 heterodimer in nature and sought to investigate its role in immune regulation. METHODS: The presence of the p40-EBI3 heterodimer was confirmed by ELISA, immunoprecipitation, and western blotting. A p40-EBI3 vector and p40-EBI3-Fc protein were synthesized to confirm the immunological role of this protein in mice with collagen-induced arthritis (CIA). The anti-inflammatory effects of p40-EBI3 were analyzed with regard to clinical, histological, and immune cell-regulating features in mice with CIA. RESULTS: Clinical arthritis scores and the expression levels of proinflammatory cytokines (e.g., IL-17, IL-1ß, IL-6, and TNF-α) were significantly attenuated in p40-EBI3-overexpressing and p40-EBI3-Fc-treated mice with CIA compared to vehicle-treated mice with CIA. Structural joint damage and vessel formation-related gene expression were also reduced by p40-EBI3 heterodimer treatment. In vitro, the p40-EBI3-Fc protein significantly suppressed the differentiation of Th17 cells and reciprocally induced CD4+CD25+Foxp3+ (regulatory T) cells. p40-EBI3 also inhibited osteoclast formation in a concentration-dependent manner. CONCLUSION: In this study, p40-EBI3 ameliorated proinflammatory conditions both in vivo and in vitro. We propose that p40-EBI3 is a novel anti-inflammatory cytokine involved in suppressing the immune response through the expansion of Treg cells and suppression of Th17 cells and osteoclastogenesis.

Metformin rescues rapamycin-induced mitochondrial dysfunction and attenuates rheumatoid arthritis with metabolic syndrome.

BACKGROUND: Rapamycin, an inhibitor of the serine/threonine protein kinase mTOR, is an immunosuppressant used to treat renal transplant recipients, but it can cause endothelial and mitochondrial dysfunction. Metformin is used for the treatment of type 2 diabetes and was reported to exert therapeutic effects against rheumatoid arthritis and obesity by improving mitochondrial dysfunction via the activation of fibroblast growth factor 21. We investigated the therapeutic effects of rapamycin-metformin combination therapy in obese mice with collagen-induced arthritis (CIA). METHODS: Mouse embryonic fibroblasts were treated with rapamycin, metformin, or rapamycin-metformin, and their respiratory level and mitochondrial gene expression were assayed. Mice were fed a high-fat diet, immunized with type II collagen, and subsequently treated with rapamycin-metformin daily for 10 weeks. RESULTS: Rapamycin-treated cells exhibited dysfunction of mitochondrial respiration and decreased mitochondrial gene expression compared with rapamycin-metformin-treated cells. Moreover, rapamycin-metformin reduced the clinical arthritis score and the extent of histological inflammation and improved the metabolic profile in obese mice with CIA. Rapamycin-metformin enhanced the balance between T helper 17 and regulatory T cells in vitro and in vivo. CONCLUSIONS: These results suggest that rapamycin-metformin is a potential therapeutic option for autoimmune arthritis.

Inhibition of IL-17 ameliorates systemic lupus erythematosus in Roquinsan/san mice through regulating the balance of TFH cells, GC B cells, Treg and Breg.

Systemic lupus erythematosus (SLE) is mediated by a chronic and dysregulated inflammatory response. Interleukin (IL)-17, a proinflammatory cytokine, and T helper (Th)17 cells are associated with chronic autoimmune diseases. We hypothesized that inhibition of IL-17 would decrease the numbers of T cell subsets that function as B-cell helpers, as well as B-cell differentiation into plasma cells and autoantibody expression. The IL-17 level was increased markedly in Roquinsan/san mice. Loss of IL-17 in Roquinsan/san mice improved nephritis by downregulating immunoglobulin (Ig)G, IgG1, and IgG2a production. Formation of germinal centers (GCs), and follicular B- and T-cell differentiation was reduced, whereas the number of regulatory T (Treg) cells and immature B cells was increased, by IL-17 deficiency in Roquinsan/san mice. These results suggest that IL-17 inhibition can ameliorate SLE by inhibiting B-cell differentiation into GCs. Therefore, IL-17-producing Th17 cells show promise as a target for development of novel therapeutics for SLE.

Metformin improves salivary gland inflammation and hypofunction in murine Sjögren's syndrome.

BACKGROUND: Activated T and B cells participate in the development and progression of Sjögren's syndrome (SS). Metformin, a first-line anti-diabetic drug, exerts anti-inflammatory and immunomodulatory effects by activating AMPK. We investigated the therapeutic effect of metformin in non-obese diabetic (NOD)/ShiLtJ mice, an animal model of SS. METHODS: Metformin or vehicle was administered orally to the mice for 9 weeks. The salivary flow rate was measured at 11, 13, 15, 17, and 20 weeks. Histological analysis of the salivary glands from vehicle- and metformin-treated mice was conducted. CD4+ T and B cell differentiation in the peripheral blood and/or spleen was determined by flow cytometry. Serum total IgG, IgG1, and IgG2a levels were determined by enzyme-linked immunosorbent assay. RESULTS: Metformin reduced salivary gland inflammation and restored the salivary flow rate. Moreover, metformin reduced the interleukin (IL)-6, tumor necrosis factor-α, IL-17 mRNA, and protein levels in the salivary glands. Metformin reduced the Th17 and Th1 cell populations and increased the regulatory T cell population in the peripheral blood and spleen and modulated the balance between Tfh and follicular regulatory T cells. In addition, metformin reduced B cell differentiation into germinal center B cells, decreased the serum immunoglobulin G level, and maintained the balance between IL-10- and IL-17-producing B cells. CONCLUSION: Metformin suppresses effector T cells, induces regulatory T cells, and regulates B cell differentiation in an animal model of SS. In addition, metformin ameliorates salivary gland inflammation and hypofunction, suggesting that it has potential for the treatment of SS.

Attenuation of Rheumatoid Inflammation by Sodium Butyrate Through Reciprocal Targeting of HDAC2 in Osteoclasts and HDAC8 in T Cells.

Rheumatoid arthritis (RA) is a systemic autoimmune disease caused by both genetic and environmental factors. Recently, investigators have focused on the gut microbiota, which is thought to be an environmental factor that affects the development of RA. Metabolites secreted by the gut microbiota maintain homeostasis in the gut through various mechanisms [e.g., butyrate, which is one of the major metabolites of gut microbiota, exerts an anti-inflammatory effect by activating G-protein-coupled receptors and inhibiting histone deacetylases (HDACs)]. Here, we focused on the inhibition of the HDACs by butyrate in RA. To this end, we evaluated the therapeutic effects of butyrate in an animal model of autoimmune arthritis. The arthritis score and incidence were lower in the butyrate-treated group compared to the control group. Also, butyrate inhibited HDAC2 in osteoclasts and HDAC8 in T cells, leading to the acetylation of glucocorticoid receptors and estrogen-related receptors α, respectively. Additionally, control of the TH17/Treg cell balance and inhibition of osteoclastogenesis were confirmed by the changes in target gene expression. Interleukin-10 (IL-10) produced by butyrate-induced expanded Treg cells was critical, as treatment with butyrate did not affect inflammatory arthritis in IL-10-knockout mice. This immune-cell regulation of butyrate was also detected in humans. These findings suggest that butyrate is a candidate agent for the treatment of RA.

YinYang1 deficiency ameliorates joint inflammation in a murine model of rheumatoid arthritis by modulating Th17 cell activation.

Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor that functions in cooperation with various cofactors to regulate gene expression. In the immune system, YY1 enhances cytokine production and T helper (Th) 2 effector cell differentiation, resulting in the activation of inflammation. However, no studies have reported the role of YY1 in Th17 cell regulation, which is implicated in rheumatoid arthritis (RA). We investigated the expression of YY1 in Th17 cells in vitro and revealed increased levels of YY1 mRNA and protein. To elucidate the function of YY1 pathogenesis in RA, we used a collagen-induced arthritis (CIA) mouse model with YY1 deficiency. Deficiency of YY1 reduced the severity of arthritis and joint destruction. Moreover, Th17 cells were dramatically reduced in YY1-deficient mice. The cytokine interleukin (IL)-17 was decreased in YY1-deficient CD4+ T cells ex vivo and in vivo. Interestingly, the level of signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α, IL-17, IL-6, and IL-1ß were markedly decreased in YY1-deficient mice with CIA. The cytokine-inducing function of YY1 was more specific to IL-17 than to interferon-γ. YY1 plays a role in Th17 cell differentiation and RA pathogenesis. Our findings suggest that future RA therapies should target the regulatory mechanism involved in Th17 cell differentiation, in which YY1 may cooperate with the STAT3 signaling pathway.

The Fos-Related Antigen 1-JUNB/Activator Protein 1 Transcription Complex, a Downstream Target of Signal Transducer and Activator of Transcription 3, Induces T Helper 17 Differentiation and Promotes Experimental Autoimmune Arthritis.

Dysfunction of T helper 17 (Th17) cells leads to chronic inflammatory disorders. Signal transducer and activator of transcription 3 (STAT3) orchestrates the expression of proinflammatory cytokines and pathogenic cell differentiation from interleukin (IL)-17-producing Th17 cells. However, the pathways mediated by STAT3 signaling are not fully understood. Here, we observed that Fos-related antigen 1 (FRA1) and JUNB are directly involved in STAT3 binding to sites in the promoters of Fosl1 and Junb. Promoter binding increased expression of IL-17 and the development of Th17 cells. Overexpression of Fra1 and Junb in mice resulted in susceptibility to collagen-induced arthritis and an increase in Th17 cell numbers and inflammatory cytokine production. In patients with rheumatoid arthritis, FRA1 and JUNB were colocalized with STAT3 in the inflamed synovium. These observations suggest that FRA1 and JUNB are associated closely with STAT3 activation, and that this activation leads to Th17 cell differentiation in autoimmune diseases and inflammation.

Epigallocatechin-3-gallate ameliorates autoimmune arthritis by reciprocal regulation of T helper-17 regulatory T cells and inhibition of osteoclastogenesis by inhibiting STAT3 signaling.

The green tea polyphenol epigallocatechin-3-gallate is a potent antioxidant. Here, we describe the effects of epigallocatechin-3-gallate on T cell differentiation and osteoclast differentiation in an animal model of arthritis. Mice with collagen-induced arthritis were injected intraperitoneally with epigallocatechin-3-gallate, 3 times/wk after the primary immunization. Surface markers of T helper 17 cells and regulatory T cells were analyzed by flow cytometry. Flow cytometry, Western blotting, and enzyme-linked immunosorbent assays were used to evaluate the effect of epigallocatechin-3-gallate on cell signaling in the collagen-induced arthritis model. Epigallocatechin-3-gallate decreased the arthritis index and showed protective effects against joint destruction in collagen-induced arthritis mice. The expression of cytokines, oxidative stress proteins, and phosphorylated-signal transducer and activator of transcription-3, 705 and 727, were significantly less in mice treated with epigallocatechin-3-gallate than it was in controls. Epigallocatechin-3-gallate reduced the expression of osteoclast markers in vitro and in vivo relative to the control, and the antiosteoclastic activity was observed in epigallocatechin-3-gallate-treated, interferon-γ knockout mice. The proportion of forkhead box protein 3-positive regulatory T cells was increased in the spleens of mice treated with epigallocatechin-3-gallate compared with control mice, whereas the proportion of T helper 17 cells was reduced. In vitro, the expression of nuclear respiratory factor 2, heme oxygenase-1, and extracellular signal-regulated kinase was increased significantly by epigallocatechin-3-gallate. We demonstrated that the administration of epigallocatechin-3-gallate attenuated the symptoms of arthritis, inhibited osteoclastogenesis and T helper 17 cell activation, and increased the number of regulatory T cells. At the molecular level, the antiarthritic effects of epigallocatechin-3-gallate may be due to induction of phosphorylated-extracellular signal-regulated kinase, nuclear respiratory factor 2, and heme oxygenase-1 and inhibition of signal transducer and activator of transcription-3 activation.

Treatment of IL-21R-Fc control autoimmune arthritis via suppression of STAT3 signal pathway mediated regulation of the Th17/Treg balance and plasma B cells.

Interleukin-21 (IL-21) is a T cell-derived cytokine modulating T cell, B cell, and natural killer cell responses. To determine whether IL-21 contributes to pathologic processes, recombinant IL-21 receptor (R) fusion protein (rhIL-21R-Fc) was examined in mice models of autoimmune arthritis (collagen-induced arthritis). DBA/1J mice were immunized with chicken type II collagen and then treated intraperitoneally with rhIL-21R-Fc, which was initiated after the onset of arthritis symptoms in 20% of the cohort. The mice were assessed 3 times per week for signs of arthritis and histologic features as well as serum immunoglobulin. Cytokine messenger RNA levels in the spleen were also examined. STAT3 phosphorylation is dose dependently activated by IL-21 and inhibited by rhIL-21R-Fc in vitro using T cells. Treatment of DBA/1J mice with rhIL-21R-Fc reduced the clinical and histologic signs of CIA. The IL-17 and STAT3-expressing CD4(+) splenocytes dramatically decreased in the rhIL-21R-Fc treated mice. IL-21R-Fc treated mice also decreased the production of IgG, STAT3 phosphorylation, and plasma cell transcription factor (Blimp1). These findings demonstrate a pathogenic role of IL-21 in animal models of RA, suggesting IL-21 as a promising therapeutic target among human RA.

Gene associated with retinoid-interferon-induced mortality 19 attenuates murine autoimmune arthritis by regulation of th17 and treg cells.

OBJECTIVE: STAT-3 is a key transcriptional factor in the interleukin-6 (IL-6)-mediated differentiation of Th17 cells. Because Th17 is believed to be a central player in rheumatoid arthritis (RA), we sought to evaluate whether an endogenous inhibitor of the STAT3 gene, GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), could attenuate the progression and severity of murine collagen-induced arthritis (CIA) through suppression of Th17 cells and, reciprocally, could increase expression of Treg cells. METHODS: Overexpression of GRIM-19 was produced either by intravenous/intramuscular administration of a GRIM-19 overexpression vector in DBA1/J mice or by development of GRIM-19-transgenic (Tg) mice on a C57BL/6 background. Clinical signs were scored for arthritis severity, and mouse splenocytes, serum, and joint tissue were obtained for immunostaining and histologic analyses. RESULTS: The numbers of CD4+IL-17+ cells and CD4+pSTAT3+ cells were decreased, while the numbers of CD4+CD25+Foxp3+ cells and CD4+pSTAT5+ cells were increased, in both GRIM-19 vector-transfected and GRIM-19-Tg mice. Administration of the GRIM-19 overexpression vector into mice with CIA markedly suppressed the clinical and histologic signs of arthritis in the affected joints. Similarly, when CIA was induced in GRIM-19-Tg mice, the arthritis phenotype was markedly attenuated and the expression of inflammatory cytokines (IL-1ß, IL-6, tumor necrosis factor α, and IL-17) in the arthritic joints was also significantly reduced. Moreover, bone marrow-derived monocyte/macrophages obtained from GRIM-19-Tg mice showed attenuated RANKL-induced osteoclastogenesis in vitro. CONCLUSION: GRIM-19 improved the clinical and histologic features of CIA and also inhibited osteoclast formation. These findings suggest that GRIM-19 may be a novel treatment agent for RA.

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.

Intravenous immunoglobulin attenuates experimental autoimmune arthritis by inducing reciprocal regulation of Th17 and Treg cells in an interleukin-10-dependent manner.

OBJECTIVE: Intravenous immunoglobulin (IVIG) is used as a therapeutic agent in various autoimmune diseases. The aims of this study were to investigate the therapeutic effects of IVIG on collagen-induced arthritis (CIA) and identify the mechanism responsible for any therapeutic effects. METHODS: IVIG was administered to mice with CIA, and the in vivo effects were determined. Th17 and Treg cell frequencies were analyzed by flow cytometry, and cytokine levels in the supernatant were measured by enzyme-linked immunosorbent assay. Subpopulations of T cells and B cells in the spleen were assessed by confocal microscopy. RESULTS: The arthritis severity score and incidence of arthritis were lower in mice treated with IVIG compared with untreated mice. Histopathologic analysis showed less joint damage in mice treated with IVIG. The expression of proinflammatory cytokines, specific type II collagen antibodies, and osteoclast markers was significantly reduced in mice treated with IVIG. Administration of IVIG induced increased FoxP3 expression and inhibited Th17 cell development. The number of FoxP3+ Treg cells was increased, and the number of Th17 cells was decreased in the spleens of mice treated with IVIG. The number of FoxP3+ follicular helper T cells was increased, and subsequent maturation of germinal center B cells was inhibited by IVIG. In addition, IVIG up-regulated interleukin-10 (IL-10) and Fcγ receptor IIB expression. The treatment effects of IVIG on arthritis were lost in IL-10-knockout mice. CONCLUSION: These results showed that IVIG has therapeutic effects by modulating CD4+ T cell differentiation. The therapeutic effects of IVIG are dependent on IL-10.

IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation.

INTRODUCTION: Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process. METHODS: Bcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay. RESULTS: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP). CONCLUSIONS: Our data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.