Jun-Geol Ryu

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

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).

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

IntroductionFibroblast-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.MethodsBcl-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.ResultsThe 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).ConclusionsOur 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.

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.

In vivo action of IL-27: reciprocal regulation of Th17 and Treg cells in collagen-induced arthritis

Interleukin (IL)-27 is a novel cytokine of the IL-6/IL-12 family that has been reported to be involved in the pathogenesis of autoimmune diseases and has a pivotal role as both a pro- and anti-inflammatory cytokine. We investigated the in vivo effects of IL-27 on arthritis severity in a murine collagen-induced arthritis (CIA) model and its mechanism of action regarding control of regulatory T (Tregs) and IL-17-producing T helper 17 (Th17) cells. IL-27-Fc-treated CIA mice showed a lower severity of arthritis. IL-17 expression in the spleens was significantly decreased in IL-27-Fc-treated CIA mice compared with that in the CIA model. The Th17 population was decreased in the spleens of IL-27-Fc-treated CIA mice, whereas the CD4+CD25+Foxp3+ Treg population increased. In vitro studies revealed that IL-27 inhibited IL-17 production in murine CD4+ T cells, and the effect was associated with retinoic acid-related orphan receptor γT and signal transducer and activator of transcription 3 inhibition. In contrast, fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) and IL-10 were profoundly augmented by IL-27 treatment. Regarding the suppressive capacity of Treg cells, the proportions of CTLA-4+ (cytotoxic T-lymphocyte antigen 4), PD-1+ (programmed cell death protein 1) and GITR+ (glucocorticoid-induced tumor necrosis factor receptor) Tregs increased in the spleens of IL-27-Fc-treated CIA mice. Furthermore, in vitro differentiated Treg cells with IL-27 exerted a more suppressive capacity on T-cell proliferation. We found that IL-27 acts as a reciprocal regulator of the Th17 and Treg populations in CD4+ cells isolated from healthy human peripheral blood mononuclear cells (PBMCs), as well as from humans with rheumatoid arthritis (RA) PBMCs. Our study suggests that IL-27 has the potential to ameliorate overwhelming inflammation in patients with RA through a reciprocal regulation of Th17 and Treg cells.

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.

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-1β, 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.

Gene Associated With Retinoid-Interferon-Induced Mortality 19 Attenuates Murine Autoimmune Arthritis by Regulation of Th17 and Treg Cells

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.

Intravenous Immunoglobulin Attenuates Experimental Autoimmune Arthritis by Inducing Reciprocal Regulation of Th17 and Treg Cells in an Interleukin-10–Dependent Manner

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.

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.

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.