Small heterodimer partner interacting leucine zipper protein (SMILE) ameliorates autoimmune arthritis via AMPK signaling pathway and the regulation of B cell activation.

Rheumatoid arthritis (RA) is an autoimmune disease that causes joint swelling and inflammation and can involve the entire body. RA is characterized by the increase of pro-inflammatory cytokines such as interleukin (IL) and tumor necrosis factor, and the over-activation of T lymphocytes and B lymphocytes, which may lead to severe chronic inflammation of joints. However, despite numerous studies the pathogenesis and treatment of RA remain unresolved. This study investigated the use of small heterodimer partner-interacting leucine zipper protein (SMILE) overexpression to treat a mouse model of RA. SMILE is an insulin-inducible corepressor through adenosine monophosphate-activated kinase (AMPK) signaling pathway. The injection of a SMILE overexpression vector to mice with collagen induced-arthritis resulted in a milder clinical pathology and a reduced incidence of arthritis, less joint tissue damage, and lower levels of Th17 cells and plasma B cells in the spleen. Immunohistochemistry of the joint tissue showed that SMILE decreased B-cell activating factor (BAFF) receptor (BAFF-R), mTOR, and STAT3 expression but increased AMPK expression. In SMILE-overexpressing transgenic mice with collagen antibody-induced arthritis (CAIA), a decrease in the arthritis score and reductions in tissue damage, the number of B cells, and antibody production were observed. The treatment of immune cells in vitro with curcumin, a known SMILE-inducing agent, led to decreases in plasma B cells, germinal center B cells, IL-17-producing B cells, and BAFF-R-positive B cells. Taken together, our findings demonstrate the therapeutic potential of SMILE in RA, based on its inhibition of B cell activation mediated by the AMPK/mTOR and STAT3 signaling pathway and BAFF-R expression. Video abstract.

Rebamipide treatment ameliorates obesity phenotype by regulation of immune cells and adipocytes.

Obesity is a medical term used to describe an over-accumulation of adipose tissue. It causes abnormal physiological and pathological processes in the body. Obesity is associated with systemic inflammation and abnormalities in immune cell function. Rebamipide, an amino acid derivative of 2-(1H)-quinolinone, has been used as a therapeutic for the protection from mucosal damage. Our previous studies have demonstrated that rebamipide treatment regulates lipid metabolism and inflammation, leading to prevention of weight gain in high-fat diet mice. In this study, mice were put on a high calorie diet for 11 weeks while receiving injections of rebamipide. Rebamipide treatment reduced the body weight, liver weight and blood glucose levels compared to control mice and reduced both glucose and insulin resistance. Fat accumulation has been shown to cause pro-inflammatory activity in mice. Treatment with rebamipide decreased the prevalence of inflammatory cells such as Th2, Th17 and M1 macrophages and increased anti-inflammatory Treg and M2 macrophages in epididymal fat tissue. Additionally, rebamipide addition inhibited adipocyte differentiation in 3T3-L1 cell lines. Taken together, our study demonstrates that rebamipide treatment is a novel and effective method to prevent diet-induced obesity.

Terahertz Spectral Properties of PEO-Based Anti-Adhesion Films Cross-Linked by Electron Beam Irradiation.

We investigated the spectral property changes in anti-adhesion films, which were cross-linked and surface-modified through electron beam irradiation, using terahertz time-domain spectroscopy (THz-TDS). Polyethylene oxide (PEO), which is a biocompatible and biodegradable polymer, was the main component of these anti-adhesion films being manufactured for testing. The terahertz characteristics of the films were affected by the porosity generated during the freeze-drying and compression processes of sample preparation, and this was confirmed using optical coherence tomography (OCT) imaging. An anti-adhesion polymer film made without porosity was measured by using the THz-TDS method, and it was confirmed that the refractive index and absorption coefficient were dependent on the crosslinking state. To our knowledge, this is the first experiment on the feasibility of monitoring cross-linking states using terahertz waves. The THz-TDS method has potential as a useful nondestructive technique for polymer inspection and analysis.

Correction: The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines.

[This corrects the article DOI: 10.1371/journal.pone.0245986.].

Enhanced Polymerization and Surface Hardness of Colloidal Siloxane Films via Electron Beam Irradiation.

Electron beam (EB) curing is a foldable hard coating process and has attracted significant research attention in the field of flexible electronic devices. In this study, we report a method for enhancing material surface hardness with low-energy EB curing in a short time. The low-energy EB improved the coating hardness of films by inducing cross-linking polymerization of the silicon-containing monomer. The hardness of the cured coating layer was measured as 3 H using a pencil hardness tester, and the transparency of the coating was higher than 90%. Owing to a series of cross-linking reactions between Si-O-C and Si-OH groups under EB curing and the formation of Si-Si bonds, the cured layer exhibited remarkable durability in the 100000-flexible cycle test. Additionally, the natural oxidation of the C-O groups on the surface of the coating formed carboxyl groups that improved the hydrophilic properties of the coating layer. To the best of our knowledge, this is the first study to propose that the hardness of polyethylene terephthalate films can be improved using low-energy EBs to rapidly cure silicon-containing coatings. Our results provide a novel and commercially viable approach for improving the hardness of touch screens and foldable displays.

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor that plays a pivotal role in cellular defense against oxidative injury. Nrf2 signaling is involved in attenuating autoimmune disorders such as rheumatoid arthritis (RA). B cells play several roles in the pathogenesis of RA, such as in autoantibody production, antigen presentation, and T-cell activation. We investigated the anti-arthritic mechanisms of sulforaphane, an activator of Nrf2, in terms of its effect on B cells. To investigate the effect of sulforaphane on collagen-induced arthritis (CIA), sulforaphane was administered intraperitoneally after CIA induction. Hematoxylin and eosin-stained sections were scored for inflammation, pannus invasion, and bone and cartilage damage. We assessed the expression levels of inflammation-related factors by real-time PCR and the levels of various IgG subclasses by enzyme-linked immunosorbent assay. Sulforaphane treatment reduced the arthritis score and the severity of histologic inflammation in CIA mice. The joints from sulforaphane-treated CIA mice showed decreased expression of interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, receptor activator of NF-κB ligand, and tartrate-resistant acid phosphatase. Sulforaphane-treated mice showed lower circulating levels of type-II-collagen-specific IgG, IgG1, and IgG2a. In vitro, sulforaphane treatment significantly reduced the differentiation of lipopolysaccharide-stimulated murine splenocytes into plasma B cells and germinal-center B cells. Finally, sulforaphane significantly inhibited the production of IL-6, TNF-α, and IL-17 by human peripheral blood mononuclear cells stimulated with an anti-CD3 monoclonal antibody in a dose-dependent manner. Inhibition of differentiation into plasma B and Germinal Center B cells may be the mechanism underlying the anti-arthritic effect of sulforaphane.

Effectiveness and safety of biologic and targeted synthetic disease-modifying anti-rheumatic drugs in elderly patients with rheumatoid arthritis: real-world data from the KOBIO Registry.

OBJECTIVES: We aimed to evaluate the clinical outcomes and safety of biologic and targeted synthetic disease-modifying anti-rheumatic drugs (b/tsDMARDs) and to identify predictors of treatment responses to b/tsDMARDs in elderly patients with rheumatoid arthritis (RA). METHODS: Data from the nationwide cohort of elderly (≥ 65 years) patients enrolled in the KOBIO Registry were analysed. Clinical outcomes were assessed, including changes in the Simplified Disease Activity Index, after treatment. Adverse events and reasons for drug discontinuation were assessed. Multivariable logistic regression analyses were performed to determine which baseline variables affected treatment responses and adverse events (AE). RESULTS: Elderly patients treated with b/tsDMARDs (n=355) or conventional synthetic DMARDs (csDMARDs) (n=104) were included. The median age was 70 years and 77% were female. After 1 year, 63% of patients in the b/tsDMARD group and 68% in the csDMARD group achieved remission or low disease activity (LDA). Overall, 27% of patients in the b/tsDMARDs group and 24% in the csDMARDs group experienced AE. A total of 43.4% of patients on b/tsDMARDs discontinued therapy due to lack of effectiveness (27%), AE (34%), or other reasons (35%). The estimated median retention of b/tsDMARDs was 2.5 years. Male sex and non-exposure to tobacco at baseline were independent factors associated with achieving remission or LDA after 1 year. Interstitial lung disease (ILD) was the most prominent comorbidity associated with AE. CONCLUSIONS: Treatment with b/tsDMARDs is effective and well tolerated in elderly patients with RA; nonetheless, ILD is a key comorbidity that should be monitored carefully.

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

OBJECTIVE: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. METHODS: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1ß (IL-1ß) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. RESULTS: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of ß-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between ß-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of ß-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, ß-carotene reduced IL-1ß secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. CONCLUSION: Our results present ß-carotene as a selective and direct inhibitor of NLRP3, and the binding of ß-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.

Retinoic Acid Receptor-Related Receptor Alpha Ameliorates Autoimmune Arthritis via Inhibiting of Th17 Cells and Osteoclastogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis characterized by progressive joint destruction. IL-17-producing CD4+ T (Th17) cells play pivotal roles in RA development and progression. Retinoic acid receptor-related orphan receptor alpha (RORα) is a negative regulator of inflammatory responses, whereas RORγt, another member of the ROR family, is a Th17 lineage-specific transcription factor. Here, we investigated the immunoregulatory potential of RORα in collagen-induced arthritis (CIA) mice, an experimental model of RA. Cholesterol sulfate (CS) or SR1078, a ligand of RORα, inhibited RORγt expression and Th17 differentiation in vitro. In addition, fortification of RORα in T cells inhibited the expression levels of glycolysis-associated genes. We found that RORα overexpression in CIA mice attenuated the clinical and histological severities of inflammatory arthritis. The anti-arthritic effect of RORα was associated with suppressed Th17 differentiation and attenuated mTOR-STAT3 signaling in T cells. Furthermore, altered RORα activity could directly affect osteoclastogenesis implicated in progressive bone destruction in human RA. Our findings defined a critical role of RORα in the pathogenesis of RA. These data suggest that RORα may have novel therapeutic uses in the treatment of RA.

Daurinol Attenuates Autoimmune Arthritis via Stabilization of Nrp1-PTEN-Foxp3 Signaling in Regulatory T Cells.

Optimizing Treg function and improving Treg stability are attractive treatment strategies for treating autoimmune rheumatoid arthritis (RA). However, the limited number of circulating Tregs and questions about the functional stability of in vitro-expanded Tregs are potential limitations of Treg-based cell therapy. The aim of this study was to analyze the regulatory effect of daurinol, a catalytic inhibitor of topoisomerase IIα, on Th cell differentiation and to evaluate their therapeutic potential in a preclinical experimental model of RA. We investigated the effect of daurinol on T cell differentiation by flow cytometry. Foxp3 stability and methylation were analyzed by suppression assays and bisulfite pyrosequencing. Daurinol was treated in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. We found that daurinol can promote Treg differentiation and reciprocally inhibit Th17 differentiation. This Treg-inducing property of daurinol was associated with decreased activity of Akt-mTOR and reciprocally increased activity of neuropilin-1 (Nrp1)-PTEN. Daurinol treatment inhibited aerobic glycolysis in Th17 conditions, indicating the metabolic changes by daurinol. We found that the daurinol increase the Treg stability was achieved by Foxp3 hypomethylation. In vivo daurinol treatment in CIA mice reduced the clinical arthritis severity and histological inflammation. The Treg population frequency increased and the Th17 cells decreased in the spleens of arthritis mice treated with daurinol. These results showed the anti-arthritic and immunoregulating properties of daurinol is achieved by increased differentiation and stabilization of Tregs. Our study provides first evidence for daurinol as a treatment for RA.

Metformin Augments Anti-Inflammatory and Chondroprotective Properties of Mesenchymal Stem Cells in Experimental Osteoarthritis.

Mesenchymal stem cells (MSCs) can protect against cartilage breakdown in osteoarthritis (OA) via their immunomodulatory capacities. However, the optimization strategy for using MSCs remains challenging. This study's objective was to identify the in vivo effects of metformin-stimulated adipose tissue-derived human MSCs (Ad-hMSCs) in OA. An animal model of OA was established by intra-articular injection of monosodium iodoacetate into rats. OA rats were divided into a control group and two therapy groups (treated with Ad-hMSCs or metformin-stimulated Ad-hMSCs). Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Our data show that metformin increased IL-10 and IDO expression in Ad-hMSCs and decreased high-mobility group box 1 protein, IL-1ß, and IL-6 expression. Metformin increased the migration capacity of Ad-hMSCs with upregulation of chemokine expression. In cocultures, metformin-stimulated Ad-hMSCs inhibited the mRNA expression of RUNX2, COL X, VEGF, MMP1, MMP3, and MMP13 in IL-1ß-stimulated OA chondrocytes and increased the expression of TIMP1 and TIMP3. The antinociceptive activity and chondroprotective effects were greater in OA rats treated with metformin-stimulated Ad-hMSCs than in those treated with unstimulated Ad-hMSCs. TGF-ß expression in subchondral bone of OA joints was attenuated more in OA rats treated with metformin-stimulated Ad-hMSCs. Our findings suggest that metformin offers a promising option for the clinical application of Ad-hMSCs as a cell therapy for OA.

Soaking Properties of Integrated Gasification Combined Cycle Slag-Based-Geopolymers and Their Nanometer-Scale Microstructures.

In this study, non-foamed and foamed geopolymers based on IGCC (integrated gasification combined cycle) slag were prepared and their water resistance properties were examined in an immersion test in order to assess their suitability for water applications such as bridge pier, seawall, tetrapod. Particularly, the immersion characteristics were investigated according to the aging time at room temperature during the production process of the geopolymer. The pH changes of the immersion liquid were analyzed considering the immersion time, and the microstructural changes on the nanometer level of the surface of the geopolymer were observed before and after the immersion. Various shapes of crystals of the zeolite phase formed in the geopolymers prepared in this study, such as needle-like crystals with thicknesses ranging from 20 to 40 nm and lengths of 200 to 300 nm, and plate-shaped crystals overlapped with each other, which is thought to be due to the uneven composition of the slag used as a raw material or to local compositional changes of the alkali activator. By measuring the pH of immersion liquid, the following facts can be seen: foamed geopolymers synthesized from IGCC slag require a room-temperature curing period of at least 7 days, whereas non-foamed specimens require a room-temperature curing period of at least 28 days to consume alkaline activators. This is due to the increase in the specific surface area of the foamed specimen as the specimen is expanded, which thereby increases the contact area between the alkali activator and the slag. The foamed specimens, however, produced larger cracks and pores when immersed in water, while a smaller cracks and pores occurred in the non-foamed specimens.

RIPK1 inhibition attenuates experimental autoimmune arthritis via suppression of osteoclastogenesis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease characterized by upregulation of inflammatory cell death and osteoclastogenesis. Necrostatin (NST)-1s is a chemical inhibitor of receptor-interacting serine/threonine-protein kinase (RIPK)1, which plays a role in necroptosis. METHODS: We investigated whether NST-1s decreases inflammatory cell death and inflammatory responses in a mouse model of collagen-induced arthritis (CIA). RESULTS: NST-1s decreased the progression of CIA and the synovial expression of proinflammatory cytokines. Moreover, NST-1s treatment decreased the expression of necroptosis mediators such as RIPK1, RIPK3, and mixed lineage kinase domain-like (MLKL). In addition, NST-1s decreased osteoclastogenesis in vitro and in vivo. NST-1s downregulated T helper (Th)1 and Th17 cell expression, but promoted Th2 and regulatory T (Treg) cell expression in CIA mice. CONCLUSIONS: These results suggest that NST-1s attenuates CIA progression via the inhibition of osteoclastogenesis and might be a potential therapeutic agent for RA therapy.

IL-1-IL-17 Signaling Axis Contributes to Fibrosis and Inflammation in Two Different Murine Models of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a progressive fibrotic disease that affects the skin and internal organs. Despite evidence implicating increased interleukin-17 (IL-17) activity in SSc, the role of IL-17 in SSc remains uncertain. The purpose of this study was to investigate whether IL-17 plays a pathophysiological role in SSc in two different murine models of SSc. METHODS: Bleomycin (BLM)-induced fibrosis and chronic graft-versus-host disease (cGVHD) models were used. Histological analysis was performed using Masson's trichrome and immunohistochemical staining. Quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunoassays were used to quantify the messenger RNA and protein levels of inflammatory mediators in dermal fibroblasts. RESULTS: IL-1 receptor antagonist-deficient (IL-1Ra-KO) mice were more severely affected by BLM injection, as shown by dermal and pulmonary fibrosis, compared with wild-type (WT) mice. Increased tissue fibrosis was reversed by knocking down IL-17. In vitro experiments showed that IL-1 and IL-17 exerted synergistic effects on the expression of profibrotic and inflammatory mediators. In the cGVHD model, C57BL/6 mice receiving splenocytes of IL-1Ra-KO BALB/c mice developed more severe cGVHD than did those receiving cells from WT mice. Knockdown of IL-17 in IL-1Ra-KO donor mice significantly attenuated the IL-1-induced acceleration of cGVHD severity. CONCLUSION: Targeting IL-1 and its downstream IL-17 activity may be a novel treatment strategy for inhibiting inflammation and tissue fibrosis in SSc.

Differences in quality of life determinants according to the presence of fibromyalgia in middle-aged female patients with systemic lupus erythematosus: a multicenter, cross-sectional, single-ethnicity cohort.

OBJECTIVE: The purpose of this study was to identify whether determinants of health-related quality of life (HRQoL) in middle-aged female patients with systemic lupus erythematosus (SLE) differed according to the presence or absence of fibromyalgia. METHODS: One hundred and fifty-two patients with SLE and 139 healthy controls (HCs) completed the Medical Outcomes Study 36-Item Short Form (SF-36) and EuroQol EQ-5D questionnaires about HRQoL. Disease activity and cumulative disease damage were assessed with standard indices. Sleep quality was assessed using the Korean version of the Pittsburgh Sleep Quality Index (K-PSQI). RESULT: The mean EQ-5D and physical and mental components of SF-36 were lower in SLE patients with fibromyalgia (n = 41) than in those without fibromyalgia (n = 111) and HCs. The scores in all eight domains of the SF-36 were lower in SLE patients with fibromyalgia than in patients without fibromyalgia and HCs. Poor sleep (defined as a K-PSQI > 5) was reported by 85% of SLE patients with fibromyalgia, by 51% of patients without fibromyalgia, and by 33% of HCs. Multivariate logistic regression analysis showed that lower educational level, cumulative organ damage severity and poor sleep quality were independent determinants of HRQoL in SLE patients with fibromyalgia, whereas disease activity, sleep quality and depressive mood were independent determinants of HRQoL in those without fibromyalgia. CONCLUSION: Poor sleep quality is the common independent risk factor for poor HRQoL in both middle-aged SLE patients with fibromyalgia and without fibromyalgia. Sleep quality improvement may improve HRQoL in female SLE patients, even in those without fibromyalgia.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3-RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin ß3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3-RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

Therapeutic Effect of Exogenous Truncated IK Protein in Inflammatory Arthritis.

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562, a leukemia cell line. It is known to be an inhibitory regulator of interferon-γ-induced major histocompatibility complex class (MHC) II expression. Previously, we found that transgenic (Tg) mice constitutively expressing truncated IK (tIK) showed reduced numbers of pathogenic Th1 and Th17 cells, which are known to be involved in the development of rheumatoid arthritis (RA). Here, we investigated whether exogenous tIK protein has a therapeutic effect in arthritis in disease models and analyzed its mechanism. Exogenous tIK protein was produced in an insect expression system and applied to the collagen antibody-induced arthritis (CAIA) mouse disease model. Injection of tIK protein alleviated the symptoms of arthritis in the CAIA model and reduced Th1 and Th17 cell populations. In addition, treatment of cultured T cells with tIK protein induced expression of A20, a negative regulator of nuclear factor-κB (NFκB)-induced inflammation, and reduced expression of several transcription factors related to T cell activation. We conclude that exogenous tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action to biopharmaceutical agents, such as tumor necrosis factor antagonists, that are currently used to treat RA.

Regulator of Calcineurin 3 Ameliorates Autoimmune Arthritis by Suppressing Th17 Cell Differentiation.

Regulator of calcineurin 3 (RCAN3), an endogenous regulator of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, inhibits the phosphatase activity of calcineurin, the nuclear translocation of NFAT, and the NFAT downstream pathway. To investigate the effects of RCAN3 on T-cell regulatory function and the development and progression of inflammatory arthritis, we studied the effects of RCAN3 transfection on regulation of Th17 cell differentiation in a murine T-lymphoma cell line and primary splenic CD4+ T cells. Overexpression of RCAN3 suppressed Th17 cell differentiation through the down-regulation of RAR receptor orphan receptor γT mRNA and up-regulation of forkhead box P3 mRNA. In mice with collagen-induced arthritis, injection of an RCAN3-overexpression vector controlled arthritis development in vivo. Injection of RCAN3 reduced the formation of osteoclasts and expression of inflammatory cytokines in vivo. Antioxidants stimulated the expression of RCAN3 in vitro, and combination therapy with pcDNA-RCAN3 had a synergistic suppressive effect on the development of arthritis. These data suggest that RCAN3 may be an effective treatment for rheumatoid arthritis.

Metformin Suppresses Systemic Autoimmunity in Roquinsan/san Mice through Inhibiting B Cell Differentiation into Plasma Cells via Regulation of AMPK/mTOR/STAT3.

Circulating autoantibodies and immune complex deposition are pathological hallmarks of systemic lupus erythematosus (SLE). B cell differentiation into plasma cells (PCs) and some T cell subsets that function as B cell helpers can be therapeutic targets of SLE. Mechanistic target of rapamycin (mTOR) signaling is implicated in the formation of B cells and germinal centers (GCs). We assessed the effect of metformin, which inhibits mTOR, on the development of autoimmunity using Roquinsan/san mice. Oral administration of metformin inhibited the formation of splenic follicles and inflammation in kidney and liver tissues. It also decreased serum levels of anti-dsDNA Abs without affecting serum glucose levels. Moreover, metformin inhibited CD21highCD23low marginal zone B cells, B220+GL7+ GC B cells, B220-CD138+ PCs, and GC formation. A significant reduction in ICOS+ follicular helper T cells was found in the spleens of the metformin-treated group compared with the vehicle-treated group. In addition, metformin inhibited Th17 cells and induced regulatory T cells. These alterations in B and T cell subsets by metformin were associated with enhanced AMPK expression and inhibition of mTOR-STAT3 signaling. Furthermore, metformin induced p53 and NF erythroid-2-related factor-2 activity in splenic CD4+ T cells. Taken together, metformin-induced alterations in AMPK-mTOR-STAT3 signaling may have therapeutic value in SLE by inhibiting B cell differentiation into PCs and GCs.

The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1.

BACKGROUND: Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice. METHODS: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1ß, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting. RESULTS: A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1ß. The serum levels of TNF-α, IL-1ß, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups. CONCLUSION: The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.