Metformin ameliorates experimental-obesity-associated autoimmune arthritis by inducing FGF21 expression and brown adipocyte differentiation

Rheumatoid arthritis (RA) is a systemic autoimmune disease involving excessive inflammation. Recently, RA associated with a metabolic disorder was revealed to be non-responsive to RA medications. Metformin has been reported to have a therapeutic effect on RA and obesity. The aim of this investigation was to study the therapeutic effect and the underlying mechanism of metformin's action in an experimental model of collagen-induced arthritis (CIA) associated with obesity. Metformin was administered daily for 13 weeks to mice with CIA that had been fed a high-fat diet. Metformin ameliorated the development of CIA in obese mice by reducing autoantibody expression and joint inflammation. Furthermore, metformin decreased the expression levels of pSTAT3 and pmTOR and had a small normalizing effect on the metabolic profile of obese CIA mice. In addition, metformin increased the production of pAMPK and FGF21. Metformin also induced the differentiation of brown adipose tissue (BAT), which led to a reciprocal balance between T helper (Th) 17 and regulatory T (Treg) cells in vitro and in vivo. These results suggest that metformin can dampen the development of CIA in obese mice and reduce metabolic dysfunction by inducing BAT differentiation. Thus, metformin could be a therapeutic candidate for non-responsive RA.

Interleukin-17 Enhances Germinal Center Formation and Immunoglobulin G1 Production in Mice

OBJECTIVE: Interleukin (IL)-17 is a pro-inflammatory cytokine that has pleiotropic effects on multiple target cells and thereby contributes to the development of immune-mediated inflammatory disorders. However, the role of IL-17 in the humoral immune response has not been clearly elucidated. METHODS: Mice deficient in IL-17A (IL-17A knockout [KO] mice) and wild type (WT) C57BL/6 mice were compared. Distinct B cell (mature/precursor and marginal zone/follicular) and plasma cell populations were compared using fluorescence-activated cell sorting (FACS) and confocal immunostaining. Immunoglobulin production was assessed by enzyme-linked immunosorbent assay. RESULTS: There was no difference in B cell and plasma cell populations between IL-17A KO and WT mice. However, after T cell-dependent antigen challenge, IL-17A KO mice produced lower levels of immunoglobulin (Ig)G1 than wild-type animals. IL-17A KO mice also showed reduced germinal center (GC) formation and lower expression of activation-induced cytidine deaminase, the essential enzyme for class switch recombination (CSR). IL-17 had no effect on the proliferation or survival of naïve B cells in in vitro functional studies. However, IL-17 treatment promoted naïve B cell differentiation into plasma cells in synergy with IL-4, although IL-17 alone had no effect. CONCLUSION: Our findings suggest that IL-17 contributes to the humoral immune response by enhancing GC formation, CSR to IgG1, and plasma cell differentiation in synergy with IL-4.

Ursodeoxycholic Acid Ameliorates Pain Severity and Cartilage Degeneration in Monosodium Iodoacetate-Induced Osteoarthritis in Rats

Osteoarthritis (OA) is a degenerative joint disease characterized by a progressive loss of cartilage. And, increased oxidative stress plays a relevant role in the pathogenesis of OA. Ursodeoxycholic acid (UDCA) is a used drug for liver diseases known for its free radical-scavenging property. The objectives of this study were to investigate the in vivo effects of UDCA on pain severity and cartilage degeneration using an experimental OA model and to explore its mode of actions. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration UDCA was initiated on the day of MIA injection. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Samples were analyzed macroscopically and histologically. Immunohistochemistry was used to investigate the expression of interleukin-1beta (IL-1beta), IL-6, nitrotyrosine and inducible nitric oxide synthase (iNOS) in knee joints. UDCA showed an antinociceptive property and attenuated cartilage degeneration. OA rats given oral UDCA significantly exhibited a decreased number of osteoclasts in subchondral bone legion compared with the vehicle-treated OA group. UDCA reduced the expression of IL-1beta, IL-6, nitrotyrosine and iNOS in articular cartilage. UDCA treatment significantly attenuated the mRNA expression of matrix metalloproteinase-3 (MMP-3), -13, and ADAMTS5 in IL-1beta-stimulated human OA chondrocytes. These results show the inhibitory effects of UDCA on pain production and cartilage degeneration in experimentally induced OA. The chondroprotective properties of UDCA were achieved by suppressing oxidative damage and inhibiting catabolic factors that are implicated in the pathogenesis of cartilage damage in OA.

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-alpha. 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.

Regulation of B cell activating factor (BAFF) receptor expression by NF-kappaB signaling in rheumatoid arthritis B cells

B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of B cell activating factor (BAFF) are detected in autoimmune diseases. BAFF and BAFF receptor (BAFF-R) are expressed in B and T cells of RA synovium. The study was undertaken to identify the NF-kappaB signal pathway involved in the induction of BAFF-R in human B cells. Immunohistochemical staining of NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R was performed on sections of synovium from severe and mild RA and osteoarthritis (OA) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from control and RA patients and B cells were isolated from controls. BAFF-R was analyzed by flow cytometry, realtime PCR and confocal staining after treatment with NF-kappaB inhibitors. NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R were highly expressed in severe RA synovium relative to mild RA synovium or OA synovium. BAFF-R expression was reduced by NF-kappaB inhibitors in PBMCs and B cells from normal controls. We also showed reduction in expression of BAFF-R via inhibition of the NF-kappaB pathway in PBMCs of RA patients. BAFF/BAFF-R signaling is an important mechanism of pathogenesis in RA and that BAFF-R reduction by NF-kappaB blocking therapy is another choice for controlling B cells in autoimmune diseases such as RA.

The Marginal Zone B Cells have an Increased Antibody Expression in Mice with Collagen-induced Arthritis / 대한류마티스학회지

OBJECTIVE: Mature B cells in the spleen of mouse can be divide into two main subsets: the follicular (FO) B cells and the marginal zone (MZ) B cells. In this study, we investigated which subtype of B cells is involved in the production of costimulatory molecules, cytokine and antibody during the induction of autoimmune arthritis. METHODS: The MZB and FOB cells isolated from DBA/1J induced- and collagen-induced arthritis (CIA) mice were stimulated with LPS or CpG. The costimulatory molecules were measured by flow cytometry (FACs). The cytokines were measured by ELISA. Production of antibodies by the MZB cells or FOB cells was measured by ELISA and the results were observed by confocal microscopy. RESULTS: The expression of co-stimulatory molecules was stronger in the MZB cells than that in the FOB cells. The production of cytokines (IL-10, IL-6) and antibodies was higher in the MZB cells. The IgG expression of the MZB cells, which is known to be associated with the acceleration of autoimmunity, was higher in the CIA mice than that in the DBA/1J mice. CONCLUSION: We observed that the MZB cells were increased in the CIA mice. The costimulatory molecules, cytokine and auto-antibodies were increased in the MZB cells compared to that of the FOB cells. Our results suggest that MZB cells mainly produce autoantibodies, and they play a key role in development of autoimmune arthritis.

Expression of CCR2A, an isoform of MCP-1 receptor, is increased by MCP-1, CD40 ligand and TGF-beta in fibroblast like synoviocytes of patients with RA

Cytokine and chemokine receptors play a key role in inflammation caused by rheumatoid arthritis (RA). Two isoforms of human CC chemokine receptor R2 (CCR2), the receptor of monocyte chemoattractant protein 1 (MCP-1), have been identified but their relative expression in fibroblast-like synoviocytes (FLS) and their contribution to inflammatory responses mediated by MCP-1 or inflammatory cytokines in patients with RA remain uncertain. We examined the pattern of expression of two CCR2 isoforms upon stimulation by proinflammatory cytokines and CD40 ligation. FLS were prepared from the synovial tissues of RA patients and cultured in the presence of MCP-1, soluble CD40 ligand (sCD40L), TGF-beta, IL-1beta, IL-18, IL-15, and LPS. CCR2A and CCR2B expression was examined by immunohistochemistry, RT-PCR and western blot analysis. IL-15, TNF-alpha and MCP-1 production was determined by ELISA. Immunohistochemistry showed that CCR2A is highly expressed in RA synovium compared with OA synovium. Transcripts of both CCR2A and CCR2B were detected in FLS. Exogenous MCP-1, CD40L, TGF-beta, and IL-15 significantly increased the expression of CCR2A but not CCR2B. Exposure of FLS to sCD40L caused strong upregulation of CCR2A but not of CCR2B protein expression. MCP-1 increased the proliferation of FLS and the production of IL-15, TNF-alpha, and IL-18. Because CCR2A is the main target of regulation by cytokines and CD40 ligation, the relatively higher expression of CCR2A on the cell surface suggests that this isoform of MCP-1 receptor functions as the principal mediator of inflammatory signals in RA FLS.

Characterization of FSC(high) Memory B Cells from Patients with Active Systemic Lupus Erythematosus / 대한류마티스학회지

OBJECTIVE: To determine phenotypic and functional characteristics of memory B cells in patients with systemic lupus erythematosus (SLE). METHODS: The percentage of memory B cell subsets in peripheral blood mononuclear cells (PBMC) from normal control (n=11), inactive (n=15) and active (n=10) SLE patients was determined by Fluorescence Activated Cell Sorter (FACS). In addition, the activation status of memory B cells was measured by the surface expression of CD86 (B7-2). The production of antibodies to chromatin and dsDNA (IgG and IgM type) by isolated memory B cell subsets was examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In this study, we analyzed 2 subtypes of memory B cells: FSC (Forward Side Scatter)(low) and FSC(high) memory B cell. The percentage of both subtypes from active and inactive SLE patients was significantly reduced compared to that of normal controls (p<0.01). In addition, the expression of activation markers, CD86 on FSC(high) memory B cells from active SLE patients was higher than those of inactive SLE patients and normal controls (p=0.014). Upon stimulation with CpG and IL-15 in vitro for 8 days, isolated FSC(high) memory B cells from active SLE patients revealed augmented production of autoantibodies to chromatin and dsDNA. CONCLUSION: Our results suggest that abnormally activated FSC(high) memory B cells from active SLE patients might be involved in spontaneous production of autoantibodies and induce transition from inactive to active phase of the patients.

Anti-CD3 Antibody Induces IL-10-producing CD4+CD25+ Regulatory T Cells, Which Suppress T Cell Response in Rheumatoid Arthritis Patients

BACKGROUND: Regulatory T cells (Tregs) have been investigated intensively for some decades. These cells regulate the immune system, prevent overactivated immune responses and can be used therapeutically. For rheumatoid arthritis (RA), understanding the functions and status of Tregs is an important step for understanding immune regulation in this autoimmune disease. METHODS: We investigated the percentages, phenotypes and suppressive functions of CD4+CD25+ Tregs in peripheral blood (PB) of patients with RA. RESULTS: The percentages were higher in the patients (n=12) than in healthy controls (n=10), and the cells expressed the CD45RBlow, CTLA-4 and CCR7 phenotypes. We also investigated the expression of Foxp3 and secretion of interleukin (IL)-10 induced CD4+CD25+ Tcells by anti-CD3 antibody treatment. A suppressive function of the patients' cells was shown through coculture with CD4+CD25+ T cells in vitro. CONCLUSION: We suggest that, despite their increased numbers and suppressive function, they manage the ongoing inflammation ineffectively. It might be possible to apply IL-10 to induce the proliferation of IL-10-producing Tregs as therapy for RA.