Interleukin-17 increases the expression of Toll-like receptor 3 via the STAT3 pathway in rheumatoid arthritis fibroblast-like synoviocytes.

We examined the effect of interleukin-17 (IL-17) on the expression of Toll-like receptors (TLRs) in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). We investigated the region downstream of IL-17 for TLR expression. We also investigated the downstream signals responsible for the effect of IL-17 in TLR expression. Levels of IL-17 protein in the serum and synovial fluid of RA and OA patients were measured by ELISA. The IL-17 mRNA expression in peripheral blood mononuclear cells and synovial fluid mononuclear cells was measured by RT-PCR. RA and OA FLS were incubated with IL-17 and/or IL-23 for 24 hr. To block the signal transducer and activator of transcription 3 (STAT3) pathway, FLS were treated with S3I-201 before incubation with IL-17 and IL-23. Synovial tissue samples from RA and OA patients were stained with antibodies to IL-17, TLR2, TLR3, TLR4, STAT3 and phospho-STAT3. Levels of IL-17 protein were higher in the serum and synovial fluid from RA patients compared with those from OA patients. The IL-17 mRNA expression in synovial fluid monocytes was also higher in RA than in OA patients. Immunohistochemical staining showed greater expression of IL-17, TLR2, TLR3 and TLR4 in synovial samples from RA compared with OA patients. Interleukin-17 increased the expression of TLR2, TLR3 and TLR4 in RA FLS; IL-23 augmented the IL-17-induced expression of TLR2, TLR3 and TLR4 in RA FLS. Blocking STAT3 with S3I-201 reduced IL-17-induced TLR3 expression in RA FLS. Our results suggest that IL-17 is a major cytokine in pathogenesis on RA. The IL-17 influences the innate immune system by increasing the synovial expression of TLR2, TLR3 and TLR4. We may control TLR3 expression via the STAT3 pathway in RA FLS.

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts.

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNAmediated knockdown of MyD88.

Grape seed proanthocyanidin extract (GSPE) differentially regulates Foxp3(+) regulatory and IL-17(+) pathogenic T cell in autoimmune arthritis.

Grape seed proanthocyanidin extract (GSPE), which is the antioxidant derived from grape seeds, has been reported to possess a variety of potent properties. We have previously shown that GSPE attenuates collagen-induced arthritis. However the mechanism by which GSPE regulates the immune response remains unclear, although it may involve effects on the regulation of pathogenic T cells in autoimmune arthritis. To clarify this issue, we have assessed the effects of GSPE on differential regulation of Th17 and regulatory T (Treg) cells subsets in vitro in mouse and human CD4(+) T cells. We observed that GSPE decreased the frequency of IL-17(+)CD4(+)Th17 cells and increased induction of CD4(+)CD25(+)forkhead box protein 3 (Foxp3)(+) Treg cells. In vivo, GSPE effectively attenuated clinical symptoms of established collagen-induced arthritis in mice with concomitant suppression of IL-17 production and enhancement of Foxp3 expression (type II collagen-reactive Treg cells) in CD4(+) T cells of joints and splenocytes. The presence of GSPE decreased the levels of IL-21, IL-22, IL-26 and IL-17 production by human CD4(+) T cells in a STAT3-dependent manner. In contrast, GSPE induces Foxp3(+) Treg cells in humans. Our results suggest that GSPE possesses a reciprocal control over IL-17 and Foxp3. By potently regulating inflammatory T cell differentiation, GSPE may serve as a possible novel therapeutic agent for inflammatory and autoimmune diseases, including rheumatoid arthritis.

Induction of macrophage migration inhibitory factor in ConA-stimulated rheumatoid arthritis synovial fibroblasts through the P38 map kinase-dependent signaling pathway.

BACKGROUND/AIMS: This study was undertaken to identify the intracellular signaling pathway involved in induction of macrophage migration inhibitory factor (MIF) in human rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Human RA synovial fibroblasts were treated with concanavalin A (ConA), various cytokines, and inhibitors of signal transduction molecules. The production of MIF by synovial fibroblasts was measured in culture supernatants by ELISA. The expression of MIF mRNA was determined using reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. Phosphorylation of p38 mitogen-activated protein (MAP) kinase in synovial fibroblasts was confirmed using Western blotting. The expression of MIF and p38 MAP kinase in RA synovium was determined using dual immunohistochemistry. RESULTS: The production of MIF by RA synovial fibroblasts increased in a dose-dependent manner after ConA stimulation. MIF was also induced by interferon-γ, CD40 ligand, interleukin-15, interleukin-1ß, tumor necrosis factor-α, and transforming growth factor-ß. The production of MIF by RA synovial fibroblasts was significantly reduced after inhibition of p38 MAP kinase. The expression of MIF and p38 MAP kinase was upregulated in the RA synovium compared with the osteoarthritis synovium. CONCLUSIONS: These results suggest that MIF production was induced through a p38 MAP-kinase-dependent pathway in RA synovial fibroblasts.

IL-10 suppresses Th17 cells and promotes regulatory T cells in the CD4+ T cell population of rheumatoid arthritis patients.

Interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including autoimmune arthritis. IL-10 promotes the generation of Foxp3(+) regulatory T cells via the IL-10 receptor signal. The objective of this study was to examine whether IL-10, which acts as an anti-inflammatory cytokine, has a suppressive effect on the activation of human Th17 cells. Expression of IL-17 and IL-10 was examined immunohistochemically in tissue obtained from rheumatoid arthritis patients. Human peripheral blood CD4(+) T cells were isolated and cultured under various stimulatory conditions. Th17 cells and regulatory T (Treg) cells were detected by flow cytometry. The gene expression of related cytokines and transcription factors were assessed by ELISA and RT-PCR. IL-17 was overexpressed in rheumatoid arthritis patients. IL-10 treatment significantly decreased the numbers of IL-17-producing and RORc-expressing cells among human CD4(+) T cells that had been activated in vitro by Th17-differentiating conditions in autoimmune arthritis patients. IL-10 induced Foxp3(+) regulatory T cells in the human CD4(+) T cell population. Our results demonstrate that IL-17 is overexpressed in autoimmune disease patients and that IL-10 suppresses IL-17 expression. IL-10 may be useful in the treatment of autoimmune diseases.

TLR-3 enhances osteoclastogenesis through upregulation of RANKL expression from fibroblast-like synoviocytes in patients with rheumatoid arthritis.

This study was undertaken to determine the effect of toll-like receptor-3 (TLR3) on the regulation of osteoclastogenic activity in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). The expression of receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and protein in RA-FLS after TLR3 activation was determined using RT-PCR, real-time PCR, western blot analysis, and immunohistochemistry. Human monocytes were cocultured with RA-FLS that had been prestimulated by the TLR3 ligand polyriboinosinic-polyribocytidylic acid and then stained for tartrate-resistant acid phosphatase (TRAP) activity. Other markers of osteoclasts were measured using RT-PCR and real-time PCR. The expression of TLR3 and RANKL was much higher in the RA synovium than in the osteoarthritis (OA) synovium. TLR3 activation induced RANKL expression in RA-FLS, but not in OA-FLS or in normal skin fibroblasts. TLR3 activation also induced the production of IL-1beta but had no effect on IL-17 or TNF-alpha production in RA-FLS. Inhibition of IL-1beta reversed the TLR3-induced upregulation of RANKL expression. Coculture of human monocytes with TLR3-activated RA-FLS or TLR3 ligand-stimulated human monocytes increased the expression of TRAP, RANK, cathepsin K, calcitonin receptor, and MMP-9, reflecting the differentiation of monocytes into osteoclasts. Our results suggest that TLR3 promotes osteoclastogenesis in the RA synovium both directly and indirectly. TLR3 stimulates human monocytes directly to promote osteoclast differentiation. TLR3 induces RANKL expression indirectly in RA-FLS, and the expression of RANKL promotes the differentiation of osteoclasts in the RA synovium. Targeting the TLR3 pathway may be a promising approach to preventing inflammatory bone destruction in RA.