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Objective We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. Methods Peripheral blood samples from RA patients were assessed using 3H-thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme-linked immunosorbent assay to determine Treg cell suppression of interferon-γ production. CTLA-4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin-2 (IL-2), IL-6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. Results Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX-exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA-4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. Conclusion This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA-4 expression. [ABSTRACT FROM AUTHOR]

Although strong binding interactions between protein receptor and ligand do not require the participation of a large number of amino acids in either site, short peptide chains are generally poor at recreating the types of protein-protein interactions which take place during cell recognition and signalling process, probably because their flexible backbones prevent the side chains from forming sufficiently rigid and stable epitopes, which can take part in binding with the desired strength and specificity. In a recently-reported study, it was shown that a proto-epitope containing F, R and S amino acids has the ability to down-regulate TNF secretion by macrophages. This paper extends these findings, putting those amino acids into a short cyclic peptide scaffold, and determining the optimal configuration required to overcome the problems of conformational instability, and give rise to molecules which have potential as therapeutic agents in human disease, such as rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Treg-cell function is compromised in rheumatoid arthritis (RA). As the master regulator of Treg cells, FOXP3 controls development and suppressive function. Stable Treg-cell FOXP3 expression is epigenetically regulated; constitutive expression requires a demethylated Treg-specific demethylated region. Here, we hypothesised that methylation of the FOXP3 locus is altered in Treg cells of established RA patients. Methylation analysis of key regulatory regions in the FOXP3 locus was performed on Treg cells from RA patients and healthy controls. The FOXP3 Treg-specific demethylated region and proximal promoter displayed comparable methylation profiles in RA and healthy-donor Treg cells. We identified a novel differentially methylated region (DMR) upstream of the FOXP3 promoter, with enhancer activity sensitive to methylation-induced silencing. In RA Treg cells we observed significantly reduced DMR methylation and lower DNA methyltransferase (DNMT1/3A) expression compared with healthy Treg cells. Furthermore, DMR methylation negatively correlated with FOXP3 mRNA expression, and Treg cells isolated from rheumatoid factor negative RA patients were found to express significantly higher levels of FOXP3 than Treg cells from RhF-positive patients, with an associated decrease in DMR methylation. In conclusion, the novel DMR is involved in the regulation of Treg-cell FOXP3 expression, but this regulation is lost post-transcriptionally in RA Treg cells. [ABSTRACT FROM AUTHOR]

Objective Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. Methods CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction. Results Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway. Conclusion We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients. [ABSTRACT FROM AUTHOR]

Objective To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. Methods A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). Results TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50] 4 n M) and HUVEC VCAM-1 up-regulation (IC50 12 n M) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: −23.5% (95% CI −12.4, −33.2 [ P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [ P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [ P = 0.0880]) for IL-1β, and -19.0% (95% CI -3.4, -32.1 [ P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [ P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [ P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [ P = 0.0656]) for monocyte chemotactic protein 1. Conclusion In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling. [ABSTRACT FROM AUTHOR]