Objective. Selective serotonin reuptake inhibitors (SSRIs), in addition to their antidepressant effects, have been reported to have antiinflammatory effects. The aim of this study was to assess the antiarthritic potential of 2 SSRIs, fluoxetine and citalopram, in murine collagen-induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA).Methods. Following therapeutic administration of SSRIs, paw swelling was assessed and clinical scores were determined daily in DBA/1 mice with CIA. Joint architecture was examined histologically at the end of the treatment period. Cultures of human RA synovial membranes were treated with SSRIs, and cytokine production was measured. Toll-like receptor (TLR) function was examined in murine and human macrophages, human B cells, and human fibroblast-like synovial cells treated with SSRIs.

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5Rα, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3Rα was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor α was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3Rα by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5Rα down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.

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. 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. Methods. CTLA-4 expression in

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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.

These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

BackgroundLentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming.ResultsHere we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction.ConclusionThe optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.