Patricia Green

Treg cell function in rheumatoid arthritis is compromised by ctla-4 promoter methylation resulting in a failure to activate the indoleamine 2,3-dioxygenase pathway.

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.

Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis.

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.

Indoleamine 2,3-dioxygenase-1 is protective in atherosclerosis and its metabolites provide new opportunities for drug development.

Significance Inflammation is an important component of the pathogenesis of cardiovascular disease, the world’s biggest killer. No antiinflammatory treatments have yet been developed to treat cardiovascular disease. Indoleamine 2,3-dioxygenase (IDO) is a critical enzyme in the metabolism of tryptophan that has been shown to be immune-regulatory in many diseases. ApoE−/− mice deficient in IDO (ApoE−/−Indo−/−) developed larger atherosclerotic lesions and an unfavorable lesion phenotype that may predispose to cardiovascular complications. Furthermore, administration of an orally active synthetic tryptophan metabolite (3,4-DAA) reduced disease development in mice and cytokine production in human atheroma. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation, defining a path toward the development of new therapeutics. Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE−/− mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE−/− mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE−/− mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.

A novel upstream enhancer of FOXP3, sensitive to methylation-induced silencing, exhibits dysregulated methylation in rheumatoid arthritis Treg cells.

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.

Selective Blockade of Tumor Necrosis Factor Receptor I Inhibits Proinflammatory Cytokine and Chemokine Production in Human Rheumatoid Arthritis Synovial Membrane Cell Cultures

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 nM) and HUVEC VCAM-1 up-regulation (IC50 12 nM) 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.

Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis.

Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. Results: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-&bgr;3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Immune cell census in murine atherosclerosis: cytometry by time of flight illuminates vascular myeloid cell diversity

Abstract Aims Atherosclerosis is characterized by the abundant infiltration of myeloid cells starting at early stages of disease. Myeloid cells are key players in vascular immunity during atherogenesis. However, the subsets of vascular myeloid cells have eluded resolution due to shared marker expression and atypical heterogeneity in vascular tissues. We applied the high-dimensionality of mass cytometry to the study of myeloid cell subsets in atherosclerosis. Methods and results Apolipoprotein E-deficient (ApoE−/−) mice were fed a chow or a high fat (western) diet for 12 weeks. Single-cell aortic preparations were probed with a panel of 35 metal-conjugated antibodies using cytometry by time of flight (CyTOF). Clustering of marker expression on live CD45+ cells from the aortas of ApoE−/− mice identified 13 broad populations of leucocytes. Monocyte, macrophage, type 1 and type 2 conventional dendritic cell (cDC1 and cDC2), plasmacytoid dendritic cell (pDC), neutrophil, eosinophil, B cell, CD4+ and CD8+ T cell, γδ T cell, natural killer (NK) cell, and innate lymphoid cell (ILC) populations accounted for approximately 95% of the live CD45+ aortic cells. Automated clustering algorithms applied to the Lin-CD11blo-hi cells revealed 20 clusters of myeloid cells. Comparison between chow and high fat fed animals revealed increases in monocytes (both Ly6C+ and Ly6C−), pDC, and a CD11c+ macrophage subset with high fat feeding. Concomitantly, the proportions of CD206+ CD169+ subsets of macrophages were significantly reduced as were cDC2. Conclusions A CyTOF-based comprehensive mapping of the immune cell subsets within atherosclerotic aortas from ApoE−/− mice offers tools for myeloid cell discrimination within the vascular compartment and it reveals that high fat feeding skews the myeloid cell repertoire toward inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis.

Design and optimisation of bioactive cyclic peptides: generation of a down-regulator of TNF secretion.

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.