HOXA5 is a key regulator of class 3 semaphorins expression in the synovium of rheumatoid arthritis patients.

OBJECTIVE: Class 3 semaphorins are reduced in the synovial tissue of RA patients and these proteins are involved in the pathogenesis of the disease. The aim of this study was to identify the transcription factors involved in the expression of class 3 semaphorins in the synovium of RA patients. METHODS: Protein and mRNA expression in synovial tissue from RA and individuals at risk (IAR) patients, human umbilical vein endothelial cells (HUVEC) and RA fibroblast-like synoviocytes (FLS) was determined by ELISA, immunoblotting and quantitative PCR. TCF-3, EBF-1 and HOXA5 expression was knocked down using siRNA. Cell viability, migration and invasion were determined using MTT, calcein, wound closure and invasion assays, respectively. RESULTS: mRNA expression of all class 3 semaphorins was significantly lower in the synovium of RA compared with IAR patients. In silico analysis suggested TCF-3, EBF-1 and HOXA5 as transcription factors involved in the expression of these semaphorins. TCF-3, EBF-1 and HOXA5 silencing significantly reduced the expression of several class 3 semaphorin members in FLS and HUVEC. Importantly, HOXA5 expression was significantly reduced in the synovium of RA compared with IAR patients and was negatively correlated with clinical disease parameters. Additionally, TNF-α down-regulated the HOXA5 expression in FLS and HUVEC. Finally, HOXA5 silencing enhanced the migratory and invasive capacities of FLS and the viability of HUVEC. CONCLUSION: HOXA5 expression is reduced during the progression of RA and could be a novel therapeutic strategy for modulating the hyperplasia of the synovium, through the regulation of class 3 semaphorins expression.

Central Role of Semaphorin 3B in a Serum-Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis.

OBJECTIVE: Semaphorin 3B (Sema3B) decreases the migratory and invasive capacities of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and suppresses expression of matrix metalloproteinases. We undertook this study to examine the role of Sema3B in a mouse model of arthritis and its expression in RA patients. METHODS: Clinical responses, histologic features, and FLS function were examined in wild-type (WT) and Sema3B-/- mice in a K/BxN serum transfer model of arthritis. Protein and messenger RNA expression of Sema3B in mouse joints and murine FLS, as well as in serum and synovial tissue from patients with arthralgia and patients with RA, was determined using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and RNA sequencing. FLS migration was determined using a wound closure assay. RESULTS: The clinical severity of serum-induced arthritis was significantly higher in Sema3B-/- mice compared to WT mice. This was associated with increased expression of inflammatory mediators and increased migratory capacity of murine FLS. Administration of recombinant mouse Sema3B reduced the clinical severity of serum-induced arthritis and the expression of inflammatory mediators. Sema3B expression was significantly lower in the synovial tissue and serum of patients with established RA compared to patients with arthralgia. Serum Sema3B levels were elevated in patients with arthralgia that later progressed to RA, but not in those who did not develop RA; however, these levels drastically decreased 1 and 2 years after RA development. CONCLUSION: Sema3B expression plays a protective role in a mouse model of arthritis. In RA patients, expression levels of Sema3B in the serum depend on the disease stage, suggesting different regulatory roles in disease onset and progression.

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α.

OBJECTIVE: SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production. METHODS: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays. RESULTS: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2α (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs. CONCLUSION: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.

Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways.

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

cDC1 are required for the initiation of collagen-induced arthritis.

Rheumatoid arthritis (RA) is chronic autoimmune disease which etiology remains unknown. Several cell types have been described to potentiate/aggravate the arthritic process however the initiating event in synovial inflammation is still elusive. Dendritic cells (DCs) are essential for the initiation of primary immune responses and thus we hypothesized that these cells might be crucial for RA induction. DCs are a heterogeneous population of cells comprising different subsets with distinct phenotype and function. Here we investigated which DC subset(s) is/are crucial for the initiation of the arthritic process. We have previously demonstrated that Flt3-/- mice, with reduced DCs, were protected from collagen induced arthritis (CIA). Here we have shown that GM-CSF derived DCs in Flt3L-/- mice are functional but not sufficient to induce arthritis. Batf3-/- mice lacking both CD103+ and CD8α+ cDC1 were resistant to collagen induced arthritis (CIA), demonstrating that this DC subset is crucial for arthritis development. CEP-701 (a Flt3L inhibitor) treatment prevented CIA induction, and reduced dramatically the numbers CD103+ cDC1s present in the lymph nodes and synovium. Hence this study identified cDC1 as the main subset orchestrating the initiation of cell-mediated immunity in arthritis.

Promotion of macrophage activation by Tie2 in the context of the inflamed synovia of rheumatoid arthritis and psoriatic arthritis patients.

OBJECTIVE: To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA. METHODS: Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex. RESULTS: Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients. CONCLUSION: Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.

Extracellular SPARC cooperates with TGF-ß signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts.

OBJECTIVES: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. METHODS: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-ß1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. RESULTS: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-ß and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-ß signalling. CONCLUSION: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-ß dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

Induction of Inflammation and Fibrosis by Semaphorin 4A in Systemic Sclerosis.

OBJECTIVE: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc). METHODS: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay. RESULTS: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors. CONCLUSION: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

BACKGROUND AND OBJECTIVE: Systemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. METHODS: Chromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression. RESULTS: 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. CONCLUSION: SSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

New insights into the genetics and epigenetics of systemic sclerosis.

Systemic sclerosis (SSc) is a severe autoimmune disease that is characterized by vascular abnormalities, immunological alterations and fibrosis of the skin and internal organs. The results of genetic studies in patients with SSc have revealed statistically significant genetic associations with disease manifestations and progression. Nevertheless, genetic susceptibility to SSc is moderate, and the functional consequences of genetic associations remain only partially characterized. A current hypothesis is that, in genetically susceptible individuals, epigenetic modifications constitute the driving force for disease initiation. As epigenetic alterations can occur years before fibrosis appears, these changes could represent a potential link between inflammation and tissue fibrosis. Epigenetics is a fast-growing discipline, and a considerable number of important epigenetic studies in SSc have been published in the past few years that span histone post-translational modifications, DNA methylation, microRNAs and long non-coding RNAs. This Review describes the latest insights into genetic and epigenetic contributions to the pathogenesis of SSc and aims to provide an improved understanding of the molecular pathways that link inflammation and fibrosis. This knowledge will be of paramount importance for the development of medicines that are effective in treating or even reversing tissue fibrosis.

Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes: beyond transcriptional regulation.

BACKGROUND: Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS). METHODS: The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1ß-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36+/+ and ZFP36-/- mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting. RESULTS: ITF2357 reduced the expression of 85% of the analyzed IL-1ß-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment. CONCLUSIONS: Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.

Class 3 semaphorins modulate the invasive capacity of rheumatoid arthritis fibroblast-like synoviocytes.

Objective: Class 3 semaphorins regulate diverse cellular processes relevant to the pathology of RA, including immune modulation, angiogenesis, apoptosis and invasive cell migration. Therefore, we analysed the potential role of class 3 semaphorins in the pathology of RA. Methods: Protein and mRNA expression in RA synovial tissue, SF and fibroblast-like synoviocytes (FLS) were determined by immunoblotting and quantitative PCR (qPCR). RA FLS migration and invasion were determined using wound closure and transwell invasion assays, respectively. PlexinA1, neuropilin-1 and neuropilin-2 expression was knocked down using small interfering RNA (siRNA). Activation of FLS intracellular signalling pathways was assessed by immunoblotting. Results: mRNA expression of semaphorins (Sema)3B, Sema3C, Sema3F and Sema3G was significantly lower in the synovial tissue of early arthritis patients at baseline who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3B and Sema3F expression was significantly lower in arthritis patients fulfilling classification criteria for RA compared with those who did not. FLS expression of Sema3A was induced after stimulation with TNF, IL-1ß or lipopolysaccharides (LPS), while Sema3B and Sema3F expression was downregulated. Exogenously applied Sema3A induced the migration and invasive capacity of FLS, while stimulation with Sema3B or Sema3F reduced spontaneous FLS migration, and platelet-derived growth factor induced cell invasion, effects associated with differential regulation of MMP expression and mediated by the PlexinA1 and neuropilin-1 and -2 receptors. Conclusion: Our data suggest that modulation of class 3 semaphorin signaling could be a novel therapeutic strategy for modulating the invasive behaviour of FLS in RA.

Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: Endoplasmic reticulum (ER) stress has proinflammatory properties, and transgenic animal studies of rheumatoid arthritis (RA) indicate its relevance in the process of joint destruction. Because currently available studies are focused primarily on myeloid cells, we assessed how ER stress might affect the inflammatory responses of stromal cells in RA. METHODS: ER stress was induced in RA fibroblast-like synoviocytes (FLS), dermal fibroblasts, and macrophages with thapsigargin or tunicamycin alone or in combination with Toll-like receptor (TLR) ligands, and gene expression and messenger RNA (mRNA) stability was measured by quantitative polymerase chain reaction. Cellular viability was measured using cell death enzyme-linked immunosorbent assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and signaling pathway activation was analyzed by immunoblotting. RESULTS: No cytotoxicity was observed in FLS exposed to thapsigargin, despite significant induction of ER stress markers. Screening of 84 proinflammatory genes revealed minor changes in their expression (fold change 90th percentile range 2.8-8.3) by thapsigargin alone, but the vast majority were hyperinduced during combined stimulation with thapsigargin and TLR ligands (35% greater than fivefold vs lipopolysaccharide alone). The synergistic response could not be explained by quantitative effects on nuclear factor-κB and mitogen-activated protein kinase pathways alone, but it was dependent on increased mRNA stability. mRNA stabilization was similarly enhanced by ER stress in dermal fibroblasts but not in macrophages, correlating with minimal cooperative effects on gene induction in macrophages. CONCLUSIONS: RA FLS are resistant to apoptosis induced by ER stress, but ER stress potentiates their activation by multiple TLR ligands. Interfering with downstream signaling pathway components of ER stress may be of therapeutic potential in the treatment of RA.

Insight into the Endocrine System and the Immune System: A Review of the Inflammatory Role of Prolactin in Rheumatoid Arthritis and Psoriatic Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects females three times more frequently than males. A potential role for hormones, such as prolactin (PRL), may in part explain this phenomenon. The risk of developing RA is increased in women who are lactating after the first pregnancy, which might be related to breastfeeding and the release of PRL. Other studies found a protective effect of PRL on RA development. Some studies have reported that hyperprolactinemia is more common in RA and serum PRL levels are correlated with several disease parameters, although others could not confirm these findings. Overall the plasma PRL levels are on average not elevated in RA. Previously, a small number of open-label clinical trials using bromocriptine, which indirectly decreases PRL levels, were performed in RA patients and showed clinical benefit, although others found the opposite effect. Locally produced PRL at the site of inflammation may have a crucial role in RA as well, as it has been shown that PRL can be produced by synovial macrophages. Locally produced PRL has both pro-inflammatory and anti-inflammatory effects in arthritis. Psoriatic arthritis (PsA) is also an autoinflammatory disease, in which the prolactin receptor is also expressed in macrophages. The aim of this review is to provide an overview of the potential role of PRL signaling in inflammatory joint diseases (RA and PsA) and its potential as a therapeutic target.

Rheumatoid arthritis and psoriatic arthritis synovial fluids stimulate prolactin production by macrophages.

Prolactin (PRL) is a neuroendocrine hormone that can promote inflammation. We examined the synovial tissue and fluid levels of PRL in patients with inflammatory arthritis, PRL expression in differentiated Mϕs from patients with arthritis and from healthy donors, and the effects of different stimuli on PRL production by Mϕs. PRL levels were measured in paired synovial fluid (SF) and peripheral blood of patients with rheumatoid arthritis (RA, n = 19), psoriatic arthritis (PsA, n = 11), and gout (n = 11). Synovial-tissue PRL mRNA expression was measured by quantitative PCR in patients with RA (n = 25), PsA (n = 11), and gout (n = 12) and in Mϕs differentiated in SF of patients with RA, PsA, other subtypes of spondyloarthritis (SpA), and gout. Synovial-tissue PRL mRNA expression correlated significantly with clinical disease parameters in patients with RA and PsA, including erythrocyte sedimentation rate (ESR, r = 0.424; P = 0.049) and disease activity score evaluated in 28 joints (DAS28, r = 0.729; P = 0.017). Synovial-tissue PRL expression was similar in RA, PsA, and gout. PRL mRNA expression was detected in monocyte-derived Mϕs from patients with RA and was significantly higher (P ≤ 0.01) in Mϕs differentiated in pooled SF from patients with RA and PsA compared with SpA or gout. PRL production by Mϕ differentiation in the SF from patients with RA was not further regulated by stimulation with CD40L, IgG, LPS, or TNF. PRL is produced locally in the synovium of patients with inflammatory arthritis. The production of PRL by Mϕs was increased by unknown components of RA and PsA SF, where it could contribute to disease progression.

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis.

OBJECTIVE: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNα, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNα, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

The acetyl code in rheumatoid arthritis and other rheumatic diseases.

Growing evidence supports the idea that aberrancies in epigenetic processes contribute to the onset and progression of human immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA). Epigenetic regulators of histone tail modifications play a role in chromatin accessibility and transcriptional responses to inflammatory stimuli. Among these, histone deacetylases (HDACs) regulate the acetylation status of histones and nonhistone proteins, essential for immune responses. Broad-spectrum HDAC inhibitors are well-known anti-inflammatory agents and reduce disease severity in animal models of arthritis; however, selective HDAC inhibitors remain poorly studied. In this review, we describe emerging findings regarding the aberrant acetyl code in RA and other rheumatic disorders which may help identify not only novel diagnostic and prognostic clinical biomarkers for RA, but also new targets for epigenetic pharmacological applications.

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVES: Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). METHODS: RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/ß receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1ß-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. RESULTS: HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1ß-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. CONCLUSIONS: Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

BET bromodomain inhibition reduces maturation and enhances tolerogenic properties of human and mouse dendritic cells.

Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histone tails. An inhibitor of the acetylated-lysine reader bromodomain and extra-terminal domain (BET) proteins, I-BET151, is known to counteract the induction of expression of inflammatory genes in macrophages. We have investigated the effects of I-BET151 on dendritic cell function, including expression of co-stimulatory molecules and cytokines, and capacity for T cell activation. Treatment of mouse bone marrow derived dendritic cells (BMDC) and human monocyte derived DCs (mdDC) with I-BET151 reduced LPS-induced expression of co-stimulatory molecules, as well as the production of multiple cyokines and chemokines. Most strikingly, secretion of IL-6, IL-12 and IL-10 was significantly reduced to 89.7%, 99.9% and 98.6% respectively of that produced by control cells. I-BET151-treated mdDC showed a reduced ability to stimulate proliferation of autologous Revaxis-specific T cells. Moreover, while I-BET151 treatment of BMDC did not affect their ability to polarise ovalbumin specific CD4+ CD62L+ naive T cells towards Th1, Th2, or Th17 phenotypes, an increase in Foxp3 expressing Tregs secreting higher IL-10 levels was observed. Suppression assays demonstrated that Tregs generated in response to I-BET151-treated BMDC displayed anti-proliferative capacity. Finally, evidence that I-BET151 treatment can ameliorate inflammation in vivo in a T cell dependent colitis model is shown. Overall, these results demonstrate marked effects of BET inhibition on DC maturation, reducing their capacity for pro-inflammatory cytokine secretion and T cell activation and enhancing the potential of DC to induce Foxp3 expressing Treg with suppressive properties.

The prolactin receptor is expressed in rheumatoid arthritis and psoriatic arthritis synovial tissue and contributes to macrophage activation.

OBJECTIVES: Prolactin (PRL) is a lactation-inducing hormone with immunomodulatory properties and is found at elevated levels in the serum of patients with RA and other rheumatic diseases. The PRL receptor (PRLR) has been shown to be expressed by macrophages in atherosclerotic plaques. The aim of this study was to examine PRLR expression by synovial macrophages and its role in the regulation of macrophage activation. METHODS: Serum monomeric 23 kDa PRL levels were measured in 119 RA patients using a fluoroimmunometric assay. PRLR expression was assessed in synovial tissue of 91 RA, 15 PsA and 8 OA patients by immunohistochemistry and digital image analysis. Double IF was used to identify PRLR-expressing cells. The effects of PRL on monocyte-derived macrophage gene expression were examined by quantitative real-time PCR and ELISA. RESULTS: Serum PRL levels were similar in female and male RA patients. Median (interquartile range) PRLR expression was significantly higher (P < 0.05) in RA and PsA synovial tissue compared with OA. PRLR colocalized with synovial CD68+ macrophages and von Willebrand factor+ endothelial cells. In vitro, PRLR was prominently expressed in IFN-γ-and IL-10-polarized macrophages compared with other polarizing conditions. PRL by itself had negligible effects on macrophage gene expression, but cooperated with CD40L and TNF to increase expression of pro-inflammatory genes including IL-6, IL-8 and IL-12ß. CONCLUSIONS: Synovial PRLR expression is enhanced in patients with inflammatory arthritis compared with OA, and PRL cooperates with other pro-inflammatory stimuli to activate macrophages. These results identify PRL and PRLR as potential new therapeutic targets in inflammatory arthritis.