Chiara Angiolilli

Inflammatory cytokines epigenetically regulate rheumatoid arthritis fibroblast-like synoviocyte activation by suppressing HDAC5 expression

Objectives Epigenetic modifications play an important role in the regulation of gene transcription and cellular function. Here, we examined if pro-inflammatory factors present in the inflamed joint of patients with rheumatoid arthritis (RA) could regulate histone deacetylase (HDAC) expression and function in fibroblast-like synoviocytes (FLS). Methods Protein acetylation in synovial tissue was assessed by immunohistochemistry. The mRNA levels of HDAC family members and inflammatory mediators in the synovial tissue and the changes in HDAC expression in RA FLS were measured by quantitative (q) PCR. FLS were either transfected with HDAC5 siRNA or transduced with adenoviral vector encoding wild-type HDAC5 and the effects of HDAC5 manipulation were examined by qPCR arrays, ELISA and ELISA-based assays. Results Synovial class I HDAC expression was associated with local expression of tumour necrosis factor (TNF) and matrix metalloproteinase-1, while class IIa HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1β or TNF stimulation selectively suppressed HDAC5 expression in RA FLS, which was sufficient and required for optimal IFNB, CXCL9, CXCL10 and CXCL11 induction by IL-1β, associated with increased nuclear accumulation of the transcription factor, interferon regulatory factor 1(IRF1). Conclusions Inflammatory cytokines suppress RA FLS HDAC5 expression, promoting nuclear localisation of IRF1 and transcription of a subset of type I interferon response genes. Our results identify HDAC5 as a novel inflammatory mediator in RA, and suggest that strategies rescuing HDAC5 expression in vivo, or the development of HDAC inhibitors not affecting HDAC5 activity, may have therapeutic applications in RA treatment.

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/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-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-1β-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.

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.

JNK-dependent downregulation of FoxO1 is required to promote the survival of fibroblast-like synoviocytes in rheumatoid arthritis

Background Forkhead box O (FoxO) transcription factors integrate environmental signals to modulate cell proliferation and survival, and alterations in FoxO function have been reported in rheumatoid arthritis (RA). Objectives To examine the relationship between inflammation and FoxO expression in RA, and to analyse the mechanisms and biological consequences of FoxO regulation in RA fibroblast-like synoviocytes (FLS). Methods RNA was isolated from RA patient and healthy donor (HD) peripheral blood and RA synovial tissue. Expression of FoxO1, FoxO3a and FoxO4 was measured by quantitative PCR. FoxO1 DNA binding, expression and mRNA stability in RA FLS were measured by ELISA-based assays, immunoblotting and quantitative PCR. FLS were transduced with adenovirus encoding constitutively active FoxO1 (FoxO1ADA) or transfected with small interfering RNA targeting FoxO1 to examine the effects on cell viability and gene expression. Results FoxO1 mRNA levels were reduced in RA patient peripheral blood compared with HD blood, and RA synovial tissue FoxO1 expression correlated negatively with disease activity. RA FLS stimulation with interleukin 1β or tumour necrosis factor caused rapid downregulation of FoxO1. This effect was independent of protein kinase B (PKB), but dependent on c-Jun N-terminal kinase (JNK)-mediated acceleration of FoxO1 mRNA degradation. FoxO1ADA overexpression in RA FLS induced apoptosis associated with altered expression of genes regulating cell cycle and survival, including BIM, p27Kip1 and Bcl-XL. Conclusions Our findings identify JNK-dependent modulation of mRNA stability as an important PKB-independent mechanism underlying FoxO1 regulation by cytokines, and suggest that reduced FoxO1 expression is required to promote FLS survival in RA.

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

BackgroundEndoplasmic 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.MethodsER 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.ResultsNo 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.ConclusionsRA 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.

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

BackgroundHistone 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).MethodsThe effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1β-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.ResultsITF2357 reduced the expression of 85% of the analyzed IL-1β-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.ConclusionsOur 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.

A2.19 HDAC3 is required for the inflammatory gene expression program in fibroblast-like synoviocytes

Background and objectives Chemical inhibitors of histone deacetylases (HDACis) possess anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Currently available HDACis show little selectivity for class I (HDAC 1–3, 8) and class II (HDAC 4–7, 9, 10) HDACs. However, preliminary evidence suggests that targeting specific class I HDACs could result in amelioration of disease in models of RA. The present study aimed to better characterise potential differential contributions of class I and class II HDAC family members to the inflammatory activation of RA fibroblast-like synoviocytes (FLS). Materials and methods The effects of pan-specific and selective HDAC1/2, HDAC3/6, HDAC6, and HDAC8 inhibitors on RA FLS global protein and histone lysine acetylation was analysed by Western blot, while effects on gene expression were analysed by qPCR. FLS were transfected with either control siRNA or with specific HDAC3 siRNA or IFNAR1 siRNA and gene expression was analysed by qPCR. Analysis of transcription factor expression and activity was performed using Western blotting and ELISA-based DNA-binding assays. Results Impairment of class I HDAC3 and class IIb HDAC6 activity by the use of a selective HDAC3/6 inhibitor (HDAC3/6i) reduced the expression of IL-beta-induced inflammatory genes suppressed by pan-HDACi in FLS. In contrast, selective inhibition of class I HDAC1/2 and HDAC8 showed poor effects on gene modulation. Silencing of HDAC3 reproduced the effects of HDAC3/6i on gene regulation (p = 0.0156), contrary to HDAC6 specific inhibition. Screening of candidate transcription factors enriched in these gene targets, as well as IFNAR1 silencing, revealed that HDAC3/6i selectively abrogates STAT1 Tyr701 phosphorylation and STAT1 activity, but does not affect STAT1 acetylation. Impairment of STAT1 activity was a consequence of HDAC3-mediated suppression of IFNβ production. Conclusions Our results demonstrate that impairment of class I HDAC3, either through pharmacological inhibition or gene silencing, reproduces the effects of pan-HDACi in suppressing inflammatory gene expression in RA FLS. A subset of these genes are dependent upon HDAC3 regulation of IFNβ production and subsequent STAT1 phosphorylation. Taken together our data demonstrate an essential role of HDAC3 in the modulation of the inflammatory gene expression program in RA FLS, and suggests that specific epigenetic intervention may be of therapeutic benefit in the treatment of inflammatory diseases such as RA.

A1.20 HDAC5 regulates CXCL chemokine expression in RA FLS via the transcription factor IRF1

Background and Objectives HDAC inhibitors (HDACi) display anti-inflammatory properties in animal and in vitro models of rheumatoid arthritis (RA), as well as initial safety and efficacy in the treatment of systemic onset juvenile idiopathic arthritis. However, most currently available HDACi display relatively little selectivity for class I (HDAC 1-3, 8) and class II (HDAC 4-7, 9, 10) HDACs, let alone individual HDACs. The present study aims to better characterise the different contributions of HDAC family members to the inflammatory activation of RA fibroblast-like synoviocytes (FLS). Materials and Methods HDAC mRNA expression in RA FLS was measured by quantitative PCR, and the activity of class I, IIa and IIb HDACs was assessed by enzymatic assays. FLS were either transduced with adenoviral GFP-HDAC5 or transfected with HDAC5 siRNA, and gene expression was analysed by qPCR array, while chemokine production was measured by ELISA. Analysis of transcription factors was performed using DNA-binding and ELISA-based assays. Results Class IIa HDAC5 mRNA expression, but not class IIa HDAC activity, was significantly and selectively reduced after RA FLS stimulation with TNFα and IL-1β. Mimicking of HDAC5 downregulation by use of siRNA, and adenoviral overexpression of HDAC5, demonstrated that of 84 inflammation-related gene products, HDAC5 downregulation was sufficient and required to allow optimal expression of IFN-B, CXCL9, CXCL10, and CXCL11. Promoter analysis of these genes compared to non-regulated genes indicated a significant enrichment in this gene set for NF-KB, STAT1 and IRF1 binding sites. Silencing of HDAC5 expression had no effect on DNA-binding activity of MEF2 transcription factors, the best described targets of class IIa HDACs, nor NF-KB or STAT1. However, HDAC5-silencing significantly enhanced IL-1β-dependent nuclear accumulation of IRF1. Conclusions Our results show that mRNA expression and activity of distinct HDAC members in RA FLS are regulated in a different fashion by inflammatory stimuli. Silencing of class IIa HDAC5 increases CXCL chemokine production, associated with enhanced nuclear accumulation of IRF1, a transcription factor required for induction of these genes. Our results suggest that the design of HDACi targeting specific HDACs can be used to selectively modulate inflammatory gene expression in RA.

A7.21 Suppression of HDAC5 Expression by Inflammatory Cytokines is Required to Promote CXCL Chemokine Production in RA FLS

Background and Objectives Histone deacetylases (HDACs) are important regulators of gene expression and protein function in the immune system. HDAC inhibitors (HDACi) display anti-inflammatory properties in animal and in vitro models of rheumatoid arthritis (RA), as well as initial safety and efficacy in the treatment of systemic onset juvenile idiopathic arthritis. However, as most of the currently available HDACi display little selectivity or specificity for class I (HDAC 1–3, 8) and class II (HDAC 4–6, 9, 10) HDACs, the role of specific HDACs in RA is unclear. Here we examined the relationship between HDACs and inflammation in RA synovial tissue and fibroblast-like synoviocytes (FLS). Materials and Methods RNA was isolated from arthroscopic synovial biopsies from 19 RA patients. MMP-1, TNFα, IL-6, and HDAC 1–10 expression was measured by quantitative PCR (qPCR). RA FLS were stimulated with IL-1β, TNFα and LP, S and HDAC expression was measured by qPCR RA FLS were transduced with adenovirus encoding control GFP or GFP-HDAC5 or transfected with control siRNA or siRNA targeting HDAC5. Effects of HDAC5 modulation on RA FLS gene expression were analysed by custom qPCR array. Results Positive correlations were observed between RA synovial tissue expression of TNFα and HDAC1 (R = 0.651, P = 0.003) HDAC2 (R = 0.523, P = 0.022) and HDAC3 (R = 0.570, P = 0.011) and between MMP-1 and HDAC1 (R = 0.501, P = 0.029) and HDAC2 (R = 0.512, P = 0.025). A significant negative correlation was observed between synovial tissue expression of IL-6 and HDAC5 (R = –0.477, P = 0.039) and between clinical parameters of disease activity and HDAC5 (CRP: R = –0.664, P = 0.007; ESR: R = –0.556, P = 0.013: DAS28: R = –0.567, P = 0.011). HDAC5 mRNA expression was significantly and selectively reduced after RA FLS stimulation with TNFα and IL-1β, but not LPS. Of 84 genes regulated in RA FLS by IL-1β or TNFα, mRNA expression of CXCL9, CXCL10, and CXCL11 was selectively upregulated following silencing of HDAC5 expression. Conversely, mRNA expression of these chemokines was suppressed by overexpression of HDAC5 in RA FLS. Conclusions RA synovial expression of HDAC 1 and 2, but not class II HDACs, positively correlates with local inflammatory mediators, while HDAC5 expression negatively correlates with IL-6 mRNA expression and with disease activity. HDAC5 mRNA is decreased after inflammatory stimulation, and silencing of HDAC5 leads to an increase of CXCL chemokine expression in RA. FLS, an effect reversed by HDAC5 overexpression. Our results suggest a protective role for HDAC5 in RA, and that HDACi which fail to target HDAC5 may be more promising for therapeutic applications.

A10.16 Inflammatory Cytokines Downregulate FoxO1 by JNK-Dependent Acceleration of mRNA Degradation to Promote Survival and Proliferation of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Background and Objectives Aberrant regulation of proliferation and survival of immune and stromal cells contributes to the pathogenesis of rheumatoid arthritis (RA). Forkhead box O (FoxO) transcription factors integrate extracellular signals to modulate expression of genes regulating cell cycle and apoptosis, and alterations in activity and expression of FoxOs have been reported in several inflammatory diseases, including RA. In this study, we examined the relationships between inflammation and FoxO expression in RA, and analysed the mechanisms and biological consequences of cytokine-mediated regulation of FoxO1 expression in RA fibroblast-like synoviocytes (FLS). Materials and Methods RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients and expression of FoxO1, FoxO3a, FoxO4 and IL-6 was measured by quantitative PCR (qPCR). FoxO1 DNA binding, FoxO1 expression and mRNA stability were measured by ELISA-based assays and qPCR in RA FLS stimulated with IL-1β, TNFα, or LPS in the absence or presence of mitogen-activated protein kinase (MAPK) or protein kinase B (PKB) inhibitors. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on cell viability and gene expression. Results In RA synovial tissue expression of FoxO1 negatively correlated with clinical parameters of disease activity: serum C-reactive protein (R = –0.771, P = 0.0008), erythrocyte sedimentation rate (R = –0.739, P = 0.0003), and DAS28 (R = –0.575, P = 0.01), as well as synovial IL-6 mRNA levels (R = –0.628, P = 0.004). In vitro, RA FLS stimulation with IL-1β or TNFα caused rapid downregulation of FoxO1 mRNA levels, followed by reduction of FoxO1 protein expression and DNA binding. This effect was independent of PKB signalling, and was associated with acceleration of FoxO1 mRNA degradation in the presence of IL-1β. Inhibition of c-Jun N-terminal kinase (JNK), but not other MAPKs, prevented downregulation of FoxO1 expression and binding by IL-1β, and blocked IL-1β-induced reduction of FoxO1 mRNA stability. Overexpression of constitutively active FoxO1 in RA FLS induced apoptosis associated with altered expression of genes regulating cell cycle and apoptosis: BIM and p27Kip1 were induced while expression of Bcl-XL was suppressed in cells expressing active FoxO1. Conclusions Collectively, our findings suggest that suppressed synovial FoxO1 expression is strongly associated with RA pathology and demonstrate that reduction of FoxO1 expression might contribute to perpetuation of inflammation in RA by promoting FLS survival and proliferation. Our data also identify JNK-mediated modulation of FoxO1 mRNA stability as an important mechanism underlying regulation of FoxO1 by inflammatory cytokines.