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.

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.

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.

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.

Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis.

BACKGROUND: CSF-1 or IL-34 stimulation of CSF1R promotes macrophage differentiation, activation and osteoclastogenesis, and pharmacological inhibition of CSF1R is beneficial in animal models of arthritis. The objective of this study was to determine the relative contributions of CSF-1 and IL-34 signaling to CSF1R in RA. METHODS: CSF-1 and IL-34 were detected by immunohistochemical and digital image analysis in synovial tissue from 15 biological-naïve rheumatoid arthritis (RA) , 15 psoriatic arthritis (PsA) and 7 osteoarthritis (OA) patients . Gene expression in CSF-1- and IL-34-differentiated human macrophages was assessed by FACS analysis and quantitative PCR. RA synovial explants were incubated with CSF-1, IL-34, control antibody (Ab), or neutralizing/blocking Abs targeting CSF-1, IL-34, or CSF1R. The effect of a CSF1R-blocking Ab was examined in murine collagen-induced arthritis (CIA). RESULTS: CSF-1 (also known as M-CSF) and IL-34 expression was similar in RA and PsA synovial tissue, but lower in controls (P < 0.05). CSF-1 expression was observed in the synovial sublining, and IL-34 in the sublining and the intimal lining layer. CSF-1 and IL-34 differentially regulated the expression of 17 of 336 inflammation-associated genes in macrophages, including chemokines, extra-cellular matrix components, and matrix metalloproteinases. Exogenous CSF-1 or IL-34, or their independent neutralization, had no effect on RA synovial explant IL-6 production. Anti-CSF1R Ab significantly reduced IL-6 and other inflammatory mediator production in RA synovial explants, and paw swelling and joint destruction in CIA. CONCLUSIONS: Simultaneous inhibition of CSF1R interactions with both CSF-1 and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA.

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)-1ß 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-1ß, 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.

The bromodomain protein inhibitor I-BET151 suppresses expression of inflammatory genes and matrix degrading enzymes in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF). METHODS: The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays. RESULTS: BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1ß. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium. CONCLUSIONS: Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA.

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants.

OBJECTIVES: Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). MATERIALS AND METHODS: Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. RESULTS: Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. CONCLUSIONS: Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases.

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 1ß 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, p27(Kip1) 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.

Inhibiting epigenetic enzymes to improve atherogenic macrophage functions.

Macrophages determine the outcome of atherosclerosis by propagating inflammatory responses, foam cell formation and eventually necrotic core development. Yet, the pathways that regulate their atherogenic functions remain ill-defined. It is now apparent that chromatin remodeling chromatin modifying enzymes (CME) governs immune responses but it remains unclear to what extent they control atherogenic macrophage functions. We hypothesized that epigenetic mechanisms regulate atherogenic macrophage functions, thereby determining the outcome of atherosclerosis. Therefore, we designed a quantitative semi-high-throughput screening platform and studied whether the inhibition of CME can be applied to improve atherogenic macrophage activities. We found that broad spectrum inhibition of histone deacetylases (HDACs) and histone methyltransferases (HMT) has both pro- and anti-inflammatory effects. The inhibition of HDACs increased histone acetylation and gene expression of the cholesterol efflux regulators ATP-binding cassette transporters ABCA1 and ABCG1, but left foam cell formation unaffected. HDAC inhibition altered macrophage metabolism towards enhanced glycolysis and oxidative phosphorylation and resulted in protection against apoptosis. Finally, we applied inhibitors against specific HDACs and found that HDAC3 inhibition phenocopies the atheroprotective effects of pan-HDAC inhibitors. Based on our data, we propose the inhibition of HDACs, and in particular HDAC3, in macrophages as a novel potential target to treat atherosclerosis.

Tie2 signaling cooperates with TNF to promote the pro-inflammatory activation of human macrophages independently of macrophage functional phenotype.

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 -differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 -differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

Local synovial engagement of angiogenic TIE-2 is associated with the development of persistent erosive rheumatoid arthritis in patients with early arthritis.

OBJECTIVE: To examine the role of vascular endothelial growth factor (VEGF) and angiopoietin signaling in the diagnosis and disease outcome of patients with early arthritis. METHODS: Fifty patients with early arthritis (disease duration <1 year) who had not been treated with disease-modifying antirheumatic drugs (DMARDs) were monitored prospectively and were classified at baseline and after 2 years as having undifferentiated arthritis (UA), rheumatoid arthritis (RA), or spondyloarthritis (SpA). All patients underwent arthroscopic synovial biopsy at baseline. Synovial expression of VEGF, VEGF receptor, angiopoietin 1 (Ang-1), Ang-2, TIE-2, and activated p-TIE-2 was evaluated by immunohistochemistry. Serum levels of VEGF, Ang-1, and Ang-2 were measured by enzyme-linked immunosorbent assay. Secreted products of macrophages stimulated with Ang-1 and Ang-2 were measured using a multiplex system. RESULTS: Expression of Ang-1 was comparable between the patients with RA at baseline and patients with UA who fulfilled the criteria for RA over time (UA/RA), and it was significantly higher in patients with RA (P < 0.05) or UA/RA (P < 0.005) than in patients with SpA. TIE-2 and p-TIE-2 were more highly expressed in patients with RA (P < 0.005) or UA/RA (P < 0.05) than in patients with SpA. Ang-1 significantly enhanced the tumor necrosis factor-dependent macrophage production of cytokines and chemokines that are known to be elevated in the synovial fluid of patients with early RA. In RA, relative TIE-2 activation predicted the development of erosive disease (R(2) = 0.35, P < 0.05). CONCLUSION: Local engagement of synovial TIE-2 is observed during the earliest phases of RA, suggesting that TIE-2 signaling may contribute to disease development and progression or may indicate an attempt to protect against these processes. Early therapeutic targeting of TIE-2 signaling may be useful in improving outcome in arthritis.

Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane ß subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

Angiopoietin-2 promotes inflammatory activation of human macrophages and is essential for murine experimental arthritis.

BACKGROUND: Angiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue, but the cellular targets of Ang signalling and the relative contributions of Ang-1 and Ang-2 to arthritis are poorly understood. OBJECTIVES: To determine the cellular targets of Ang signalling in RA synovial tissue, and the effects of Ang-2 neutralisation in murine collagen-induced arthritis (CIA). METHODS: RA and psoriatic arthritis (PsA) synovial biopsies were examined for expression of Tie2 and activated phospho (p)-Tie2 by quantitative immunohistochemistry and immunofluorescent double staining. Human monocyte and macrophage Tie2 expression was determined by flow cytometry and quantitative PCR. Regulation of macrophage intracellular signalling pathways and gene expression were examined by immunoblotting and ELISA. CIA was assessed in mice treated with saline, control antibody, prednisolone or neutralising anti-Ang-2 antibody. RESULTS: Expression of synovial Tie2 and p-Tie2 was similar in RA and PsA. Tie2 activation in RA patient synovial tissue was predominantly localised in synovial macrophages and was expressed by human macrophage. Ang-1 and Ang-2 stimulated activation of multiple intracellular signalling pathways, and cooperated with tumour necrosis factor to induce macrophage interleukin 6 and macrophage inflammatory protein 1α production. Ang-2 selectively suppressed macrophage thrombospondin-2 production. Ang-2 neutralisation significantly decreased disease severity, synovial inflammation, neo-vascularisation and joint destruction in established CIA. CONCLUSIONS: The authors identify synovial macrophages as primary targets of Ang signalling in RA, and demonstrate that Ang-2 promotes the pro-inflammatory activation of human macrophages. Ang-2 makes requisite contributions to pathology in CIA, indicating that targeting Ang-2 may be of therapeutic benefit in the treatment of RA.

Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay.

BACKGROUND: Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue. OBJECTIVES: To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model. METHODS: RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1ß, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays. RESULTS: HDACi (0.25-1.0 µM) suppressed RA FLS IL-6 production induced by IL-1ß, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1ß stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1ß stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages. CONCLUSIONS: Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.

Apoptosis induced by overall metabolic stress converges on the Bcl-2 family proteins Noxa and Mcl-1.

Apoptosis provoked by glucose shortage in dividing T cells is mediated via the BH3-only protein Noxa and inhibition of its binding partner Mcl-1. It is unknown how signals from cellular metabolism can affect the balance between Mcl-1 and Noxa and to what extent other Bcl-2 members are involved in this apoptosis cascade. Here, we defined the mechanism underlying apoptosis in relation to various types of metabolic stress. First, we established that the Noxa/Mcl-1 balance is regulated by glucose deprivation as well as by general metabolic stress, via changes in proteasome-mediated degradation of Mcl-1. Second, in contrast with cytokine-deprivation, no transcriptional modulation of Mcl-1, Puma, Bim or Noxa was observed during glucose deprivation. Third, no changes in PKB or GSK3 activity occurred and no clear role for AMPK was detected. Fourth, apoptosis triggered by nutrient deprivation was executed without signs of overt autophagy and independent of ROS production or p38 MAP kinase activity. Lastly, apoptosis under nutrient limitation could also be delayed by knock-down of Bim or overexpression of Bcl-2. In conclusion, Noxa functions in a specific apoptotic pathway that integrates overall nutrient stress, independent from attenuated PI3K/PKB signaling and without clear involvement of autophagy.

Histone deacetylases in RA: epigenetics and epiphenomena.

Reduced synovial expression of histone deacetylases (HDACs) is proposed to contribute to pathology in rheumatoid arthritis (RA) by enhancing histone-dependent access of transcription factors to promoters of inflammatory genes. In the previous issue of Arthritis Research & Therapy, Kawabata and colleagues provided independent evidence that HDAC activity is increased in the synovium and fibroblast-like synoviocytes (FLSs) of patients with RA and is paralleled by increased HDAC1 expression and synovial tumor necrosis factor-alpha (TNFα) production. Remarkably, stimulation of RA FLSs with TNFα specifically increases HDAC activity and HDAC1 expression, suggesting that changes in synovial HDAC activity and expression may be secondary to local inflammatory status.

Silencing the expression of Ras family GTPase homologues decreases inflammation and joint destruction in experimental arthritis.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis. Each Ras protein was expressed in synovial tissue and cultured FLS. Each homolog was also activated following FLS stimulation with tumor necrosis factor-α or interleukin (IL)-1ß. Constitutively active mutants of each Ras protein enhanced IL-1ß-induced FLS matrix metalloproteinase-3 production, while only active H-Ras enhanced IL-8 production. Gene silencing demonstrated that each Ras protein contributed to IL-1ß-dependent IL-6 production, while H-Ras and N-Ras supported IL-1ß-dependent matrix metalloproteinase-3 and IL-8 production, respectively. The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GTPases in vivo suppresses global inflammation and joint destruction in arthritis. Consistent with this, simultaneous silencing of H-Ras, K-Ras, and N-Ras expression significantly reduces inflammation and joint destruction in murine collagen-induced arthritis, while specific targeting of N-Ras alone is less effective in providing clinical benefits.

Sustained T cell Rap1 signaling is protective in the collagen-induced arthritis model of rheumatoid arthritis.

OBJECTIVE: Defective activation of T cell receptor-proximal signaling proteins, such as the small GTPase Rap1, is thought to contribute to the pathologic behavior of rheumatoid arthritis (RA) synovial T cells. This study was undertaken to determine whether maintaining Rap1 signaling in murine T cells modifies disease onset or severity in collagen-induced arthritis (CIA). METHODS: CIA experiments were conducted using wild-type and RapV12-transgenic mice, which express an active mutant of Rap1 in the T cell compartment. Mice were assessed using macroscopic, microscopic, and radiologic measures, and serum levels of anticollagen antibodies were measured by enzyme-linked immunosorbent assay. Phenotypic and functional characterization of wild-type and RapV12-transgenic T cells under homeostatic conditions and during disease onset was performed by flow cytometry. RESULTS: Disease incidence and severity, synovial infiltration, joint destruction, and anticollagen antibody production were significantly reduced in RapV12-transgenic mice. Although the numbers and percentages of CD3+, CD4+, and CD8+ (naive, effector, and memory) T cells, Treg cells, and Th17 cells were equivalent in wild-type and RapV12-transgenic mice, a significant decrease in the percentage of tumor necrosis factor α-secreting CD8+ T cells was observed in RapV12-transgenic mice during CIA. RapV12-transgenic T cells also inefficiently expressed inducible costimulator and CD40L costimulatory proteins involved in B cell immunoglobulin class switching. CONCLUSION: Our findings indicate that maintenance of T cell Rap1 signaling in murine T cells reduces disease incidence and severity in CIA, which are associated with specific defects in T cell effector function. Therefore, the restoration of Rap1 function in RA synovial T cells may have therapeutic benefit in RA.