Linda M. Hartkamp

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

BackgroundCSF-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.MethodsCSF-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).ResultsCSF-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.ConclusionsSimultaneous 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.

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.

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

Objectives Brutons 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.

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)-1β. Constitutively active mutants of each Ras protein enhanced IL-1β-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-1β-dependent IL-6 production, while H-Ras and N-Ras supported IL-1β-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.

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.

THU0536 The AGC Kinases Protein Kinase B (PKB) and Serum and Glucocorticoid Kinase (SGK) Differentially Regulate the Metabolic Activity and Inflammatory Activation of Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Human Macrophages

Background Phosphatidylinositol 3-kinase (PI3K) –dependent activation of PKB, and subsequent PKB phosphorylation and inactivation of FoxO transcription factors, is a critical regulator of cellular proliferation and survival. Constitutive activation of the PI3K/PKB pathway, and inactivation of FoxO proteins is observed in rheumatoid arthritis (RA) patient synovial tissue, and rescue of FoxO1 activity induces RA fibroblast-like synoviocyte (FLS) apoptosis. However, SGK, an AGC kinase related to PKB, can also target the same residues of FoxO proteins. Objectives Here, we sought to determine the relative contributions of PKB and SGK to RA FLS survival and inflammatory activation. Methods PKB and SGK isoform expression was detected by qRT-PCR and immunoblotting experiments on isolated cell populations. Effects of specific PKB (Akt inhibitor VIII) and SGK (GSK 650394) inhibitors on metabolic activity and IL6 production was assessed by MTT assay and ELISA, respectively. The influence of pharmacological inhibition of PKB or SGK on expression of IL1 responsive genes in RA FLS and human macrophages was determined using low density qPCR arrays. Furthermore, effects of pharmacological targeting of PKB or SGK on RA FLS IL-1β- or PDGF- induced FoxO1 phosphorylation was assessed by immunoblotting. Results All PKB isoforms (AKT1, AKT2, and AKT3) were expressed at equivalent levels in RA FLS, while only SGK1 and SGK3, but not SGK2, were detected. PKBi, but not SGKi, significantly decreased FLS mitochondrial activity. In contrast, only SGKi significantly reduced IL-1β-induced IL-6 production. Examining 84 genes induced by IL-1β in RA FLS, we found that PKBi suppressed transcription of CCL7, CXCL6 CXCL9, CXCL10, CXCL11, IL1RN, and MMP13, but enhanced IL23A and TNFα expression. SGKi enhanced EREG expression, but suppressed CXCL10, CCL4, CCL5, CSF3, IL6, MMP3, MMP7 and VCAM1 expression. In human macrophages we found that PKBi suppressed transcription of IL36A and PGLYRP1, SGKi suppressed IL6 and TNF. Neither compound interfered with IL-1β-induced FoxO1 phosphorylation at AGC kinase consensus sites. Conclusions Our results provide the first evidence for a role of SGK in the inflammatory activation of RA FLS and human macrophages, point to an unidentified third AGC kinase regulating FoxO1, and suggest that targeting distinct PI3K-dependent AGC kinases can preferentially modulate specific components of cellular activation. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5096

A1.51 The AGC kinases protein kinase B (PKB) and serum and glucocorticoid kinase (SGK) differentially regulate the metabolic activity and inflammatory activation of rheumatoid arthritis fibroblast-like synoviocytes

Background and Objectives Phosphatidylinositol 3-kinase (PI3K) – dependent activation of PKB, and subsequent PKB phosphorylation and inactivation of FoxO transcription factors, is a critical regulator of cellular proliferation and survival. Constitutive activation of the PI3K/PKB pathway, and inactivation of FoxO proteins is observed in rheumatoid arthritis (RA) patient synovial tissue, and rescue of FoxO1 activity induces RA fibroblast-like synoviocyte (FLS) apoptosis. However, SGK, an AGC kinase related to PKB, can also target the same residues of FoxO proteins. Here, we sought to determine the relative contributions of PKB and SGK to RA FLS survival and inflammatory activation. Materials and Methods PKB and SGK isoform expression was detected by qRT-PCR and immunoblotting experiments on isolated cell populations. Effects of specific PKB (Akt inhibitor VIII) and SGK (GSK 650394) inhibitors on metabolic activity and IL6 production was assessed by MTT assay and ELISA, respectively. The influence of pharmacological inhibition of PKB or SGK on RA FLS expression of IL1 responsive genes was determined using low density qPCR arrays. Furthermore, effects of pharmacological targeting of PKB or SGK on RA FLS IL-1β- or PDGF-induced FoxO1 phosphorylation was assessed by immunoblotting. Results All PKB isoforms (AKT1, AKT2, and AKT3) were expressed at equivalent levels in RA FLS, while only SGK1 and SGK3, but not SGK2, were detected. PKBi, but not SGKi, significantly decreased FLS mitochondrial activity. In contrast, only SGKi significantly reduced IL-1β-induced IL-6 production. Examining 84 genes induced by IL-1β in RA FLS, we found that PKBi suppressed transcription of CCL7, CXCL6 CXCL9, CXCL10, CXCL11, IL1RN, and MMP13, but enhanced IL23A and TNFα expression. SGKi enhanced EREG expression, but suppressed CXCL10, CCL4, CCL5, CSF3, IL6, MMP3, MMP7 and VCAM1 expression. Neither compound interfered with IL-1β-induced FoxO1 phosphorylation at AGC kinase consensus sites. Conclusions Our results provide the first evidence for a role of SGK in the inflammatory activation of RA FLS, point to an unidentified third AGC kinase regulating FoxO1, and suggest that targeting distinct PI3K-dependent AGC kinases can preferentially modulate specific components of RA FLS activation.

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.

SAT0066 NF-κB signaling in rheumatoid arthritis drives inflammation and joint destruction in part through inactivation of forkhead box class O transcription factors

Background Rheumatoid arthritis (RA) stromal fibroblast-like synoviocytes (FLS) are key effector cells in the pathology of RA, and share many phenotypic characteristics with transformed cancer cells. In many cancer models, inflammatory NF-κB signaling promotes transformation via inactivation of forkhead box class O (FoxO) transcription factors, classically regulated by phosphatidylinositol 3-kinase (PI3-K). Objectives As both NF-κB and PI3-K pathways are activated in RA synovial tissue, we examined potential functional interactions between these pathways. Methods Expression and/or phosphorylation of IKKβ, IκBα, PTEN, a negative regulator of PI3-K, protein kinase B (PKB) and FoxO1, downstream targets of PI3-K, was detected by immunohistochemistry combined with digital image analysis in synovial tissue from 15 disease-modifying antirheumatic drug (DMARD)-naive RA patients. Biopsies were obtained at baseline, and x-rays made of hands and feet at baseline and at 2 years follow-up. At 2 years, patients were classified as having non-erosive or erosive disease (Sharp van der Heijde score >2). Effects of PI3-K isoform specific inhibitors on RA FLS IL-1β- induced IκBα phosphorylation and NF-κB p65 activation were assessed by immunoblotting and ELISA, respectively. Furthermore, effects of NF-κB inhibition on LPS induced FoxO nuclear export was assessed by immunoblotting. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on RA FLS NF-κB dependent gene sets using low density qPCR arrays. Results In vivo we observed a strong correlation between expression of IKKβ and phosphorylation of its target IκBα (R=0.70, p<0.01). Similarly, activation of PKB correlated with phosphorylation of FoxO1 (R=0.66, p<0.01). Reciprocal positive correlations were also observed between activation of NF-κB and PI3-K pathways. In vitro, pan-PI3-K inhibitor LY294002 and inhibitors specific for α/β, γ and δ isoforms of PI3-K failed to influence IL-1β and LPS-induced activation of NF-κB transcriptional activity and cytokine production. However, inhibitors of IKKβ significantly reduced nuclear exclusion of FoxO proteins in FLS and macrophages, and increased FoxO DNA-binding activity. qPCR array analysis of NF-κB-dependent gene sets induced by IL-1β in RA FLS identified several genes, including CCL2, ICAM1 and BCL2A1, which were suppressed by introduction of constitutively active FoxO1. Conclusions Our data suggest that NF-κB signaling drives inflammation and joint destruction in RA at least in part via inactivation of FoxO transcription factors. Disclosure of Interest None Declared

OP0304 BTK Inhibition Suppresses Inflammatory Cytokine Production and Affects Gene Expression in Human Macrophages and RA Synovial Tissue Explants

Background Rheumatoid arthritis (RA) is a chronic and progressive autoinflammatory disorder characterized by the infiltration of inflammatory cells, including B-cells, T-cells and macrophages, in the synovial membrane ultimately leading to cartilage destruction and bone erosion. Clinical disease activity in RA correlates strongly with macrophage numbers in synovial tissue and expression of macrophage-derived cytokines. Bruton’s tyrosine kinase (Btk) is important not only in B cell activation, but also mediates immune complex-dependent activation of monocytes. Pharmacological inhibition of Btk has been shown to be effective in suppressing pathology in murine models of arthritis Objectives The objective of this study was to determine the potential role of Btk in the pathology of RA. Methods Btk expression was detected by immunohistochemistry and digital image analysis in synovial tissue from 16 RA and 12 PsA patients, naïve to treatment with biologicals. Immunofluorescent double labelling confocal microscopy was performed to identify which cell types express Btk in RA synovial tissue. Validating qRT-PCR and immunoblotting experiments were performed on isolated relevant cell populations. Effects of a specific Btk inhibitor, RN486, on activation-dependent human macrophage IL-6 production (n=8) and RA synovial tissue explant cultures (n=6) were assessed by ELISA. RN486 influence on macrophage expression of genes related to angiogenesis, cellular adhesion, tissue remodelling and innate immune responses was determined using low density qPCR arrays. Results Btk was expressed at equivalent levels in patients with RA and PsA. No relationship was observed between expression levels and patient clinical characteristics (CRP, ESR, DAS28, RF-positivity). In RA, but not PsA, Btk expression was significantly related to numbers of synovial macrophages (R= 0.63, p< 0.01) and T cells (R= 0.79, p< 0.001), but not fibroblast-like synoviocytes (FLS), B cells, plasma cells, or endothelial cells. qPCR and immunoblotting experiments confirmed that Btk was expressed in B cells, monocytes, and macrophages, but not T cells or RA FLS. RN486 (1μM) inhibited macrophage IL-6 production induced by Fc receptor stimulation (40% inhibition, p< 0.01) and anti-CD40 antibodies (50%, p< 0.05), but not TNFα or LPS stimulation. qPCR analysis of human macrophages demonstrated that RN486 inhibited by more than 2-fold 12 of 21 genes induced by IgG, 11 of 52 genes induced by CD40 stimulation, and 6 of 25 genes induced by RA SF in 3 independent experiments. RN486 also inhibited spontaneous IL-6 production by cultured RA synovial explants (65%, p< 0.01). Conclusions Btk is expressed in RA synovial tissue and macrophages would be prominent synovial targets of strategies aimed at inhibiting Btk in RA. Btk activity is needed to drive macrophage activation in response to multiple stimuli relevant to RA, and drives IL-6 production in RA synovial tissue. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA. Disclosure of Interest L. Hartkamp: None Declared, I. van Es: None Declared, J. Fine Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, M. Smith Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. Woods Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, S. Narula Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. DeMartino Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, P. Tak Consultant for: GlaxoSmithKline, Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Hoffmann-La Roche