13 research outputs found

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

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    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). 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). 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. 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, p27(Kip1) and Bcl-XL. 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 R

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

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    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). 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. 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. 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 disease

    Regulation of Tie2 and Tie1 expression during macrophage polarization.

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    <p>(A) FACS analysis of macrophages polarized for 7 d with the indicated cytokines, using Ig control (filled grey area) and Tie2 antibodies (open area). Experiments shown are representative of 7 independent experiments. (B) Quantification of relative Tie2 surface expression (geomean) and Tie2 mRNA expression (C) by macrophages differentiated in medium alone or with the indicated cytokine. (D) Quantification of relative Tie1 mRNA expression and (E) Tie2 mRNA expression relative to Tie1 by macrophages differentiated in medium alone or with indicated cytokine. qPCR data is shown as relative quantity, as described in material and methods Values are the mean ± SEM of 5–7 independent experiments. <sup>*</sup>P<0.05. <sup>**</sup>P<0.01 versus macrophages differentiated in medium alone. Kruskal-Wallis test was used for statistical analyses.</p

    Ang-1 and Ang-2 stimulation of macrophages cooperates with TNF to induce monocyte recruitment.

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    <p>Monocyte migration assays in response to (A) chemotaxis buffer alone (medium) or in combination with Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml), or chemotaxis buffer supplemented with conditioned medium of macrophages polarized in (B) GM-CSF, (C) IFN-γ or (D) IL-10 in the absence (unstimulated) or presence of 24 h stimulation with TNF (10 ng/ml) alone or in combination with Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml). Bars represent the means and SEM of 7–8 independent experiments. Experiments with each polarization condition were performed in parallel, but for ease of analysis are presented as 3 independent graphs. *P<0.05, * P<0.01, between stimulatory conditions, #P<0.05, ##P<0.01, ### P<0.001, compared to conditioned medium. Friedman test was used for statistical analyses.</p

    Ang-1 and Ang-2 enhance the TNF-induced expression of chemokines and cytokines.

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    <p>(A) mRNA expression profiles angiogenesis related genes in macrophages differentiated with GM-CSF, IFN-γ or IL-10 after 4 h incubation in medium alone or TNF-α (10 ng/ml) in the absence or presence of Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml) (n = 3). Data is presented as a heat map where lowest mRNA expression is showed in dark blue and highest in yellow. (B) Analyses of chemokines and cytokines mRNA expression levels of selected genes analyzed in (A). Data is shown as log<sub>2</sub> relative quantity respect to unstimulated cells, as described in material and methods. Bars represent the means and SEM of 3 independent experiments. *P<0.05, between stimulatory conditions. Friedman test was used for statistical analyses. (C) Multiplex analysis of protein production by macrophages differentiated in GM-CSF, IFN-γ, or IL-10 after 24 h incubation in medium alone or TNF (10 ng/ml) in the absence or presence of Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml). Bars represent the means and SEM of 6 independent experiments. *P<0.05, * P<0.01, between stimulatory conditions, #P<0.05, ##P<0.01, ### P<0.001, compared to unstimulated cells. Friedman test was used for statistical analyses.</p

    Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype

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    <div><p>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 <i>CXCL3</i>, <i>CXCL5</i>, <i>CXCL8</i>, <i>IL6</i>, and <i>IL12B</i> 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.</p></div

    Ang-1 and Ang-2 regulation of cytokine production in polarized macrophages.

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    <p>Analyses of (A,B) IL-6, (C,D) TSP-2, and (E,F) IL-10 production in supernatants of macrophages differentiated in (A,C,E) IFN-γ or (B,D,F) IL-10 after 24 h incubation in medium alone or TNF-α (10 ng/ml) in the absence (white bars) or presence of Ang-1 (200 ng/ml, gray bars) or Ang-2 (200 ng/ml, black bars). Bars represent the means and SEM of 4–7 independent experiments. N.D., not detectable. *P<0.05, **P<0.01 versus cells not exposed to Ang-1 or Ang-2. Friedman test was used for statistical analyses.</p

    Macrophage polarization influences angiogenic expression profile.

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    <p>(A) mRNA expression profiles of 84 angiogenesis related genes in macrophages differentiated in GM-CSF, M-CSF, IFN-γ and IL-10 7 d (n = 3). Data is presented as an unsupervised clustergram. (B) Heat map analysis of mRNA levels of angiogenesis-related genes in macrophages differentiated in GM-CSF after 4 h incubation in medium alone or Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml).</p

    Ang-1 and Ang-2 fail to induce macrophage polarization.

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    <p>(A–D) FACS analysis of expression of macrophage surface markers (A) CD16, (B) CD163, (C) CD200R and (D) CD64 in macrophages differentiated for 7 d in medium alone, or medium containing the indicated polarizing cytokines, Ang-1 (200 ng/ml) or Ang-2 (200 ng/ml). Data in A–D are presented as the geomean normalized to values obtained for macrophages cultured in medium alone, and represent the mean ± SEM of 4–5 independent experiments per marker. (E) Surface protein expression (geomean) and mRNA expression (F) of Tie2, as determined by FACS analysis (n = 5) and qPCR (n = 3), respectively, in human macrophages cultured for 7 d in the absence (medium) or presence of 200 ng/ml Ang-1 or Ang-2. *P<0.05, **P<0.01, ***P<0.001, versus macrophages differentiated in medium alone. Kruskal-Wallis test was used for statistical analyses.</p
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