Publications by authors named "Kris A Reedquist"

56 Publications

Semaphorin3B plays a central role in serum-induced arthritis model and is reduced in patients with rheumatoid arthritis.

Arthritis Rheumatol 2022 Jan 9. Epub 2022 Jan 9.

Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands.

Objectives: Sema(phorin)3B decreases the migrative and invasive capacities of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and suppress expression of matrix-metalloproteinases (MMPs). Here, we determined the role of Sema3B in an arthritis mouse model and its expression in RA patients.

Methods: Clinical responses, histological analysis and FLS function were examined in wild-type (WT) and Sema3B-deficient (Sema3b ) mice in the K/BxN serum transfer model of arthritis. Protein and mRNA expression from mice joints and murine FLS, as well as from serum and synovial tissue of arthralgia and RA patients, was determined by ELISA, immunoblotting, qPCR and RNA sequencing. FLS migration was determined using the wound closure assay.

Results: The clinical severity of serum-induced arthritis was significantly higher in Sema3b mice compared to WT mice. This was associated with an increased expression of inflammatory mediators and migratory capacity of mFLS. The administration of recombinant mouse Sema3B reduced the clinical severity of serum-induced arthritis and the expression of inflammatory mediators. Sema3B expression was significantly lower in the synovial tissue and serum of established RA patients compared to arthralgia patients. Sema3B serum levels were elevated in arthralgia patients that later-on progressed to RA, but not in those that did not develop RA; however these levels drastically decreased 1 and 2 years after RA development.

Conclusions: Sema3B expression has a protective role in a mouse model of arthritis. In RA patients, expression levels depend on the disease stage, suggesting different regulatory roles in disease onset and progression.
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http://dx.doi.org/10.1002/art.42065DOI Listing
January 2022

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

Rheumatology (Oxford) 2021 Sep 24. Epub 2021 Sep 24.

Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Objective: Systemic sclerosis (SSc) is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified CXCL4 as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production.

Methods: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several TLR-agonists. Further, pro-inflammatory cytokine production, CXCL4, HIF-1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays.

Results: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (p < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (p = 0.0079) leading to stabilization of HIF-2α (p = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord (uc)CD34 derived pDCs.

Conclusion: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.
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http://dx.doi.org/10.1093/rheumatology/keab532DOI Listing
September 2021

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

Proc Natl Acad Sci U S A 2021 03;118(11)

Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, 3584 EA Utrecht, The Netherlands;

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.
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http://dx.doi.org/10.1073/pnas.2009647118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980471PMC
March 2021

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

J Transl Autoimmun 2020 16;3:100066. Epub 2020 Sep 16.

Department of Clinical Immunology and Rheumatology.

Rheumatoid arthritis (RA) is chronic autoimmune disease which etiology remains unknown. Several cell types have been described to potentiate/aggravate the arthritic process however the initiating event in synovial inflammation is still elusive. Dendritic cells (DCs) are essential for the initiation of primary immune responses and thus we hypothesized that these cells might be crucial for RA induction. DCs are a heterogeneous population of cells comprising different subsets with distinct phenotype and function. Here we investigated which DC subset(s) is/are crucial for the initiation of the arthritic process. We have previously demonstrated that Flt3-/- mice, with reduced DCs, were protected from collagen induced arthritis (CIA). Here we have shown that GM-CSF derived DCs in Flt3L-/- mice are functional but not sufficient to induce arthritis. Batf3 mice lacking both CD103 and CD8α cDC1 were resistant to collagen induced arthritis (CIA), demonstrating that this DC subset is crucial for arthritis development. CEP-701 (a Flt3L inhibitor) treatment prevented CIA induction, and reduced dramatically the numbers CD103 cDC1s present in the lymph nodes and synovium. Hence this study identified cDC1 as the main subset orchestrating the initiation of cell-mediated immunity in arthritis.
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http://dx.doi.org/10.1016/j.jtauto.2020.100066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522802PMC
September 2020

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

Rheumatology (Oxford) 2020 09;59(9):2258-2263

Department of Rheumatology and Clinical Immunology.

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-β1, 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-β 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-β signalling.

Conclusion: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.
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http://dx.doi.org/10.1093/rheumatology/kez583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449812PMC
September 2020

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

Rheumatology (Oxford) 2020 02;59(2):426-438

Department of Rheumatology and Clinical Immunology, University of Utrecht, Utrecht.

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.
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http://dx.doi.org/10.1093/rheumatology/kez315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571483PMC
February 2020

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

Arthritis Rheumatol 2019 10 27;71(10):1711-1722. Epub 2019 Aug 27.

University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands.

Objective: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc).

Methods: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay.

Results: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors.

Conclusion: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.
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http://dx.doi.org/10.1002/art.40915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790618PMC
October 2019

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

Ann Rheum Dis 2019 04 6;78(4):529-538. Epub 2019 Feb 6.

Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Background And Objective: Systemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes.

Methods: Chromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression.

Results: 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression.

Conclusion: SSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.
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http://dx.doi.org/10.1136/annrheumdis-2018-214295DOI Listing
April 2019

New insights into the genetics and epigenetics of systemic sclerosis.

Nat Rev Rheumatol 2018 11;14(11):657-673

Department of Rheumatology and Clinical Immunology, University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands.

Systemic sclerosis (SSc) is a severe autoimmune disease that is characterized by vascular abnormalities, immunological alterations and fibrosis of the skin and internal organs. The results of genetic studies in patients with SSc have revealed statistically significant genetic associations with disease manifestations and progression. Nevertheless, genetic susceptibility to SSc is moderate, and the functional consequences of genetic associations remain only partially characterized. A current hypothesis is that, in genetically susceptible individuals, epigenetic modifications constitute the driving force for disease initiation. As epigenetic alterations can occur years before fibrosis appears, these changes could represent a potential link between inflammation and tissue fibrosis. Epigenetics is a fast-growing discipline, and a considerable number of important epigenetic studies in SSc have been published in the past few years that span histone post-translational modifications, DNA methylation, microRNAs and long non-coding RNAs. This Review describes the latest insights into genetic and epigenetic contributions to the pathogenesis of SSc and aims to provide an improved understanding of the molecular pathways that link inflammation and fibrosis. This knowledge will be of paramount importance for the development of medicines that are effective in treating or even reversing tissue fibrosis.
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http://dx.doi.org/10.1038/s41584-018-0099-0DOI Listing
November 2018

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

Arthritis Res Ther 2018 07 20;20(1):148. Epub 2018 Jul 20.

Laboratory of Translational Immunology and Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS).

Methods: The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-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.

Results: ITF2357 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.

Conclusions: Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.
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http://dx.doi.org/10.1186/s13075-018-1638-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053802PMC
July 2018

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

Rheumatology (Oxford) 2018 05;57(5):909-920

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and Immunology Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Objective: Class 3 semaphorins regulate diverse cellular processes relevant to the pathology of RA, including immune modulation, angiogenesis, apoptosis and invasive cell migration. Therefore, we analysed the potential role of class 3 semaphorins in the pathology of RA.

Methods: Protein and mRNA expression in RA synovial tissue, SF and fibroblast-like synoviocytes (FLS) were determined by immunoblotting and quantitative PCR (qPCR). RA FLS migration and invasion were determined using wound closure and transwell invasion assays, respectively. PlexinA1, neuropilin-1 and neuropilin-2 expression was knocked down using small interfering RNA (siRNA). Activation of FLS intracellular signalling pathways was assessed by immunoblotting.

Results: mRNA expression of semaphorins (Sema)3B, Sema3C, Sema3F and Sema3G was significantly lower in the synovial tissue of early arthritis patients at baseline who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3B and Sema3F expression was significantly lower in arthritis patients fulfilling classification criteria for RA compared with those who did not. FLS expression of Sema3A was induced after stimulation with TNF, IL-1β or lipopolysaccharides (LPS), while Sema3B and Sema3F expression was downregulated. Exogenously applied Sema3A induced the migration and invasive capacity of FLS, while stimulation with Sema3B or Sema3F reduced spontaneous FLS migration, and platelet-derived growth factor induced cell invasion, effects associated with differential regulation of MMP expression and mediated by the PlexinA1 and neuropilin-1 and -2 receptors.

Conclusion: Our data suggest that modulation of class 3 semaphorin signaling could be a novel therapeutic strategy for modulating the invasive behaviour of FLS in RA.
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http://dx.doi.org/10.1093/rheumatology/kex511DOI Listing
May 2018

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

Arthritis Res Ther 2017 09 18;19(1):207. Epub 2017 Sep 18.

Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Endoplasmic reticulum (ER) stress has proinflammatory properties, and transgenic animal studies of rheumatoid arthritis (RA) indicate its relevance in the process of joint destruction. Because currently available studies are focused primarily on myeloid cells, we assessed how ER stress might affect the inflammatory responses of stromal cells in RA.

Methods: ER stress was induced in RA fibroblast-like synoviocytes (FLS), dermal fibroblasts, and macrophages with thapsigargin or tunicamycin alone or in combination with Toll-like receptor (TLR) ligands, and gene expression and messenger RNA (mRNA) stability was measured by quantitative polymerase chain reaction. Cellular viability was measured using cell death enzyme-linked immunosorbent assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and signaling pathway activation was analyzed by immunoblotting.

Results: No cytotoxicity was observed in FLS exposed to thapsigargin, despite significant induction of ER stress markers. Screening of 84 proinflammatory genes revealed minor changes in their expression (fold change 90th percentile range 2.8-8.3) by thapsigargin alone, but the vast majority were hyperinduced during combined stimulation with thapsigargin and TLR ligands (35% greater than fivefold vs lipopolysaccharide alone). The synergistic response could not be explained by quantitative effects on nuclear factor-κB and mitogen-activated protein kinase pathways alone, but it was dependent on increased mRNA stability. mRNA stabilization was similarly enhanced by ER stress in dermal fibroblasts but not in macrophages, correlating with minimal cooperative effects on gene induction in macrophages.

Conclusions: RA FLS are resistant to apoptosis induced by ER stress, but ER stress potentiates their activation by multiple TLR ligands. Interfering with downstream signaling pathway components of ER stress may be of therapeutic potential in the treatment of RA.
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http://dx.doi.org/10.1186/s13075-017-1386-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604427PMC
September 2017

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

Front Immunol 2017 23;8:720. Epub 2017 Jun 23.

Laboratory of Translational Immunology, Department of Rheumatology and Clinical Immunology, University Medical Center, Utrecht, Netherlands.

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.
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http://dx.doi.org/10.3389/fimmu.2017.00720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481306PMC
June 2017

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

J Leukoc Biol 2017 09 22;102(3):897-904. Epub 2017 Jun 22.

Department of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and Immunology Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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, = 19), psoriatic arthritis (PsA, = 11), and gout ( = 11). Synovial-tissue PRL mRNA expression was measured by quantitative PCR in patients with RA ( = 25), PsA ( = 11), and gout ( = 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, = 0.424; = 0.049) and disease activity score evaluated in 28 joints (DAS28, = 0.729; = 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 ( ≤ 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.
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http://dx.doi.org/10.1189/jlb.2A0317-115RRDOI Listing
September 2017

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

Arthritis Rheumatol 2017 09 25;69(9):1891-1902. Epub 2017 Jul 25.

University Medical Center Utrecht, Utrecht, The Netherlands.

Objective: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.

Methods: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs.

Results: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNα, mimicking the PDC phenotype observed in SSc patients.

Conclusion: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNα, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.
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http://dx.doi.org/10.1002/art.40163DOI Listing
September 2017

The acetyl code in rheumatoid arthritis and other rheumatic diseases.

Epigenomics 2017 04 19;9(4):447-461. Epub 2017 Jan 19.

Laboratory of Translational Immunology & Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.2217/epi-2016-0136DOI Listing
April 2017

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

Mol Immunol 2016 11 3;79:66-76. Epub 2016 Oct 3.

Tytgat Institute for Gastro-Intestinal and Liver Research, Academic Medical Center, Amsterdam, The Netherlands. Electronic address:

Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histone tails. An inhibitor of the acetylated-lysine reader bromodomain and extra-terminal domain (BET) proteins, I-BET151, is known to counteract the induction of expression of inflammatory genes in macrophages. We have investigated the effects of I-BET151 on dendritic cell function, including expression of co-stimulatory molecules and cytokines, and capacity for T cell activation. Treatment of mouse bone marrow derived dendritic cells (BMDC) and human monocyte derived DCs (mdDC) with I-BET151 reduced LPS-induced expression of co-stimulatory molecules, as well as the production of multiple cyokines and chemokines. Most strikingly, secretion of IL-6, IL-12 and IL-10 was significantly reduced to 89.7%, 99.9% and 98.6% respectively of that produced by control cells. I-BET151-treated mdDC showed a reduced ability to stimulate proliferation of autologous Revaxis-specific T cells. Moreover, while I-BET151 treatment of BMDC did not affect their ability to polarise ovalbumin specific CD4 CD62L naive T cells towards Th1, Th2, or Th17 phenotypes, an increase in Foxp3 expressing Tregs secreting higher IL-10 levels was observed. Suppression assays demonstrated that Tregs generated in response to I-BET151-treated BMDC displayed anti-proliferative capacity. Finally, evidence that I-BET151 treatment can ameliorate inflammation in vivo in a T cell dependent colitis model is shown. Overall, these results demonstrate marked effects of BET inhibition on DC maturation, reducing their capacity for pro-inflammatory cytokine secretion and T cell activation and enhancing the potential of DC to induce Foxp3 expressing Treg with suppressive properties.
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http://dx.doi.org/10.1016/j.molimm.2016.09.010DOI Listing
November 2016

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

Rheumatology (Oxford) 2016 Dec 10;55(12):2248-2259. Epub 2016 Sep 10.

Division of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and Immunology Center, Academic Medical Center/University of Amsterdam, Amsterdam, the Netherlands.

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-12β.

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.
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http://dx.doi.org/10.1093/rheumatology/kew316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5144667PMC
December 2016

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

Ann Rheum Dis 2017 Jan 25;76(1):277-285. Epub 2016 Jul 25.

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1136/annrheumdis-2015-209064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264225PMC
January 2017

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

Arthritis Res Ther 2016 Mar 31;18:75. Epub 2016 Mar 31.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1186/s13075-016-0973-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818474PMC
March 2016

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

Ann Rheum Dis 2016 Feb 2;75(2):422-9. Epub 2014 Dec 2.

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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)-1β 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-1β. 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.
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http://dx.doi.org/10.1136/annrheumdis-2014-205809DOI Listing
February 2016

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

Ann Rheum Dis 2016 Feb 1;75(2):430-8. Epub 2014 Dec 1.

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1136/annrheumdis-2014-205635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336378PMC
February 2016

Inhibiting epigenetic enzymes to improve atherogenic macrophage functions.

Biochem Biophys Res Commun 2014 Dec 18;455(3-4):396-402. Epub 2014 Nov 18.

Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.bbrc.2014.11.029DOI Listing
December 2014

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

Ann Rheum Dis 2015 Sep 8;74(9):1763-71. Epub 2014 May 8.

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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, 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.
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http://dx.doi.org/10.1136/annrheumdis-2013-203610DOI Listing
September 2015

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

Ann Rheum Dis 2015 Aug 24;74(8):1603-11. Epub 2014 Apr 24.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1136/annrheumdis-2013-204143DOI Listing
August 2015

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

PLoS One 2014 3;9(1):e82088. Epub 2014 Jan 3.

Department of Experimental Immunology, University of Amsterdam, Amsterdam, the Netherlands ; Department of Clinical Immunology and Rheumatology, University of Amsterdam, Amsterdam, the Netherlands.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082088PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880273PMC
September 2014

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis.

Arthritis Res Ther 2013 ;15(6):R209

Introduction: The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.

Methods: The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.

Results: Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.

Conclusions: The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.
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http://dx.doi.org/10.1186/ar4403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978611PMC
November 2014

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

Arthritis Rheum 2013 Dec;65(12):3073-83

Academic Medical Center and University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1002/art.38128DOI Listing
December 2013

The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2.

Biol Open 2013 Jun 16;2(6):569-79. Epub 2013 Apr 16.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam , Amsterdam, 1066CX , The Netherlands.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.
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http://dx.doi.org/10.1242/bio.20134382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683159PMC
June 2013

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

J Immunol 2013 Apr 27;190(7):3740-8. Epub 2013 Feb 27.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β 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.
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http://dx.doi.org/10.4049/jimmunol.1202192DOI Listing
April 2013
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