Catherine Swales

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

TSG-6 inhibits osteoclast activity via an autocrine mechanism and is functionally synergistic with osteoprotegerin

OBJECTIVE TSG-6 (the product of tumor necrosis factor [TNF]-stimulated gene 6) has a potent inhibitory effect on RANKL-mediated bone erosion. The aim of this study was to compare the activity of TSG-6 with that of osteoprotegerin (OPG) and to investigate its role as an autocrine modulator of cytokine-mediated osteoclast formation/activation. We also determined TSG-6 expression in inflammatory joint disease. METHODS The effects of TSG-6, OPG, and the inflammation mediators TNFα, interleukin-1 (IL-1), and IL-6 on the formation of osteoclasts from peripheral blood mononuclear cells and synovial fluid (SF) macrophages were determined by tartrate-resistant acid phosphatase staining. Lacunar resorption and filamentous actin ring formation were measured as indicators of osteoclast activity. The amount of TSG-6 in culture media or SF was quantified by enzyme-linked immunosorbent assay, and expression of TSG-6 in synovial tissue was assessed by immunohistochemistry. RESULTS TSG-6 acted in synergy with OPG to inhibit RANKL-mediated bone resorption and was produced by osteoclast precursors and mature osteoclasts in response to TNFα, IL-1, and IL-6. Expression of TSG-6 correlated with inhibition of lacunar resorption; this effect was ameliorated by an anti-TSG-6 antibody. The level of TSG-6 protein was determined in SF from patients with various arthritides; it was highest in patients with inflammatory conditions such as rheumatoid arthritis, in which it correlated with the amount of TSG-6 immunostaining in the synovium. TSG-6 inhibited the activation but not the formation of osteoclasts from SF macrophages. CONCLUSION In the presence of inflammatory cytokines, osteoclasts produced TSG-6 at concentrations that are sufficient to inhibit lacunar resorption. This may represent an autocrine mechanism to limit the degree of bone erosion during joint inflammation.

Independent roles for the dorsal paraflocculus and vermal lobule VII of the cerebellum in visuomotor coordination.

Two distinct areas of cerebellar cortex, vermal lobule VII and the dorsal paraflocculus (DPFl) receive visual input. To help understand the visuomotor functions of these two regions, we compared their afferent and efferent connections using the tracers wheatgerm agglutinin horseradish peroxidase (WGA-HRP) and biotinilated dextran amine (BDA). The sources of both mossy fibre and climbing fibre input to the two areas are different. The main mossy fibre input to lobule VII is from the nucleus reticularis tegmenti pontis (NRTP), which relays visual information from the superior colliculus, while the main mossy fibre input to the DPFl is from the pontine nuclei, relaying information from cortical visual areas. The DPFl and lobule VII both also receive mossy fibre input from several common brainstem regions, but from different subsets of cells. These include visual input from the dorsolateral pons, and vestibular–oculomotor input from the medial vestibular nucleus (MVe) and the nucleus prepositus hypoglossi (Nph). The climbing fibre input to the two cerebellar regions is from different subdivisions of the inferior olivary nuclei. Climbing fibres from the caudal medial accessory olive (cMAO) project to lobule VII, while the rostral MAO (rMAO) and the principal olive (PO) project to the DPFl. The efferent projections from lobule VII and the DPF1 are to all of the recognised oculomotor and visual areas within the deep cerebellar nuclei, but to separate territories. Both regions play a role in eye movement control. The DPFl may also have a role in visually guided reaching.

Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption.

Introduction Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA). The secreted adipokine angiopoietin-like 4 (ANGPTL4) specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA). Results Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL). Conclusions Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA.

Cellular and molecular mechanisms of bone damage and repair in inflammatory arthritis.

Bone remodelling relies on tightly controlled cycles of bone resorption and formation, mediated by osteoclasts and osteoblasts, respectively. The past two decades have seen a huge increase in our understanding of immune modulation and disruption of bone homeostasis in rheumatic diseases; identification of the molecular pathways responsible for accelerated bone loss in such conditions has given rise to potential novel therapeutic targets. Most recently, the role of microRNAs in inflammatory and noninflammatory bone loss raises the intriguing possibility that modification of cellular protein translation could also be a treatment strategy for bone damage.

A4.9 Angiopoietin-like 4 is over-expressed in rheumatoid arthritis: a potential role in pathological bone resorption.

Background and Objectives In contrast to normal synovial tissue, rheumatoid synovium is hypoxic, and expresses the hypoxia-inducible transcription factors HIF-1α and HIF-2α which allow the transcription of genes involved in angiogenesis, inflammation, apoptosis and regulation of immune function. Hypoxia also stimulates osteoclast differentiation and causes a HIF-1α-dependent 3-fold increase in bone resorption. Angiopoietin-like 4 (ANGPTL4) is a hypoxia- and HIF-inducible pro-angiogenic adipokine that is induced in fibroblast-like synoviocytes in rheumatoid arthritis. This study sought to investigate whether ANGPTL4 is expressed in osteoclasts and other cells within rheumatoid synovial tissue, and to compare serum and synovial fluid levels of ANGPTL4 with those in normal controls and patients with osteoarthritis. Materials and Methods Serum, synovial fluid and synovial tissue samples were derived from patients with osteoarthritis (OA) and rheumatoid arthritis (RA); serum was obtained from aged-matched normal controls. All donors were recruited from the Nuffield Orthopaedic Centre, Oxford, UK and gave written informed consent. ANGPTL4 and HIF-1α expression was assessed in OA and RA synovial sections by immunohistochemistry and immunofluorescence. Serum and synovial fluid levels of ANGPTL4 were measured by ELISA. Osteoclasts were differentiated from circulating RA monocytes using M-CSF and RANKL, and hypoxic induction of ANGPTL4 mRNA was measured by real-time PCR. Results Bone-apposing osteoclasts within the rheumatoid synovium expressed ANGPTL4 and its regulating transcription factor HIF-1α. ANGPTL4 was strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68 + macrophages and plasma cells within the RA synovium. Little ANGPTL4 was evident in normal synovium, mirroring the expression pattern of HIF-1α in rheumatoid versus normal synovial tissue. ANGPTL4 concentrations were higher in the serum and synovial fluid of RA patients than in OA patients or normal controls. High serum ANGPTL4 correlated with elevated levels of the serum bone resorption marker sRANKL. Finally, ANGPTL4 mRNA was induced 5.5-fold by hypoxia in monocyte-derived osteoclasts from RA patients. Conclusions ANGPTL4 is over-expressed in both the serum and the synovial fluid and tissue of RA patients. Expression of ANGPTL4 in bone-apposing osteoclasts and correlation of high serum ANGPTL4 with circulating sRANKL suggests ANGPTL4 as a marker for bone destruction, and a potential target for inhibition of osteoclast-mediated bone resorption in RA.

Role of LIGHT in the pathogenesis of joint destruction in rheumatoid arthritis.

AIM To characterise the role of substitutes for receptor-activator nuclear factor kappa-B ligand (RANKL) in rheumatoid arthritis (RA) joint destruction. METHODS Synovial fluid (SF) macrophages isolated from the knee joint of RA patients were incubated with 25 ng/mL macrophage-colony stimulating factor (M-CSF) and 50 ng/mL LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) in the presence and absence of 25 ng/mL RANKL and 100 ng/mL osteoprotegerin (OPG) on glass coverslips and dentine slices. Osteoclastogenesis was assessed by the formation of multinucleated cells (MNCs) expressing tartrate-resistant acid phosphatase (TRAP) on coverslips and the extent of lacunar resorption pit formation on dentine slices. The concentration of LIGHT in RA and osteoarthritis (OA) synovial fluid was measured by an enzyme-linked immunosorbent assay (ELISA) and the expression of LIGHT in RA and OA synovium was determined by immunohistochemistry using an indirect immunoperoxidase technique. RESULTS In cultures of RA SF macrophages treated with LIGHT and M-CSF, there was significant formation of TRAP + MNCs on coverslips and extensive lacunar resorption pit formation on dentine slices. SF-macrophage-osteoclast differentiation was not inhibited by the addition of OPG, a decoy receptor for RANKL. Resorption pits were smaller and less confluent than in RANKL-treated cultures but the overall percentage area of the dentine slice resorbed was comparable in LIGHT- and RANKL-treated cultures. LIGHT significantly stimulated RANKL-induced lacunar resorption compared with RA SF macrophages treated with either RANKL or LIGHT alone. LIGHT was strongly expressed by synovial lining cells, subintimal macrophages and endothelial cells in RA synovium and the concentration of LIGHT was much higher in RA compared with OA SF. CONCLUSION LIGHT is highly expressed in RA synovium and SF, stimulates RANKL-independent/dependent osteoclastogenesis from SF macrophages and may contribute to marginal erosion formation.

Thoughts of being an imposter, in medical students

...Whereas I, as I do not know anything, do not think I do either. Plato 1 Insight, as we imagine what Plato alludes to here, is vital in both learning and practising medicine. Without it, one is in danger of developing a sense of superiority, even omnipotence, that may come with the title ‘Doctor’: an individual who is likely to be respected, deeply valued and, most importantly, trusted. Being doctors, we recognise that it is only with refl ection and insight that we are humbled by the weaknesses and breaches in our knowledge. Ultimately, we are responsible for human lives, and this is not to be taken lightly.

IDENTIFICATION OF CHROMATIN MODIFYING MECHANISMS IN INFLAMMATORY MACROPHAGES IN RHEUMATOID ARTHRITIS

Background Alterations in epigenetic mechanisms have been implicated in the regulation of pro-inflammatory cytokine production, and bromodomain-containing proteins (the BET family) may have a specific role in the transcriptional regulation of inflammatory genes. Our objective was to examine specific epigenetic variation between the responses of macrophages derived from RA patients and healthy volunteers, and those derived from inflammatory synovial fluid (SF). Delineation of any epigenetic differences between different macrophage populations may highlight new therapeutic targets in inflammatory disease. Materials and methods A library of small molecule inhibitors, including BET protein inhibitors, was used to identify key epigenetic regulators of pro-inflammatory cytokine production from macrophages. Macrophages were differentiated from the blood monocytes of normal volunteers and RA patients. Synovial fluid derived macrophages were isolated from RA patients undergoing therapeutic arthrocentesis. Cell culture media was supplemented with a small molecule inhibitor, and lipopolysaccharide subsequently used to stimulate inflammatory cytokine production. Cell viability was confirmed using WST-1 assay, cytokine production was measured using an MSD platform and qPCR to assess gene expression. Each experimental condition was performed in triplicate, alongside positive and negative controls. The study was ethically approved and all samples were obtained with informed consent. Results Inhibitors of BET proteins (JQ1, I-BET151 and PFI-1) were the only class of inhibitor to show consistent down-regulation of pro-inflammatory cytokines in both healthy and RA-patient derived macrophages. BET protein inhibitors significantly suppressed the production of TNFα and IL-6 production from RA blood derived macrophages and IL-6 from corresponding SF-derived macrophages. However, only JQ1 achieved significant inhibition of TNFα production from SF-derived macrophages; it also abrogated LPS-induced cytokine and chemokine mRNA expression in the macrophages of patients with RA. Conclusions This data suggests that the BET proteins are essential regulators of pro-inflammatory cytokine and chemokine production from the macrophages of patients with RA. The differential response to BET inhibition by macrophages derived from blood and synovial fluid of RA patients could indicate alteration in BET protein distribution at pro-inflammatory cytokine promoters, influenced by the chronic inflammatory environment. This study highlights the therapeutic potential of BET inhibitors (particularly JQ1) in RA. Funding acknowledgements NIHR BRU, BBSRC and GlaxoSmithKline

SAT0193 Utility of Ultrasound in the Nuffield Orthopaedic Centre Emergency Rheumatology Clinic: Survey of Clinical Effectiveness

Background The aim of the survey was to evaluate the impact of clinic-based musculoskeletal ultrasonography (MSUS) on diagnosis and management of cases seen in the Nuffield Orthopaedic Hospital emergency rheumatology clinic. Objectives Primary: To assess the clinical utility of US in the emergency clinic: aiding diagnosis assess disease severity Secondary: Does US alter clinical management decisions? Methods MSUS was performed on a selected population of cases, which included new patients with diagnostic uncertainty and challenging follow-up patients requiring assessment of disease activity/severity. The service was provided by a consultant rheumatologist trained and experienced in MSUS; the scans were performed during the emergency clinic appointment. Clinician evaluating patient requested MSUS for confirmation of diagnosis or in cases of diagnostic uncertainty. All scans were performed on a GE Logiq E9 using a linear transducer, recording grey-scale and power Doppler findings. Data from all patients who had undergone scans during October 2011- November 2012 was reviewed for demographics, suspected or existing diagnosis of inflammatory arthritis, clinical presentation, clinical findings and management outcome as a direct result of MSUS. Results There were 62 patients studied (25 men, 38 women); their mean age was 57.17 years (range 30-88). A set joint scan was performed in all patients consisting of 10 MCP and PIP joints, radiocarpal joint and ulnar styloid. The new patient group consisted of 34 patients; all had been referred for inflammatory arthritis. Of these, at ultrasound 17 (50%) had osteophytes, 16 (47%) had grey-scale synovitis, with 15 (44%) power Doppler. In one, (3%) no abnormality was detected. This resulted in change in final diagnosis in 22 (65%) new patients and a confirmed diagnosis of active inflammatory arthritis in 12 (35%) patients. Overall, management of new patients directly influenced by ultrasound scan resulted in the discharge of 50% of patients. Of the 17/34 new patients who had a confirmed diagnosis of inflammatory arthritis, 14 (82%) started combination disease modifying anti rheumatic agents at first visit. There were 28 patients in the follow-up group who were referred for diagnostic uncertainty. Rheumatoid arthritis, psoriatic arthritis, connective tissue disease accounted for the majority, with 17 (61%), 5 (18%), 4 (14%) and 3 (11%) patients, respectively. The impact of MSUS on the follow-up group influenced change in treatment in 13/28 (47%) of patients. Specifically, all RA patients underwent scanning for disease assessment. In 15/17 (88%) patients, treatment escalation was directly influenced by MSUS findings; co-existing pathology was detected in 3/17 (18%) which included findings of gout and osteoarthritis. Ultrasound remission was identified in 5/17 (30%) with 2/17 (12%) were started on neuromodulators for pain management. Conclusions This data shows the positive impact of MSUS in the rheumatology clinic, specifically highlighting multiple benefits in daily practice of reduced visits, discharge at first encounter, immediate management decisions. Our survey shows the importance of integrating MSUS service in a one-stop clinic. References Agrawal S, Bhagat S, Dasgupta B. Improvement in diagnosis and management of musculoskeletal conditions with one-stop clinic-based ultrasonography. Mod Rheumatol. 2009;19(1):53-6. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4650