Pernille A. Usher

Urokinase Plasminogen Activator Is a Central Regulator of Macrophage Three-Dimensional Invasion, Matrix Degradation, and Adhesion

Urokinase plasminogen activator (uPA) and its receptor (uPAR) coordinate a plasmin-mediated proteolytic cascade that has been implicated in cell adhesion, cell motility, and matrix breakdown, for example, during inflammation. As part of their function during inflammatory responses, macrophages move through tissues and encounter both two-dimensional (2D) surfaces and more complex three-dimensional (3D) interstitial matrices. Based on approaches employing uPA gene–deficient macrophages, plasminogen supplementation, and neutralization with specific protease inhibitors, it is reported in this study that uPA activity is a central component of the invasion of macrophages through a 3D Matrigel barrier; it also has a nonredundant role in macrophage-mediated matrix degradation. For murine macrophages, matrix metalloproteinase-9 activity was found to be required for these uPA-mediated effects. Evidence for a unique role for uPA in the inverse relationship between macrophage adhesion and 2D migration was also noted: macrophage adhesion to vitronectin was enhanced by uPA and blocked by plasminogen activator inhibitor-1, the latter approach also able to enhance in turn the 2D migration on this matrix protein. It is therefore proposed that uPA can have a key role in the inflammatory response at several levels as a central regulator of macrophage 3D invasion, matrix remodeling, and adhesion.

Rapid‐onset clinical and mechanistic effects of anti‐C5aR treatment in the mouse collagen‐induced arthritis model

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti‐C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti‐murine C5aR (anti‐mC5aR) surrogate antibody in mouse collagen‐induced arthritis (CIA). First, efficacy was demonstrated in a multiple‐dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti‐mC5aR‐treated mice. Then, the mechanism of action was examined by looking for early effects of anti‐mC5aR treatment in single‐dose treatment studies. We found that 48 h after single‐dose treatment with anti‐mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose‐setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti‐C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice

IntroductionRheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.MethodsDTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.ResultsWe demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.ConclusionsOur findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.

Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model.

Abstract Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.

Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

Urokinase plasminogen activator and receptor promote collagen-induced arthritis through expression in hematopoietic cells

The plasminogen activation (PA) system has been implicated in driving inflammatory arthritis, but the precise contribution of PA system components to arthritis pathogenesis remains poorly defined. Here, the role of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) in the development and severity of inflammatory joint disease was determined using uPA- and uPAR-deficient mice inbred to the strain DBA/1J, a genetic background highly susceptible to collagen-induced arthritis (CIA). Mice deficient in uPA displayed a near-complete amelioration of macroscopic and histological inflammatory joint disease following CIA challenge. Similarly, CIA-challenged uPAR-deficient mice exhibited significant amelioration of arthritis incidence and severity. Reduced disease development in uPA-deficient and uPAR-deficient mice was not due to an altered adaptive immune response to the CIA challenge. Reciprocal bone marrow transplant studies indicated that uPAR-driven CIA was due to expression by hematopoietic-derived cells, as mice with uPAR-deficient bone marrow challenged with CIA developed significantly reduced macroscopic and histological joint disease as compared with mice with uPAR expression limited to non-hematopoietic-derived cells. These findings indicate a fundamental role for uPAR-expressing hematopoietic cells in driving arthritis incidence and progression. Thus, uPA/uPAR-mediated cell surface proteolysis and/or uPAR-mediated signaling events promote inflammatory joint disease, indicating that disruption of this key proteolytic/signaling system may provide a novel therapeutic strategy to limit clinical arthritis.

Sensitive and specific in situ hybridization for early drug discovery.

High-throughput analyses of gene expression such as microarrays and RNA-sequencing are widely used in early drug discovery to identify disease-associated genes. To further characterize the expression of selected genes, in situ hybridization (ISH) using RNA probes (riboprobes) is a powerful tool to localize mRNA expression at the cellular level in normal and diseased tissues, especially for novel drug targets, where research tools like specific antibodies are often lacking.We describe a sensitive ISH protocol using radiolabelled riboprobes suitable for both paraffin-embedded and cryo-preserved tissue. The riboprobes are generated by in vitro transcription using PCR products as templates, which is less time consuming compared to traditional transcription from linearized plasmids, and offers a relatively simple way to generate several probes per gene, e.g., for splice variant analyses. To ensure reliable ISH results, we have incorporated a number of specificity controls in our standard experimental setup. We design antisense probes to cover two non-overlapping parts of the gene of interest, and use the corresponding sense probes as controls for unspecific binding. Probes are furthermore tested on sections of paraffin-embedded or cryo-preserved positive and negative control cells with known gene expression. Our protocol thus provides a method for sensitive and specific ISH, which is suitable for target validation and characterization in early drug discovery.

THU0025 Expression of interleukin-20 and its receptors in osteoblasts and osteoclasts in bone tissue from patients with rheumatoid arthritis

Background Elevated expression of interleukin-20 (IL-20) and its receptors has been demonstrated in synovium from patients with rheumatoid arthritis (RA), and IL-20 is thought to be implicated in the pathogenesis of RA. Selective inhibition of IL-20 reduced disease activity and bone loss in rats with collagen-induced arthritis (1). In support of a role of IL-20 in bone erosion in RA, it has been demonstrated in murine cells that IL-20 stimulates expression of RANK-L in osteoblasts and RANK in osteoclasts, which leads to osteoclastogenesis and induces osteoclast activity (2) Objectives To analyse the expression and localisation of IL-20 and its receptor chains IL-20R1, IL-20R2 and IL-22R in bone samples from patients with RA using immunohistochemistry and cell identification with selected bone cell markers Methods RA bone and synovial samples were collected following joint replacement knee surgery. The bone samples were decalcified in ImmunoCal and paraffin embedded. Immunohistochemistry was performed with antibodies against IL-20 (rabbit polyclonal 2313b, Novo Nordisk), IL-20R1 (mouse monoclonal MAB11761, R&D), IL-20R2 (goat polyclonal AF1788, R&D) or IL-22R (goat polyclonal BAF2770, R&D) and immunoreactivity was visualised with DAB. Osteoblasts were identified with an antibody against RANK-L. Osteoclasts were identified using a histochemical staining for Tartrate-Resistant Alkaline Phosphatase (TRAP). The specificity of IL-20, IL-20R1, IL-20R2 and IL-22R antibodies was validated in sections of paraffin-embedded cells transfected with IL-20, or either of the two receptor complexes IL-20R1/IL-20R2 or IL-22R/IL-20R2 Results Immunohistochemical staining of paired synovial and bone tissue samples from the same patient revealed that IL-20 is present in synovial inflammatory cells located in both the lining layer and the sublining layer as previously reported (3). In the RA bone samples, a distinct localisation of IL-20 is noted in the osteoblasts lining both trabecular and cortical bone, whereas a weaker staining of IL-20 is detected in the adjacently located osteoclasts. Immunostaining with an antibody against RANK-L showed that IL-20 and RANK-L are expressed in osteoblasts in similar patterns. Expression of all three IL-20 receptor chains were analysed in bone samples from patients with RA. In serial sections stained with anti-IL-20, anti-IL-20R1 or TRAP, it was demonstrated that IL-20R1 is expressed in trabecular TRAP-positive osteoclasts, but not in the adjacent IL-20-positive osteoblasts Conclusions IL-20 is highly expressed in osteoblasts, and IL-20R1 is found in osteoclasts in bone samples from patients with RA. In addition to the role of IL-20 in synovial inflammation, the present data supports the hypothesis that IL-20 may also be involved in bone modulation in human disease, and that neutralisation of IL-20 may lead to inhibition of bone erosion in patients with RA. A human anti-IL-20 monoclonal antibody, NNC0109-0012, is currently being studied in patients with rheumatoid arthritis References Hsu et al, Arthritis Rheum. 62:3311-21, 2010; 2 Hsu et al, J Exp Med. 208:1849-61, 2011; 3 Rømer et al, Abstract #FRI0079 at EULAR, 2009 Disclosure of Interest J. Rømer Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, P. Usher Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, M. Nielsen Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, J. Mandelbaum Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, M. Jackerott Shareholder of: Novo Nordisk, Employee of: Novo Nordisk

AB0079 Anti-c5a receptor antibody treatment ameliorates disease activity in delayed-type hypersensitivity (dth) arthritic mice

Background C5a is an anaphylatoxin generated in response to complement activation. C5a promotes leukocyte chemotaxis and activation via the C5a receptor (C5aR), both directly on C5aR-positive cells such as neutrophils, monocytes and macrophages, and indirectly on T cells via effects on C5aR-positive antigen specific cells (APCs)1. C5a production and C5aR signalling have been implicated in a wide range of inflammatory disorders including rheumatoid arthritis (RA)2. DTH arthritis is a single-paw arthritis model, which is dependent on CD4+ T cells. Besides T cells, neutrophils and macrophages are involved in the pathology, and inflammatory mediators associated with these cells are induced locally in the arthritic paw3 Objectives The purpose of this study was to investigate the therapeutic effect of a blocking anti-C5aR antibody in a single–paw arthritis model in mice in both a 2-week multiple-dose (MD) study and in a single-dose (SD) study with read-out at 60 h post-dosing. Methods DTH arthritis was induced as described by Atkinson3. In the MD study mice were treated twice weekly with anti-C5aR antibody (25 mg/kg/dose) from the day of arthritis induction. Mechanistic studies were conducted to investigate the immune-modulating effect of a SD of 25 mg/kg anti-C5aR with read-out at 60 h post-dosing/arthritis induction. Cytokine/chemokine levels in whole–paw homogenate supernatants were analyzed using ELISA and bioplex. Paw sections were evaluated histologically, and composition of leukocyte subsets isolated from lymph nodes and arthritic paws were analyzed by flow cytometry. Statistics: unpaired two-sided t-test. Results Significant reduction in paw swelling (P<0.01) and histopathology score (P<0.0001) was observed in the MDstudy. Swelling of the arthritic paw was also significantly reduced compared to control–treated mice in the SD study (P<0.0001). Also a single dose of anti-C5aR resulted in significantly reduced levels of MIP-2, MCP-1 and TNFα (P<0.05 for each analyte) in the arthritic paw compared to the levels found in control-treated mice. However, no difference was detected in the total number of leukocytes infiltrating the arthritic paws 60 h after SD treatment. Flow cytometric analysis of leukocyte subsets isolated from arthritic pawsafter SD treatment revealed significant reductions in CD4+ T cell counts of anti-C5aR-treated mice compared to control-treated mice (P<0.05), which correlated with significantly reduced CD4+ T cell proliferation (P<0.05) in the draining popliteal lymph node. Target validation in control-treated DTH arthritic mice revealed expression of C5aR on neutrophils (blood and paw) and on a subset of macrophages (paw), but not on CD4+ T cells. Conclusions Our studies reveal a significant effect of anti-C5aR treatment on DTH arthritis disease activity, and our mechanistic SD studies strongly suggests that the effect is mediated via reduced local production of inflammatory mediators and reduced CD4+ T cell proliferation, while no effect on leukocyte chemotaxis was observed 60 h after SD anti-C5aR antibody treatment. The effect on CD4+ T cell proliferation is probably indirect via effects of an anti-C5aR antibody on APCs. References Dunkelberger JR et al.(2010) Mol Immunol 47: 2176–2186; Lee H et al. (2008) Immunol Cell Biol 86: 153–160; Atkinson SA et al. (2012) Arthritis Res Ther 14(3):R134 Disclosure of Interest A. Nansen Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, S. Atkinson Employee of: PhD student, Research performed at Novo Nordisk A/S, P. Usher Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, C. Haase Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, L. Hornum Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S