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© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ., Background: Postgraduate rheumatology training programmes are already established at a national level in most European countries. However, previous work has highlighted a substantial level of heterogeneity in the organisation and, in part, content of programmes. Objective: To define competences and standards of knowledge, skills and professional behaviours required for the training of rheumatologists. Methods: A European Alliance of Associations for Rheumatology (EULAR) task force (TF) of 23 experts, including two members of the European Union of Medical Specialists (UEMS) section of rheumatology, was convened. The mapping phase consisted of the retrieval of key documents on specialty training in rheumatology and other related specialties across a broad set of international sources. The content of these documents was extracted and represented the foundation for the document draft that underwent several rounds of online discussion within the TF, and afterwards was also distributed to a broad group of stakeholders for collecting feedback. The list of generated competences was voted on during the TF meetings, while the level of agreement (LoA) with each statement was established by anonymous online voting. Results: A total of 132 international training curricula were retrieved and extracted. In addition to the TF members, 253 stakeholders commented and voted on the competences through an online anonymous survey. The TF developed (1) an overarching framework indicating the areas that should be addressed during training, (2) 7 domains defining broad areas that rheumatology trainees should master by the end of the training programme, (3) 8 core themes defining the nuances of each domain and (4) 28 competences that trainees should acquire to cover each of the areas outlined in the overarching framework. A high LoA was achieved for all competences. Conclusion: These points to consider for EULAR-UEMS standards for the training of European rheumatologists are now defined. Their dissemination and use can hopefully contribute to harmonising training across European countries., The project was funded by the European Alliance of Associations for Rheumatology (grant number EDU049).

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL’s bioactivity.

The US Food and Drug Administration (FDA) has recently added a new ‘black box warning’ on all currently approved Janus kinase (JAK) inhibitors indicated for the treatment of arthritis and other inflammatory conditions based on results from the ORAL Surveillance study of tofacitinib versus tumour necrosis factor alpha inhibitors in rheumatoid arthritis. This is a warning difficult to ignore because the data, being from a randomised controlled trial, are of high fidelity and hard to reproach. It is especially problematic because safety data for all the other JAK inhibitors will be pending for several years. So how might we proceed, without being bound by our stasis? The lack of absolute certainty seems to require a pragmatic approach to the routine care use of JAK inhibitors. The patients who were at greatest risk were older and had other risk factors for the corresponding adverse events, in keeping with effect modification. This highlights the need to focus on risk stratification when tailoring therapy. In this viewpoint, we propose a simple illustration to guide clinical decision-making. First, identify general risk factors for venous thromboembolic event (VTE), major adverse cardiac event (MACE) and cancer (age>65 years and smoking) and whether there is a previous history of VTE, MACE or cancer. Then, evaluate risk based on the number of other risk factors for VTE and the number of other risk factors for MACE. Ultimately, ‘treat-to-target’ will in the end always be ‘treat-to-agreement’. As we have done in the past, and will do in the future, the optimal treatment strategy will have to be tailored based on individual patient risk factors and preferences in a shared-decision process.

The primary vasculitides constitute a heterogeneous group of immune mediated diseases of incompletely understood pathogenesis currently classified by the size of blood vessels affected (Chapel Hill classification). In recent years, several drugs with well-characterized immunological targets have been tested in clinical trials in large vessel vasculitis and small vessel vasculitis. Such trials provide "reverse translational" or bedside to bench information about underlying pathogenic mechanisms. Therefore, the aim of this systematic literature review was to examine the evidence base for a more refined mechanistic immunological classification of vasculitis. A total of 40 studies (20 randomized controlled trials (RCTs), 16 prospective studies, 1 retrospective cohort study and 3 case series) were included for full qualitative assessment. RCTs concerning biologic therapy for large vessel vasculitis mainly supports interleukin 6 receptor inhibition (tocilizumab). RCTs concerning biologic therapy for granulomatosis with polyangiitis and microscopic polyangiitis mainly support anti-CD20 treatment (rituximab) and complement inhibition with a small molecule C5a receptor antagonist (avacopan) is an emerging treatment option. The biologic treatment of eosinophilic granulomatosis with polyangiitis is centered around interleukin 5 inhibition (mepolizumab). Studies on tumor necrosis factor alpha inhibition (adalimumab, infliximab, and etanercept) showed negative results in giant cell arteritis but some effect in Takayasu arteritis. Taken together, clinical studies with cytokine and cell specific drugs are dissecting the heterogeneous immunopathogenic mechanisms of vasculitis and support a mechanistic immunological classification. Especially, cytokine antagonism is pointing towards immunological distinctions between eosinophilic granulomatosis with polyangiitis and granulomatosis with polyangiitis/microscopic polyangiitis and differences between giant cell arteritis and Takayasu arteritis.

Axial spondyloarthritis (axSpA), psoriatic arthritis (PsA), psoriasis, inflammatory bowel disease (IBD), and noninfectious uveitis form a distinct group among the immune mediated inflammatory diseases. Thus, many patients suffer from more than one of these disease manifestations. Here, we will use the term spondylitis-psoriasis-enthesitis-enterocolitis-dactylitis-uveitis-peripheral synovitis (SPEED-UP) spectrum disease. The aim is to review the new targeted pharmacological treatment options for all these diseases. All biological or targeted synthetic drugs with U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA) approval for any of the diagnoses axSpA, PsA, psoriasis, IBD, or non-infectious uveitis were included. Some of the drugs have documented efficacy in more than one of the diseases, e.g. tumor necrosis factor (TNF) inhibitors. However, other drugs are particularly effective for a specific inflamed tissue and approved in only one or two of the disease entities, e.g. abatacept for peripheral arthritis and vedolizumab for inflammatory bowel disease. This contributes with bedside to bench understanding of the immunology underlying this disease spectrum and provides clinicians with an overview that can assist stratified treatment decisions. We hope that this review will help guide clinicians to speed up treatment of patients with this disease spectrum. ispartof: AUTOIMMUNITY REVIEWS vol:20 issue:2 ispartof: location:Netherlands status: published

ObjectiveTo update the European League Against Rheumatism (EULAR) recommendations for the pharmacological treatment of psoriatic arthritis (PsA).MethodsAccording to the EULAR standardised operating procedures, a systematic literature review was followed by a consensus meeting to develop this update involving 28 international taskforce members in May 2019. Levels of evidence and strengths of recommendations were determined.ResultsThe updated recommendations comprise 6 overarching principles and 12 recommendations. The overarching principles address the nature of PsA and diversity of both musculoskeletal and non-musculoskeletal manifestations; the need for collaborative management and shared decision-making is highlighted. The recommendations provide a treatment strategy for pharmacological therapies. Non-steroidal anti-inflammatory drugs and local glucocorticoid injections are proposed as initial therapy; for patients with arthritis and poor prognostic factors, such as polyarthritis or monoarthritis/oligoarthritis accompanied by factors such as dactylitis or joint damage, rapid initiation of conventional synthetic disease-modifying antirheumatic drugs is recommended. If the treatment target is not achieved with this strategy, a biological disease-modifying antirheumatic drugs (bDMARDs) targeting tumour necrosis factor (TNF), interleukin (IL)-17A or IL-12/23 should be initiated, taking into account skin involvement if relevant. If axial disease predominates, a TNF inhibitor or IL-17A inhibitor should be started as first-line disease-modifying antirheumatic drug. Use of Janus kinase inhibitors is addressed primarily after bDMARD failure. Phosphodiesterase-4 inhibition is proposed for patients in whom these other drugs are inappropriate, generally in the context of mild disease. Drug switches and tapering in sustained remission are addressed.ConclusionThese recommendations provide stakeholders with an updated consensus on the pharmacological management of PsA, based on a combination of evidence and expert opinion.

Objective: A reduction in haemoglobin level is a frequent complication among rheumatoid arthritis (RA) patients. Hepcidin has been linked to disturbed erythropoiesis. The objective of this study was to investigate the longitudinal changes in hepcidin in patients with early RA. Method: Hepcidin plasma concentrations were measured by enzyme-linked immunosorbent assay in patients with early RA (n = 80) and healthy volunteers (HV, n = 40). Haemoglobin and other iron-related proteins were also measured. At baseline, all patients had active disease and were treatment naïve. Patients were treated with disease-modifying anti-rheumatic drugs (DMARDs) and with additional adalimumab (ADA, n = 42) or placebo (PLA, n = 38) during 52 weeks, using a treat-to-target strategy, aiming for a 28-joint Disease Activity Score (DAS28)

The aim was to examine anti-tumor necrosis factor α (anti-TNFα) therapy influence changes on Th17 and Th22 cells in patients with spondyloarthritis (SpA), and its correlation with changes in clinical and magnetic resonance imaging (MRI) activity and chronicity scores. The Th17 and Th22 cells were assessed at baseline, after 12 and 52 weeks of anti-TNFα therapy by flow cytometry (ClinicalTrials.gov NCT4682724). The percentages of both Th17 and Th22 cells were increased by 70% at baseline compared with healthy controls (both p

Background:Rheumatoid arthritis (RA) is an immune mediated inflammatory disease with autoimmune features, including antibodies to citrullinated proteins and peptides (ACPAs). Several in vitro studies have suggested a pathogenic role of ACPAs in RA. However, in vivo proof of this concept has been hampered by the lack of therapeutic strategies to reduce or deplete ACPA in serum and synovial fluid. Previously, we constructed a chitosan-hyaluronic acid nanoparticle formulation with the ability to use neutrophil recruitment as a delivery mechanism to inflamed joints. Specifically, nanoparticles got phagocytosed and then released to synovial fluid upon death of the short-lived neutrophilsObjectives:We hypothesized that reducing ACPA levels would have a therapeutic effect by blocking cytokine production. In this study, we prepared and tested a series of therapeutic nanoparticles for specific targeting of ACPA in synovial fluid.Methods:Nanoparticles were prepared by the microdroplet method and then decorated with synthetic cyclic citrullinated peptide aptamer PEP2, PEG/hexanoic acid and fluorophore (Cy5.5). Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Nanoparticles were then used in a series of in vitro assays, including cell uptake with flow cytometry (FACS) detection, and in vivo studies including disease activity scores, cytokine measurements and near-infrared imaging.Results:We screened a series of citrullinated peptide epitopes and identified a fibrinogen-derived 21-amino-acid-long citrullinated peptide showing high selectivity toward autoantibodies in RA samples. We incorporated this aptamer in the chitosan-hyaluronic acid nanoparticle formulation previously described. Average nanoparticle size was 230 nm ± 10 nm by DLS and SEM; z potential was -0.0012. Purity by HPLC was over 95%. Attachment efficiency of the aptamer was 92% by HPLC. FACS study showed selective uptake of Cy5.5 labelled aptamer-nanoparticle conjugates by neutrophils in the concentration range 0.5-4 nM. Similar to previous studies,1there was no apparent immunogenicity for this nanoparticle formulation measured by cytokine secretion from human peripheral blood leukocytes. In vivo, over 50% reduction of disease activity was achieved in three weeks treatment using as little as 1 nM drug candidate (dosed every 48 hours) in the collagen-induced (CIA) mouse model of RA (N=30; pConclusion:Overall, we have designed a first-in-class therapeutic nanoparticle drug for specific targeting of anti-citrullinated protein antibodies. The marked effect of this nanoparticle observed in vivo holds promise for targeting ACPAs as a therapeutic option in RA.References:[1]Khatri S, Hansen J, Mendes AC, Chronakis IS, Hung S-C, Mellins ED, Astakhova K. Bioconjug Chem. 2019 Oct 16;30(10):2584–259Disclosure of Interests:Sangita Khatri: None declared, Jonas Hansen: None declared, Mads Hartvig Clausen Shareholder of: iBio Tech ApS, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Shu-Chen Hung: None declared, Elisabeth Mellins: None declared, Kira Astakhova: None declared

Background:During cancer treatment with immune checkpoint inhibitors (ICI) such as the anti-PD-1 antibody pembrolizumab, 2-4% of patients develop inflammatory arthritis as an immune related adverse event and half of patients with pre-existing inflammatory arthritis have disease flares. This type of adverse events shows striking similarities with traditional immune mediated inflammatory arthritis. However, the underlying immunological mechanisms of inflammatory arthritis associated with ICI are not fully understood.Objectives:We aimed to develop an in vitro model of inflammatory arthritis associated with ICI, and to use this model to investigate monocyte differentiation and activation following treatment with pembrolizumab.Methods:First, synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from patients with immune mediated inflammatory arthritis (rheumatoid arthritis and peripheral spondyloarthritis, n=22) and PBMCs from healthy controls were incubated with pembrolizumab and assessed for monocyte chemoattractant protein 1 (MCP-1) secretion by ELISA. Then, cytokine production in SFMCs was studied in more detail by the multiplex V-PLEX proinflammatory panel and by intracellular flow cytometry. Finally, pembrolizumab treated SFMCs were incubated with the different disease modifying anti-rheumatic drugs adalimumab, tocilizumab, tofacitinib, and baricitinib.Results:Pembrolizumab significantly increased MCP-1 production in the SFMCcultures (P=0.0031). In contrast, pembrolizumab did not change MCP-1 production by PBMCs from neither patients nor healthy controls (P=0.77 and P=0.43). Pembrolizumab also increased the production of TNFα (P=0.049), IFNγ (P=0.047), and IL-12p70 (P=0.031), but did not change the production of IL-6 (P=0.98). Among SFMCs treated with pembrolizumab there was an increased frequency of intermediate monocytes (P=0.044). Interestingly, pembrolizumab also increased the MCP-1 production within the intermediate monocytes only (P=0.028). In contrast, among SFMCs treated with LPS, only the classical monocyte subset was increased (P=0.0045) and MCP-1 production increased in both intermediate and classical monocyte subsets. Lastly, the TNFα inhibitor adalimumab and the Janus kinase inhibitors baricitinib and tofacitinib attenuated the pembrolizumab-induced MCP-1 production (P=0.0004, P=0.033, and P=0.025, respectively) while this was not seen with the IL-6 inhibitor tocilizumab (P=0.75).Conclusion:We have developed a very simple in vitro model of inflammatory arthritis associated with ICI. Using this model, we found that pembrolizumab specifically activated intermediate monocytes and induced TNFα, IFNγ, and IL-12p70 production, whereas IL-6 was unchanged. These findings were supported by effectful reduction of MCP-1 secretion with TNFα inhibition but not with IL-6 inhibition. This model could potentially be used to further study the effects of ICIs and the underlying immunological mechanisms of inflammatory arthritis associated with ICI.References:None.Disclosure of Interests:Anne Sofie Sørensen: None declared, Morten Nørgaard Andersen: None declared, Kristian Juul-Madsen: None declared, Cæcilie Skejø: None declared, Henrik Schmidt: None declared, Thomas Vorup-Jensen: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB.

Background:Fibroblast-like synoviocytes (FLS) are central cellular components in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). Pathological subsets of FLS have been identified from synovial tissue. However, the synovial tissue obtained from arthroplasty procedures is acquired at late disease stages and the cellular yield obtained from synovial tissue biopsies is fairly low. Collectively, challenging the robustness of human RAin vivoandin vitromodels. FLS obtained from the synovial fluid (SF-FLS) are proposed as an alternative source of FLS, but a detailed phenotypical and functional characterization of FLS subsets from the synovial fluid has not been performed.Objectives:The aim of this study was to determine the phenotypical and functional characteristics of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.Methods:In the present study, paired peripheral blood mononuclear cells (PBMC) and SF-FLS from patients with RA were obtained (n=7). FLS were isolated from the synovial fluid by a strict trypsinization protocol1and their cellular characteristics and functionality were evaluated at passage 4. Monocultures (SF-FLS) and autologous co-cultures (SF-FLS and PBMC) were established from five patients with RA and subsequently evaluated by flow cytometry, Western blotting and multiplex immunoassays. Human cartilage-sponges (n=3) with SF-FLS and without SF-FLS (n=3) were co-implanted subcutaneously in SCID mice (n=15), mice with only cell-free human cartilage-sponges were used as controls (n=12). After 45 days, the implants were evaluated using stained sections to determine the SF-FLS invasion score based on perichondrocytic cartilage degradation. Data are expressed as median (25-75 percentile). P-values Results:The homogeneous subpopulations of FLS, isolated from the synovial fluid, were negative for CD34 and CD45 [98.9%, (97.5-99.7]) and positive for Thy-1 and PDPN [94.6%, (79.9-97.4]). Without stimulation, RA SF-FLS showed high and comparable levels of NFκB related pathway proteins and secreted multiple pro-inflammatory cytokines and chemokines dominated by IL-6 [2648 pg/mL, (1327-6116)] and MCP-1 [2458 pg/mL, (692-8719)]. SF-FLS increased their ICAM-1 and HLA-DR expression after encountering autologous PBMCs (pin vivowas at primary site, [1.6, (1.3-1.7)] and contralateral implantation site [1.5, (1.1-2.2)]. The invasion score of the human SF-FLS-containing implants both at primary and contralateral site were significantly higher compared with cartilage-sponges evaluated from SF-FLS-free control mice (pConclusion:This phenotypical and functional characterization of SF-FLS, acquired and activated at the site of pathology, lays a foundation for establishingin vivoandin vitroFLS models. These FLS models will be beneficial in our understanding of the role of this cellular subset in arthritis and for characterization of drugs specifically targeting this pathological RA FLS subset.References:[1]Nielsen M. A. et al. Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis. ACR Open Rheumatology, 2(1):3-10.http://doi.org/10.1002/acr2.11094Disclosure of Interests:Ditte Køster: None declared, Johanne Hovgaard Egedal: None declared, Malene Hvid: None declared, Martin Roelsgaard Jakobsen: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Bent Deleuran: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Elena Neumann: None declared, Morten Aagaard Nielsen: None declared

Objective Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. Methods Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2−/− mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n ​= ​103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. Results PD-L2−/− mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. Conclusion PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA., Highlights • PD-L2 is closely related to bone homeostasis in a mouse model. • PD-L2 inhibits osteoclastogenesis and osteoclast activation in vitro. • PD-L2 is highly expressed by cells in the synovial membrane of rheumatoid arthritis. • PD-L2 is associated with less radiographic progression in patients with early rheumatoid arthritis.

Summary The pathogenesis of sepsis involves a dual inflammatory response, with a hyperinflammatory phase followed by, or in combination with, a hypoinflammatory phase. The adhesion molecules lymphocyte function-associated antigen (LFA-1) (CD11a/CD18) and macrophage-1 (Mac-1) (CD11b/CD18) support leucocyte adhesion to intercellular adhesion molecules and phagocytosis through complement opsonization, both processes relevant to the immune response during sepsis. Here, we investigate the role of soluble (s)CD18 in sepsis with emphasis on sCD18 as a mechanistic biomarker of immune reactions and outcome of sepsis. sCD18 levels were measured in 15 septic and 15 critically ill non-septic patients. Fifteen healthy volunteers served as controls. CD18 shedding from human mononuclear cells was increased in vitro by several proinflammatory mediators relevant in sepsis. sCD18 inhibited cell adhesion to the complement fragment iC3b, which is a ligand for CD11b/CD18, also known as Mac-1 or complement receptor 3. Serum sCD18 levels in sepsis non-survivors displayed two distinct peaks permitting a partitioning into two groups, namely sCD18 ‘high’ and sCD18 ‘low’, with median levels of sCD18 at 2158 mU/ml [interquartile range (IQR) 2093–2811 mU/ml] and 488 mU/ml (IQR 360–617 mU/ml), respectively, at the day of intensive care unit admission. Serum sCD18 levels partitioned sepsis non-survivors into one group of ‘high’ sCD18 and low CRP and another group with ‘low’ sCD18 and high C-reactive protein. Together with the mechanistic data generated in vitro, we suggest the partitioning in sCD18 to reflect a compensatory anti-inflammatory response syndrome and hyperinflammation, respectively, manifested as part of sepsis.

Summary The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)-20 and IL-24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL-20 and IL-24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in-vitro cell culture studies. The plasma IL-20 and IL-24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll-like receptor 4-induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL-20 and IL-24 increased the production of monocyte chemoattractant protein-1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf-1 by SpA fibroblast-like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease-aggravating functions of IL-20 and IL-24 in SpA.

The treatment of rheumatoid arthritis (RA) and spondyloarthritis (SpA) was transformed a little over a decade ago by the introduction of agents neutralizing the pro-inflammatory cytokine tumour necrosis factor (TNF)-α. Nevertheless, some patients do not achieve remission and the inhibition of the normal immune system with current drugs increases the risk of infection. The interleukin (IL)-20 receptor (IL-20R) axis is pivotal for tissue homeostasis. By contrast, this axis does not seem to directly activate cells of the immune system. Thus, modulation of the IL-20R axis might not result in increased risk of infection. The IL-20R axis consists of the three cytokines IL-19, IL-20, and IL-24 (termed the IL-20R cytokines) and their shared receptors. All three cytokines bind the receptor complex of IL-20R2/IL-20R1 whereas only IL-20 and IL-24 also bind the receptor complex of IL-20R2/IL-22R1. This short review describes how the IL-20R axis could be a novel link between innate immune recognition and bone homeostasis. The IL-20R cytokines are produced in response to both danger-associated molecular patterns and immune complexes formed by RA-associated autoantibodies. This could be of importance because these mediators can thus be present even in situations without inflammation. IL-19 shows anti-inflammatory properties in arthritis through IL-20R1. IL-20 and IL-24 through IL-22R seem to participate in the recruitment of mononuclear cells to the synovial joint and to sites of bone erosion in particular. Our results indicate that dual inhibition of IL-20 and IL-24 or attenuation of the shared IL-22R subunit could have a beneficial effect on radiographic progression, especially in seropositive RA.

Viruses, bacteria and other infectious pathogens are the major postulated environmental triggers of autoimmunity. In the present nation-wide study we describe the association between infections and 29 autoimmune diseases. We used the Danish Civil Registration System to identify 4.5 million persons born between 1945 and 2000. Information on infections and autoimmune diseases was obtained from the Danish Hospital Register. The cohort was followed from 1977 to 2012. Incidence rate ratios for developing an autoimmune disease were estimated using poisson regression. We found an association between hospital admission for an infection and 29 autoimmune diseases. This study shows that infections are risk factors for a broad spectrum of autoimmune diseases in a dose-response and temporal manner, in agreement with the hypothesis that infections are an environmental risk factor contributing to the etiology of autoimmune diseases together with genetic factors.

In osteoimmunology, osteoclastogenesis is understood in the context of the immune system. Today, the in vitro model for osteoclastogenesis necessitates the addition of recombinant human receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The peripheral joints of patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) are characterized by an immune-mediated inflammation that can lead to bone destruction. Here, we evaluate spontaneous in vitro osteoclastogenesis in cultures of synovial fluid mononuclear cells (SFMCs) activated only in vivo. SFMCs were isolated and cultured for 21 days at 0.5-1.0 × 10(6) cells/mL in culture medium. SFMCs and healthy control peripheral blood monocytes were cultured with RANKL and M-CSF as controls. Tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells were found in the SFMC cultures after 21 days. These cells expressed the osteoclast genes calcitonin receptor, cathepsin K, and integrin β3, formed lacunae on dentin plates and secreted matrix metalloproteinase 9 (MMP9) and TRAP. Adding RANKL and M-CSF potentiated this secretion. In conclusion, we show that SFMCs from inflamed peripheral joints can spontaneously develop into functionally active osteoclasts ex vivo. Our study provides a simple in vitro model for studying inflammatory osteoclastogenesis.

Summary Spondyloarthritis (SpA) is a group of immune mediated inflammatory diseases affecting joints, gut, skin and entheses. The inflammatory process involves activation of Toll-like receptor (TLR)-2 and TLR-4 and production of cytokines and chemokines such as monocyte chemoattractant protein 1 (CCL2/MCP-1). This proinflammatory chemokine recruits monocytes to sites of inflammation and is central in the development of several immune-mediated inflammatory diseases. Interleukin (IL)-19 is a member of the IL-10 family of cytokines. IL-19-deficient mice are more susceptible to innate-mediated colitis and develop more severe inflammation in response to injury. In this work, we studied inducers of IL-19 production and effect of IL-19 on the production of CCL2/MCP-1 and proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and in PBMCs and synovial fluid mononuclear cells (SFMCs) from SpA patients. Further, we measured IL-19 in plasma from HCs and in plasma and synovial fluid from SpA patients. Constitutive IL-19 expression was present in both PBMCs and SFMCs and the secretion of IL-19 was increased by TLR-2 and TLR-4 ligands. Neutralizing IL-19 in HC PBMCs and SpA SFMCs resulted in increased production of CCL-2/MCP-1. IL-19 concentrations were decreased in synovial fluid compared with plasma and associated inversely with disease activity in SpA. SpA SFMCs produced less IL-19 in response to LPS compared with HC PBMCs. These findings indicate that IL-19 production is diminished in SpA. Taken together, impaired IL-19 control of the innate immune system might be involved in the pathogenesis of SpA.

INTRODUCTION: A key phenomenon in rheumatoid arthritis is the formation of lymphoid follicles in the inflamed synovial membrane. C-X-C motif chemokine 13 (CXCL13) is central in this process as it attracts C-X-C chemokine receptor type 5 (CXCR5)-expressing B cells and T follicular helper cells to the follicle. We here examine the role of CXCL13 and its association with disease in patients with treatment-naïve early rheumatoid arthritis.METHODS: Plasma samples from patients in the OPERA trial were examined for CXCL13 at treatment initiation and after 6 months of treatment with either methotrexate plus placebo (DMARD) (n = 37) or methotrexate plus adalimumab (DMARD + ADA) (n = 39). Treatment outcome was evaluated after 1 and 2 years. CXCL13 plasma levels in healthy volunteers (n = 38) were also examined.RESULTS: Baseline CXCL13 plasma levels were increased in early rheumatoid arthritis patients in comparison with healthy volunteers. Also, plasma CXCL13 correlated positively with disease activity parameters; swollen joint count 28 (rho = 0.34) and 40 (rho = 0.39), visual analog score (rho = 0.38) and simplified disease activity index (rho = 0.25) (all P CONCLUSIONS: In treatment-naïve early rheumatoid arthritis patients, plasma CXCL13 levels were associated with joint inflammation. Furthermore, patients with high baseline plasma CXCL13 levels had an improved chance of remission after 2 years. We propose that high CXCL13 concentrations indicate recent onset of inflammation that may respond better to early aggressive treatment. Thus, high levels of CXCL13 could reflect the 'the window of opportunity' for optimal treatment effect.TRIAL REGISTRATION: Clinicaltrial.gov NCT00660647. Registered 10 April 2008.

Background Inflammatory conditions affect bone turnover, as is seen in rheumatoid arthritis (RA). Inflammatory cytokines in RA stimulate osteoclasts and shift the balance between bone formation and resorption. Programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2, are important for maintaining self-tolerance, and this pathway plays a prominent role in RA. Recently, repulsive guidance molecule b (RGMb) and bone morphogenetic protein (BMP) was identified as a novel receptor complex for PD-L2 (1). We previously showed the PD-1 pathway to be associated with radiographic progression in RA (2). The interaction of PD-L2 with the RGMb/BMP receptor complex could potentially provide a link between bone homeostasis and the PD-1 pathway. Objectives To investigate the possible association between bone homeostasis and the PD-1 pathway in RA. Methods We generated osteoclasts from RA synovial fluid mononuclear cells (SFMCs) (3), stimulated them with recombinant (rec) PD-1 and PD-L2, and subsequently evaluated these osteoclasts for TRAP activity, and expression of PD-1, PD-L2 and RGMb. We induced RA-like disease in PD-1-and PD-L2-KO mice by immunization with Freund9s adjuvant and chick collagen, and evaluated clinical arthritis. After sacrifice, paws were paraffin embedded and stained for TRAP and osteocalcin. The femur was subjected to ex vivo DXA scanning. In addition, we stimulated normal mouse calvarial cells with the recombinant proteins in a BMP reporter assay to evaluate the potential of PD-1 and PD-L2 to affect BMP signaling. Results Recombinant PD-1 and PD-L2 increased TRAP activity in osteoclasts generated from RA SFMCs, at day 21 (p=0.046). Pre-osteoclasts in the culture expressed both PD-L2 and RGMb. The KO mice did not develop clinical signs of arthritis, ensuring intact mobility and constant bone load. DXA scan revealed that immunized PD-L2 KO mice had a higher bone mass density than other KO mice, WT and non-immunized PD-L2 KO (p=0.03). These mice did not differ histologically in TRAP and osteocalcin expression. Rec PD-L2 significantly decreased BMP signaling in the BMP reporter assay. Conclusions The clinical association between the PD-1 pathway and radiographic progression in early RA points to its importance in RA bone homeostasis. The increase in TRAP activity after incubation with recombinant PD-L2 supports this association. The increased bone mass density observed in immunized PD-L2 KO mice further supports a potential role for PD-L2 in bone homeostasis. The expression of PD-L2 in pre-osteoclasts also suggests a role for PD-L2 in bone homeostasis. These data provide a new insight into the close relation between the immune system and bone, as well as suggest a potential therapeutic target in diseases involving inflammatory bone changes. References Xiao Y, et. al Journal of Exp Med. 2014 Apr 21;3(1):81. Greisen SR, et al. Scand J Rheum. 2013 Nov 1;43(2):101–8. Greisen SR, et al. APMIS. 2015 Sep;123(9):779–86. Disclosure of Interest None declared

Background The pro-inflammatory Th17 associated cytokines IL-17A and IL-22 have been proposed as important mediators in spondyloarthritis (SpA). Treatment of SpA has been improved dramatically with the introduction of tumor necrosis factor (TNF)α inhibitors. Despite good clinical efficacy, anti-TNFα fail to affect bone formation and radiograpchic progression in SpA. Th17 cells and cytokines might be involved in this lack of response to anti TNF-α antibodies. Objectives To investigate the association of Th17 cells with changes in clinical and MRI activity in SpA patients treated with anti-TNFα. Furthermore, to investigate the involvement of the Th17 cytokines IL-17A and IL-22 on bone anabolic pathways in SpA. Methods Percentage of IL-17A and IL-22 producing T cells were assessed in peripheral blood mononuclear cells from 30 SpA patients at baseline, after 12 and 52 weeks of anti-TNFα therapy using intracellular flow cytometry. Clinical activity was evaluated using the Ankylosing Spondylitis Disease Activity Score (ASDAS) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). MRI grading was performed on the whole spine and in the sacroiliac joints (SIJ). Correlation analyses were performed to investigate the association between percentages of Th17 cells and disease progression during the course of treatment. Normal human osteoblasts and fibroblast like synoviocytes (FLS) were stimulated with IL-17A, IL-22, TNFα and supernatants from CD4+CD45RO+CCR6+ cells to determine the bone modulatory activity of Th17 cytokines. Results Percentages of IL-17A producing and IL-22 producing, and CCR6 expressing CD4+CD45RO+ T cells were increased at baseline compared with healthy controls. These subsets increased during 52 weeks of anti-TNFα therapy, despite clinical response with the composite scores ASDAS decreasing from 3.6 (3.0–4.0) to 1 (0.5–1.9) and BASDAI decreasing from 63.50 (52–71) to 15 (3- 31). High baseline percentages of IL-17A and IL-22 producing CD4+CD45RO+ T cells correlated with greater improvement in ASDAS and BASDAI score. Both baseline percentages of IL-17A and IL-22 producing CD4+CD45RO+ T cells were associated with increased levels of chronic MRI changes at baseline (both p IL-17A, IL-22 and TNFα stimulation increased mineral deposition from normal human osteoblasts. IL-22 in combination with TNFα inhibited the secretion of Dkk-1 from SpA FLS, whereas neither TNFα nor IL-17A were able to affect Dkk-1 secretion from SpA FLS. Conclusions Th17 cells increased during anti-TNFα therapy, regardless of clinical improvement. Th17 cell percentages at baseline predict clinical response and are associated with chronic MRI changes at baseline and outcome at 52 weeks. Not only IL-17A, but also IL-22 are able to affect bone formation and bone remodelling pathways, but only IL-22 induce Dkk-1 in FLS. Hereby, the consistently elevated percentages of Th17 cells, despite good disease control in this group of SpA patients, could provide a link to the ongoing ectopic bone formation seen in SpA. Disclosure of Interest T. Andersen Grant/research support from: Janssen, M. Hvid: None declared, T. Kragstrup: None declared, H. Gleerup: None declared, A. Jurik: None declared, R. Ostgard: None declared, B. Deleuran: None declared

Background New bone formation in spondyloarthritis involves myofibroblasts [1]. IL-26 is part of the IL-10 cytokine family and was recently reported to increase Th17 differentiation [2]. It was first discovered in herpesvirus saimiri–transformed T cells and thus suspected to be a T-cell cytokine [3]. However, more recent studies of RA patients show IL-26 expression in synoviolin-postive cells harvested from the synovia [2]. The knowledge about IL-26 is compromised by lack of IL-26 in mice and no studies of IL-26 in spondyloarthritis (SpA) and its regulation have been conducted. Objectives In the present study we aim to characterize IL-26 producing cells in spondyloarthritis (SpA). Methods IL-26 was measured by ELISA in plasma and synovial fluid (SF) of 14 SpA patients attending the clinic with a swollen joint and in plasma samples from 7 age and gender matched healthy controls. Synovial-fluid-mononuclear-cells (SFMC) and fibroblast-like-synoviocytes (FLS) were cultured and stimulated for 48 hours with IL-1β, TNFα, TGF-β, IL-17A and LPS. The supernatants were harvested and analyzed for IL-26 by ELISA. Tissue samples of inflamed synovial membrane were stained with a fluorochrome conjugated rabbit IL-26 antibody for immunofluorescence. Imaging of the sections was performed by confocal microscopy. For flow cytometry analysis, FLS were surface stained for CD90 and a live-dead marker, and subsequently permeabilized and stained intracellularly for α-Smooth-Muscle-Actin (αSMA) and IL-26. Samples were run on the flow cytometer within 24 hours of staining. Data are expressed as mean ± SD. Results IL-26 levels were significantly higher in synovial fluid compared with plasma; this applied to plasma from the healthy controls (HC) as well as SpA patients. (HC plasma: 5.8 ± 3.3 pg/ml, SpA plasma: 6.6 ± 1.8 pg/ml, SpA SF: 41.6 ± 6.8 pg/ml - P-values HC vs. SF: 0.0020, P-value SP vs. SF: + and CD90 + myofibroblasts by flow cytometry. Conclusions IL-26 levels in synovial fluid are significantly higher than in plasma, suggesting a local production of IL-26 in the inflamed joint. We found IL-26 positive cells in the synovial membrane with fibroblast morphology and FLS from the inflamed joints of SpA patients produce high amounts of IL-26. By flow cytometry we established that these IL-26 producing fibroblasts seem to be myofibroblasts primarily. References Beyer C, et al: Changing paradigms in spondylarthritis: The myofibroblast signature. Arthritis Rheum. 2012;65:24–7 Corvaisier M, et al: IL-26 is overexpressed in rheumatoid arthritis and induces proinflammatory cytokine production and Th17 cell generation. PLoS Biol 2012;10:e1001395 Knappe A, et al: Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri. J Vi- rol 2000;74:3881–3887 Disclosure of Interest None declared

Background The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The innate immune system is activated through pattern recognition receptors including Toll like receptor 4 (TLR4). Inflammation is a result of increased production of pro-inflammatory cytokines and chemokines including tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein 1 (MCP-1). New bone formation by osteoblasts is determined by a balance between the inhibitory effect of Dickkopf-1 (DKK1) and the stimulatory effect of bone morphogenetic proteins (BMPs) including BMP2. The two cytokines IL-20 and IL-24 have been shown to link innate immune activation and tissue homeostasis.(1) Previously, we have found increased plasma levels of IL-20 and IL-24 in SpA patients. Objectives We hypothesized that IL-20 and IL-24 are secreted as part of activation of the innate immune system and affect bone homeostasis. The aim was to describe associations of IL-20 and IL-24 and disease activity and magnetic resonance imaging (MRI) scores and to identify the sources and targets of the two cytokines in SpA. Methods Patients with axial SpA (n=83) were included in the study of associations between cytokine levels and disease activity and MRI scores. Patients with peripheral SpA (n=16) were included for studying sources and targets of IL-20 and IL-24 among synovial fluid mononuclear cells (SFMCs), peripheral blood mononuclear cells (PBMCs), and fibroblast-like synoviocytes (FLSs) using lipopolysaccharide (LPS) and TNFα as positive controls. Healthy human osteoblasts were used for studying the effect of the two cytokines on mineralization using BMP2 as a positive control. The secretion of IL-20, IL-24, MCP-1 and DKK1 were measured by enzyme linked immunosorbant assays and flow cytometry, and mineralization was measured by hydroxyapatite deposition. Plasma IL-20 and IL-24 levels were analyzed with the Mann-Whitney U test and the Spearman correlation. Supernatant cytokine concentration and flow cytometry median fluorescence intensity data were calculated as ratios, log-transformed and compared with the paired or unpaired t-test. Results The plasma IL-20 and IL-24 levels were increased in SpA patients compared with healthy controls (HCs) by 57% and 83%, respectively (both p Conclusions Taken together, our findings indicate that IL-20 and IL-24 are novel links between activation of the innate immune system and new bone formation. References Rutz S et al. The IL-20 subfamily of cytokines - from host defence to tissue homeostasis. Nat Rev Immunol. 2014. Disclosure of Interest None declared

CD18 integrins are adhesion molecules expressed on the cell surface of leukocytes and play a central role in the molecular mechanisms supporting leukocyte migration to zones of inflammation. Recently, it was discovered that CD11a/CD18 is shed from the leukocyte surface in models of inflammation. In this study, we show that shedding of human CD11/CD18 complexes is a part of synovial inflammation in rheumatoid arthritis and spondyloarthritis but not in osteoarthritis. In vivo and in vitro data suggest that the shedding is driven by TNF-α, which links the process to central events in the inflammatory response. The shed complexes contain multiple heterodimers of CD11/CD18, are variable in size, and differ according to the type of synovial inflammation. Furthermore, the differential structures determine the avidity of binding of the complexes to the ICAM-1. With the estimated concentrations of CD11/CD18 in plasma and synovial fluid a significant coverage of binding sites in ICAM-1 for CD18 integrins is expected. Based on cell adhesion experiments in vitro, we hypothesize that the large soluble complexes of CD11/CD18 act in vivo to buffer leukocyte adhesion by competing with the membrane-bound receptors for ICAM-1 binding sites. As reported here for synovial inflammation changes in the concentration or structure of these complexes should be considered as likely contributors to disease activity.

The purpose of this study was to analyze the expression of the two proinflammatory cytokines IL-20 and IL-24 and their shared receptors in rheumatoid arthritis and spondyloarthropathy. IL-20 was increased in plasma of rheumatoid arthritis patients compared with osteoarthritis patients and IL-24 was increased in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy patients compared with osteoarthritis patients. IL-20 and IL-24 mRNA was only present at low levels in the synovium. In the synovial membrane, IL-20 protein was present in mononuclear cells and neutrophil granulocytes whereas IL-24 protein was observed in endothelial cells and mononuclear cells. IL-20 receptor type 1 and IL-22 receptor were expressed by granulocytes in the synovial fluid. In synovial fluid mononuclear cell cultures, stimulation with recombinant human IL-20 or recombinant human IL-24 induced monocyte chemoattractant protein 1 (CCL2/MCP-1) secretion, but not tumour necrosis factor alpha mRNA synthesis or IL-6 secretion. Both IL-20 and IL-24 showed correlations to CCL2/MCP-1 in plasma from rheumatoid arthritis and spondyloarthropathy patients. This study associates IL-20 and IL-24 to the synovium of rheumatoid arthritis and spondyloarthropathy and results indicate that the two cytokines contribute to disease pathogenesis through recruitment of neutrophil granulocytes and induction of CCL2/MCP-1. Udgivelsesdato: 2008-Jan

Background In rheumatoid arthritis (RA) 4-1BB (TNFSFR9) is believed to be involved in the continued T cell activation within the inflamed joint. 4-1BB binds to 4-1BB ligand (4-1BBL) generating local clonal expansion and accumulation of antigen-specific effector-type T cells. Galectin-9 (Gal-9) is upregulated at site of inflammation and has recently been suggested to be a restricting factor for the inflammatory signal of 4-1BB. ADAM17 (TACE) has previously been linked to RA and inflammation. It is a sheddase with a broad substrate specificity although it is best know for its involvement in TNFα release by being the main sheddase of proTNFα. Objectives To determine the role of the interplay between 4-1BB, ADAM17 and Gal-9 in RA. Methods 4-1BB and Gal-9 expression was studied in paired peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMCs) by flow cytometry in chronic RA (cRA), and in PBMCs from healthy volunteers (HV). 4-1BBL, Gal-9, and TNFα were used to stimulate SFMCs. Surface plasmon resonance analysis was performed with human recombinant proteins 4-1BB, 4-1BBL, and Gal-9. Transfected HEK293 cells expressing 4-1BB were established to investigate potential sheddases of 4-1BB. Soluble 4-1BB was measured by ELISA in plasma from treatment naive early RA (eRA) patients obtained at treatment initiation (the OPERA regimen, n=96) 1 . Results In chronic RA (cRA), the percentage of T cells expressing 4-1BB was significantly higher in SFMCs compared with PBMCs and in cRA PBMCs compared with HV PBMCs (both p Conclusions ADAM17 induces 4-1BB shedding in RA and is connected to TNFα metabolism. Gal-9 is pivotal for the local function of 4-1BB in RA. Furthermore, high plasma levels of s4-1BB are associated with number of swollen joints, but also with a good prognosis measured by DAS28CRP and TSS. 4-1BB metabolism thereby plays a central role in the delicate network of pro- and anti-inflammatory factors in RA. References Horslev-Petersen et al. Ann rheum Dis. 2013 Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is characterized by high occurrence of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs) and bone erosions. Successful treatment with biologic drugs such as anti-tumor necrosis factor alpha compromises the normal immune response increasing the risk of infections. The interleukin 20 receptor (IL-20R) axis comprising IL-19, IL-20, and IL-24 (“the IL-20R cytokines”) and their shared receptors IL-20R2/IL-20R1 and IL-20R2/IL-22R activates tissue homeostasis processes but not the immune system ([Figure 1][1]). ![Figure][2] Objectives The objective of this study was to determine the role of the IL-20R axis in early RA (eRA) with focus on associations of the three cytokines with clinical disease activity and prognosis. Methods The IL-20R cytokines were measured in plasma samples from treatment naive early RA patients during 24 months of treatment with methotrexate, adalimumab/placebo and intra-articular glucocorticoid injections (the OPERA trial) (n=152)1. The IL-20R1 and IL-22R expression was studied in paired peripheral blood and synovial fluid cells from a different cohort of RA patients (n=15) with at least one swollen joint (for obtaining synovial fluid) by flow cytometry and confocal microscopy. Heat aggregated human gamma globulins (HAGGs) and immune complexes containing citrullinated fibrinogen (cFb-ICs) were used to stimulate myeloid cells. Osteoclasts (OCs) derived from synovial fluid cells were used to assess the effect of the IL-20R cytokines. Results The plasma concentrations of IL-20 and IL-24 (but not IL-19) were increased in eRA patients compared with HCs (both P< 0.002) and decreased after 6 months of treatment (both P

Background In rheumatoid arthritis (RA) immune activation and leukocyte migration to the joints precede clinical onset of disease. Early inhibition of inflammation is the main aim of treat-to-target strategy, but the mechanisms and time course for restoration of the immune system remains poorly understood. The family of CD18 integrins is central in the inflammatory response by allowing leukocytes to bind ICAM-1 and migrate to inflammatory foci. We have demonstrated a soluble form of CD18 (sCD18), which is shed to the systemic circulation by monocytes and increased following stimulation with TNFα. sCD18 competes with the cell-expressed receptors for binding to ICAM-1 and thus may function by limiting leukocyte adhesion to ICAM-1.(1) Recently, we found that plasma levels of sCD18 are decreased because of binding to inflammation induced ICAM-1 and associate inversely with disease activity in chronic spondyloarthritis.(2) Objectives To characterize the profile and regulation of plasma sCD18 levels in patients with early rheumatoid arthritis before and during a treat-to-target strategy (the OPERA regimen). Methods The level of sCD18 in plasma was analyzed with a time-resolved immunoflourometric assay (TRIFMA) in a study population of 152 patients with early treatment naive RA (the OPERA cohort) at baseline and after 3, 6, and 12 months of treatment with methotrexate + placebo (PLA) or methotrexate + adalimumab (ADA) and matched healthy controls (HC). In vitro, synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from 9 RA patients were cultured with or without adalimumab (5μg/ml) for 48 hours. Results Plasma levels of sCD18 were decreased in early RA patients at baseline (median=958mU/ml, IQR=766-1243mU/ml) compared with HC (median=1001mU/ml, IQR=872-1355mU/ml) ( P P P =0.6). RA patients with an ACR70 response or EULAR DAS response after 3 months of treatment had a greater increase in sCD18 plasma levels from baseline to 12 months (both P P P Conclusions The decreased plasma levels of sCD18 could be part of the deregulated immune system in early RA. Interestingly, sCD18 exhibited a biphasic course during a treat-to-target strategy with an initial decline followed by a gradual increase to HC levels. In vitro, adalimumab decreased the CD18 shedding explaining the initial decrease in sCD18 plasma levels. The late increase found especially in the patients with an early ACR70 response or EULAR DAS response could reflect latency in restoration of the normal immune system, pointing to sCD18 as a marker for such restoration. References Gjelstrup LC et al., J. Immunol., 2010 Oct. Kragstrup TW et al., Arthritis Res Ther, 2014 Jan (accepted). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5732

Background In spite of the widespred use of sandwich ELISAs in arthritis research recommendations on how to eliminate unspecific interference by rheumatoid factor and anti-animal protein antibodies frequently found in arthritis plasma are lacking. Objectives The objective of this study was to design a simple set of steps for validating and optimizing a sandwich ELISA before using it for measuring arthritis plasma. As an example of an immunoassay for arthritis research an anti-IL-24 sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody. Methods Plasma samples from patients with rheumatoid arthritis and spondyloarthritis were analyzed. A simple set of validation steps identified signals from samples in wells coated with isotype matched control antibody (false positive results) and lack of recovery after spiking samples (false negative results). ELAST ® (Perkin Elmer) amplification kit was used to amplify HRP signal before addition of substrate. Results Interference was removed after preincubating the arthritis plasma samples with goat or bovine IgG (10 µg/ml – 100 µg/ml), suggesting that anti-animal protein antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that an amplification step, using ELAST could increase the signal-to-noise ratio. Furthermore we show that repetitive freeze-thaw cycles of the samples, the anticoagulant used in the samples and the buffer used for the standard curve could affect the calculated concentration of the analyte. Conclusions This study establishes an in-house sandwich ELISA system and validates it for measuring arthritis plasma with a simple set of validation and optimization steps removing the anti-animal antibodies cross binding of capture and detection antibodies. Any sandwich ELISA to be used in arthritis research could be validated an optimized by a similar setup. Disclosure of Interest None Declared

Background Lack of measures of disease activity and prognosis is a key challenge in spondyloarthritis (SpA). Several soluble biomarkers have been proposed, but very few have been found to relate to MRI activity and add to the diagnostic value of CRP.(1) Leukocytes are key players in SpA disease activity.(2) The soluble form of CD18 (sCD18) has been found to be shed from leukocytes in inflammation and to be present in the blood.(3) Objectives This study examines the association of sCD18 in the blood and SpA disease activity. Methods Plasma from a study population with 84 SpA patients at baseline and 33 patients at three-year follow-up and a control group of age and gender matched normal healthy volunteers (NHV) were studied. The samples were analyzed for sCD18 by a time resolved immunoflourometric assay (TRIFMA) using the KIM185 antibody as capture antibody and the KIM127 antibody as detection antibody as previously described.(3) Results The level of sCD18 was decreased in plasma from SpA patients compared to plasma from NHV (p Conclusions sCD18 is associated with clinical findings, CRP and SIJ MRI activity in SpA. sCD18 could be a novel disease activity marker in SpA directly measuring leukocyte activity. References Maksymowych WP. Biomarkers in spondyloarthritis. Current rheumatology reports. 2010;12(5):318-24. Dougados M, Baeten D. Spondyloarthritis. Lancet. 2011;377(9783):2127-37. Gjelstrup LC, Boesen T, Kragstrup TW, Jorgensen A, Klein NJ, Thiel S, et al. Shedding of large functionally active CD11/CD18 Integrin complexes from leukocyte membranes during synovial inflammation distinguishes three types of arthritis through differential epitope exposure. J Immunol. 2010;185(7):4154-68. Disclosure of Interest None Declared

Spondyloarthritis (SpA) is characterized by inflammation in the axial skeleton and entheses. Integrin adhesion molecules containing the CD18 chain are pivotal regulators of leukocyte extravasation from the blood into zones of inflammation. Recently, we showed that CD18 is shed from leukocytes forming a soluble form of CD18 (sCD18) in the blood. Here we show that plasma sCD18 is a marker of inflammatory activity in SpA. The plasma levels of sCD18 were decreased in SpA patients compared with healthy controls and showed inverse correlations with disease activity scores including the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Remarkably, this situation could be simulated in vitro. Thus, the degree of CD18 shedding from SpA patient-derived PBMC correlated inversely with the BASDAI score. According to flow cytometric analyses, CD18 was primarily shed from intermediate (CD14++ CD16+) monocytes. In agreement with earlier reports identifying CD18 as a substrate for MMP-9, the concentration of this enzyme in the culture supernatants correlated with the sCD18 concentration. Also, sCD18 complexes adhered to TNFα-stimulated endothelial cells expressing high levels of ICAM-1 but not to unstimulated cells. Taken together, our data links alterations in plasma sCD18 and inflammatory activity in SpA. The low plasma level of sCD18 in SpA seems to be a combination of inadequate CD18 shedding and increased depletion of sCD18 potentially affecting leukocyte migration.