D Sieghart

Hydrogen sulphide decreases IL-1β-induced activation of fibroblast-like synoviocytes from patients with osteoarthritis.

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H2S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.

AB0061 Hydrogen Sulfide Reduces IL-1β-Induced Activation of Fibroblast-Like Synoviocytes from Patients with Osteoarthritis

Background In the pathophysiology of osteoarthritis (OA) the secretion of pro-inflammatory cytokine interleukin (IL)-1β is among the critical steps mediating aberrant degenerative processes in which activated fibroblast-like synoviocytes (FLS) play a pivotal role [1-3]. Objectives Since hydrogen sulfide (H2S) seems to positively manipulate cells that are affected during degenerative joint disease [4], it was the objective of this study to investigate the effects of exogenous H2S on IL-1β-activated FLS. Methods Primary cell lines derived from FLS of patients with OA were used throughout this study. The effects of IL-1β and NaHS treatment on the phosphorylation of MAP kinases were analyzed by proteome profiler array covering 26 different kinases. The secretion of IL-6 was monitored by ELISA in cell culture supernatants from cultured FLS treated with NaHS. In addition, FLS were grown in 3-dimensional extracellular matrix micromass cultures, stimulated with IL-1β and treated with NaHS. Results NaHS treatment significantly reduced spontaneous and IL-1β-induced IL-6 production. Data from human phospho-MAPK proteome profiler array revealed that IL-1β upregulated the phosphorylation of several MAPkinases but decreased the amount of p-Akt. Remarkably, NaHS treatment inhibited the IL-1β-induced activation of MAPkinases and induced the phosphorylation of Akt. FLS micromass cultures stimulated with IL-1β developed a hyperplastic lining layer-like structure and treatment with NaHS almost completely inhibited this process. Conclusions Our results propose anti-inflammatory properties of hydrogen sulfide on activated synovial fibroblasts that result from selective manipulation of the MAPkinase and the PI3K/Akt pathway. References Fernandes, JC, J Martel-Pelletier, and JP Pelletier, The role of cytokines in osteoarthritis pathophysiology. Biorheology, 2002. 39(1-2): p. 237-46. Roman-Blas, JA, DG Stokes, and SA Jimenez, Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes. Osteoarthritis Cartilage, 2007. 15(12): p. 1367-77. Smith, MD, S Triantafillou, A Parker, et al., Synovial membrane inflammation and cytokine production in patients with early osteoarthritis. J Rheumatol, 1997. 24(2): p. 365-71. Francon, A and R Forestier, [Spa therapy in rheumatology. Indications based on the clinical guidelines of the French National Authority for health and the European League Against Rheumatism, and the results of 19 randomized clinical trials]. Bull Acad Natl Med, 2009. 193(6): p. 1345-56. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3609

Determination of Autoantibody Isotypes Increases the Sensitivity of Serodiagnostics in Rheumatoid Arthritis

Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.

FRI0585 Prevalence of anti-acetylated protein antibodies in inflammatory arthritis, osteoarthritis, connective tissue diseases and its discriminative capacity as diagnostic marker for early rheumatoid arthritis

Background Numerous post-translationally modified proteins have been described as auto-antigens in rheumatoid arthritis (RA) patients. Antibodies (abs) against acetlyated (ac) peptides (AAPA) have recently been reported in RA patients, but not yet been evaluated in other inflammatory and non-inflammatory rheumatologic conditions; therefore their specificity (spec) and sensitivity (sens) remains unclear. Objectives To determine the prevalence of AAPA in RA, healthy subjects and other rheumatic diseases in order to evaluate their diagnostic potential for discriminating RA, healthy and other rheumatic diseases. Methods We obtained serum samples of patients with early untreated RA, established RA (>3 years), osteoarthrits (OA), systemic lupus erythematosus, granulomatosis with polyangiitis (GPA), polymyositis, axial spondyloarthritis, primary Sjögren’s syndrome and healthy subjects. AAPA were measured by ELISA using peptides derived from mutated vimentin (acetylation of lysine or ornithine in position 7 or 2 (inverse peptide), as antigen. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA. Results Areas under the curves (AUC) were significant in early RA (eRA; n=120; 75% female, mean disease duration: −0.07±0.51 years, mean symptom duration 1.49±2.01 years) versus healthy subjects for IgG-abs against ac lysine, inverse lysine and ornithine (AUC of 0.666, 0.687, 0.800, respectively). We chose a cutoff of 20 U/ml putting an emphasise on high spec, with balanced sens (ac-lysine: spec: 97.0%; sens: 32.5%;+likelihood ratio (LR) 10.7, CI: 3.4–33.7; ac-inverse-lysine: spec: 80.7%; sens: 42%;+LR 2.2, CI: 1.3–3.6; ac-ornithine: spec: 93.9%; sens: 39.2%;+LR 6.5; CI: 2.9–14.5). Analyses of positivity for multiple ab-reactivity revealed increasing +LR by number of abs, with 100% specifity when all 3 AAPAs are detected (table 1). Testing this cutoff against OA patients showed similar specificities, but with lower +LR (2 AAPA:+LR 3.48, CI: 1.9–6.6). Sens is increased when testing established RA versus healthy controls, with ac-ornithine performing best (ac-lysine: 49.2%, CI: 42.0–56.5; ac-inv-lysine: 35.2%, CI: 28.5–42.4; ac-ornithin: 53.9%, CI: 46.6–61.0) We found that practically only RA patients showed three different AAPA reactivities (in eRA: 39% positive for ac-ornithine abs, 33% for ac-lysine abs, 48% for inverse ac-lysine abs). Polymyositis and GPA patients showed the lowest prevalence of AAPA (Graph 1A). Among eRA patients 17% were found to be exclusively positive for AAPA, while 39% were also positive for rheumatoid factor (RF) and anti-citrullinated antibodies (ACPA) (distribution in Graph 1B). Also in RF- and ACPA- patients the presence of one AAPA identified RA patients vs. healthy subjects with a spec of 77.7% and those with 2 AAPA reactivities with even 97% respectively.Abstract FRI0585 – Table 1 Sensitivity, specificity, positive and negative likelihood ratio (LR) for identifying early RA patients against healthy controls by the number of AAPA reactivities Graph 1A: Prevalence (in%) of IgG antibodies against the 3 different acetylated peptides using 20 U/ml as cutoff Graph 1B: Venn diagram, outlining the distribution and overlap of AAPA (blue circle), ACPA (striped circle) and RF (rose circle) in early rheumatoid arthritis patients (n=120) Conclusions AAPA are highly prevalent autoantibodies in early RA, closing a further gap of seronegativity, with only 24.6% of early RA patients remaining negative for RF, ACPA or AAPA. In particular, multiple reactivity to AAPA increased the specificity for eRA, also adding diagnostic value beyond RF and ACPA. Disclosure of Interest P. Studenic: None declared, H. Bang Employee of: Orgentc Diagnostika GmbH, A. Alunno: None declared, D. Sieghart: None declared, D. Aletaha: None declared, S. Blüml: None declared, H. Haslacher: None declared, J. Smolen: None declared, G. Steiner: None declared

FRI0125 Rheumatoid arthritis patients with anti-acetylated peptide antibodies starting their first tumor-necrose-factor-inhibitor treatment show greater response

Background Anti-acetylated-peptide antibodies (AAPA) have recently been described in rheumatoid arthritis (RA) patients.[1] Patients that show multiple autoantibody positivity have a higher likelihood to flare when stopping biological treatment. The role of AAPA antibodies for response to Tumor-Necrose-Factor-inhibitor treatment (TNFi) has not been explored. Objectives To determine the prevalence and serological overlap of AAPA to rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) in a cohort of RA patients starting their TNF-inhibitor, and to assess their relation of autoantibody status to response to therapy. Methods We measured AAPA by ELISA using two acetylated peptides derived from vimentin, as well as RF and ACPA. We evaluated the prevalence of autoantibodies, baseline characteristics of autoantibody positive and negative patients, and their association with a 50% response by the Simplified disease activity index (SDAI50), and with achieving of SDAI low disease activity or remission at 6 months after starting the first TNFi. Likelihood ratios were calculated from logistic regression analyses. To better determine differences in SDAI change over time General Estimated Equation analyses (GEE) was used. Results Among our 93 patients starting treatment on a TNFi (85% female, mean disease duration: 7.7±7 years mean SDAI20±13), 50% were positive for AAPA, 57% for ACPA and 61% for RF (for overlap of antibodies see figure). There were no significant differences in baseline characteristics found between AAPA positive and negative patients. 61% of patients reached LDA or REM and 33% achieved an SDAI50 response after 6 months of TNFi treatment. AAPA positive patients showed a likelihood ratio (LR+) of 5.3 (p=0.022) to achieve an SDAI50 response and 3.9 (p=0.048) to reach LDA/REM. In contrast, being RF or ACPA positive did not coincide with a higher likelihood of achieving SDAI50 (LR+ of 0.08; p=0.773 and 1.09; p=0.297, respectively) or LDA/REM (LR+ of 1.83; p=0.177 and 1.55; p=0.213). Relative changes upon treatment with TNFi in composite scores were neither different between RF+ vs. RF- patients, nor between ACPA+ vs ACPA-patients; however AAPA+ showed significantly higher relative changes in SDAI (p=0.021), CDAI (p=0.025) and DAS (p=0.022) compared to AAPA- patients. Of note, relative changes in CRP were similar between the groups. GEE on up to 230 days of TNFi treatment or until change of treatment, showed no significant difference in SDAI courses comparing RF+ vs RF- or ACPA+ vs ACPA- patients; but for AAPA+ vs. AAPA- patients the difference in change in SDAI was significant (p=0.045) over time.Figure 1. Overlap of RF, ACPA and AAPA positive patients. Conclusions AAPA positivity, in contrast to RF and ACPA positivity, appears to have a tighter association with greater levels of response in patients who are initiating TNFi-treatment. Therefore, AAPA might add additional information to estimate the chances of RA patients for responding to TNF-inhibitors. References Juarez M, Bang H, Hammar F, et al. Identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis. Ann Rheum Dis 2016;75(6):1099–107. Disclosure of Interest None declared

A9.3 Hydrogen sulfide and MEK1 inhibition reduce IL-1β-induced activation of fibroblast-like synoviocytes

Background and Objectives In the pathophysiology of osteoarthritis (OA) the secretion of the pro-inflammatory cytokine interleukin (IL)-1β is among the critical steps mediating the aberrant degenerative processes in which activated fibroblast-like synoviocytes (FLS) play a pivotal role. The major goal of this study was to investigate the molecular mechanisms underlying this activation and how treatment with hydrogen sulfide (which is applied for treatment of OA in the form of sulfur spa therapy), or various mitogen-activated protein kinase (MAPK) inhibitors influences them. Methods FLS derived from OA patients were used throughout this study. The effect of IL-1β and NaHS treatment on the phosphorylation of 26 MAPkinases and other serine / threonine kinases was analysed by proteome profiler array. The secretion of IL-6 was monitored by ELISA in cell culture supernatants from conventional cultured FLS treated with NaHS or MAPK inhibitors. In addition, FLS were grown in 3-dimensional extracellular matrix micromass cultures, stimulated with IL-1β and treated with NaHS or the MAPK inhibitors PD98059 (MEK1), SB203580 (p38) and SP600125 (JNK). Results Data gained from human phospho-MAPK proteome profiler array revealed that IL-1β as well as NaHS increased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 (68 ± 16% and 90 ± 27%), mitogen- and stress activated protein kinase (MSK)2 (37 ± 5%), p38 subunits α (232 ± 51%), β (125 ± 38%) and γ (417 ± 216%) as well as c-Jun-N-terminal (JNK) 1/2 (33 ± 7% and 89 ± 3%). Interestingly, NaHS treatment inhibited the IL-1β-induced activation of ERK1/2, MSK2, p38δ and JNK2 between 10 up to 30%. Furthermore, treatment with NaHS or MEK1 inhibitor completely abolished the IL-1β-induced secretion of IL-6, whereas inhibitors of p38 and JNK did not show this effect. FLS micromass cultures stimulated with IL-1β developed a hyperplastic lining layer structure and treatment with NaHS or the MEK1 inhibitor was found to almost completely inhibit this process. Conclusion These experiments demonstrate that hydrogen sulfide decreases the IL-1β-induced phosphorylation of ERK1/2 and MSK2, which signal downstream of MEK1/2. This suggests a direct effect of hydrogen sulfide on MEK1/2 activation leading to decreased IL-6 secretion and inhibition of formation of hyperplastic lining layer structures, which might explain some of the beneficial effects of sulfur spa therapy. Gene expression studies will help to elucidate the molecular mechanisms of the inhibitory effects exerted by hydrogen sulfide.

A8.8 Hydrogen Sulphide Inhibits IL-1β Stimulation of Fibroblast-Like Synoviocytes from Osteoarthritis Patients in a 3-D Model

Objective Osteoarthritis (OA) is a degenerative and most common joint disease which shows characteristic features like loss of cartilage, formation of osteophytes and alteration of subchondral bone leading to joint impairment and pain. The pathogenesis of OA is still not fully understood. Fibroblast-like synoviocytes (FLS), occurring in the intimal lining layer of the synovial membrane, were shown to promote secondary synovitis by the release of pro-inflammatory cytokines and matrix-metalloproteinases (MMPs). The objective of this study was to analyse the possible anti-inflammatory potential of hydrogen sulphide (H2S) on activated FLS cultured in three-dimensional micromass culture. Methods Primary cell lines based on FLS derived from patients with OA were cultivated in spherical extracellular matrix micromasses. Micromass cultures were stimulated for 1 h with IL-1β (10 ng/ml) only or with IL-1β plus either 0.125 mM or 1 mM of sodium hydrogen sulphide (NaHS). As a control, cultures were treated with PBS only. Treatments were applied on day three, five, seven, nine, 12, 14, 16 and 19. Micromasses were cultured for 21 days, fixed with paraformaldehyde, sectioned and stained for hematoxylin and eosin (H&E), IL-6 or MMP-3. Secretion of IL-6 was analysed by enzyme-linked immunosorbent assay (ELISA). Results We observed the spontaneous formation of a compacted, lining layer-like architecture by OA-FLS, already described for rheumatoid arthritis (RA)-FLS. Untreated cultures, in addition, showed clusters of elongated cells underneath the condensed cell layer. A cellular response, which included increased formation of synovial lining as well as changes in cell morphology, could be seen after stimulation with the pro-inflammatory cytokine IL-1β. Treatment with 1 mM of NaHS had the potential to inhibit structural changes caused by cell activation induced by IL-1β. Furthermore, H2S treatment reduced the IL-1β stimulation-related elevated levels of IL-6 secretion. Conclusions The ability of NaHS to inhibit the development of cellular responses to pro-inflammatory IL-1β could be considered a cartilage protective effect and has to be elucidated in more detail.