J.E. Paramarta

Clinical and Imaging Signs of Spondyloarthritis in First-Degree Relatives of HLA-B27-Positive Ankylosing Spondylitis Patients: The Pre-Spondyloarthritis (Pre-SpA) Cohort Study

To investigate whether seemingly healthy first‐degree relatives of patients with ankylosing spondylitis (AS) have clinical, laboratory, or imaging features of spondyloarthritis (SpA).

OP0156 Targeting Synovial Mast Cells in Spondyloarthritis: A Proof-Of-Concept Study with Nilotinib A Tyrosine Kinase Inhibitor

Background Immunopathological studies on synovitis recently identified the mast cell as potential novel therapeutic target in spondyloarthritis (SpA).[1] Mast cells can be targeted by inhibiting the signalling of c-Kit, which is one of the targets of the tyrosine kinase inhibitor nilotinib. Objectives To evaluate the immunomodulating and clinical effects of nilotinib in the treatment of SpA. Methods 28 patients with active peripheral and/or axial SpA were included in a randomized, double-blind, placebo-controlled clinical trial. Patients were treated 1:1 with nilotinib or placebo for 12 weeks, followed by an open label extension for another 12 weeks. Paired synovial tissue biopsies, serum sampling and assessment of clinical symptoms were performed serially. Results In peripheral SpA (n=13) synovial inflammation was markedly reduced after 12 weeks of nilotinib treatment as evidenced by histopathology (decrease in number of infiltrating CD68+ and CD163+ macrophages and mast cells). Compared to placebo the mRNA expression of c-Kit as mast cell marker (p=0.037) and of pro-inflammatory cytokines such as IL-6 (p=0.024) were reduced. The improvement of synovial inflammation was paralleled by a decrease in serum biomarkers of inflammation such as C-reactive protein (CRP) from 9.2 (IQR 1.7-33.1) to 5.2 (IQR 1.7-25.1) mg/L (p=0.024) and calprotectin from 359.9 (IQR 183.3-484.9) to 287.9 (IQR 116.7-457.1) ng/mL (p=0.055). Also clinical parameters such as patients global assessment of disease activity (week 0: 52 (IQR 43-65) vs week 12: 21 (IQR 0-51) mm; p=0.031) and Ankylosing Spondylitis Disease Activity Score (ASDAS) (week 0: 2.2 (IQR 1.2-3.0) vs week 12: 1.1 (IQR 0.7-2.4); p=0.031) showed improvement upon 12 weeks of nilotinib but not placebo treatment, and this improvement was further augmented at week 24. In sharp contrast to peripheral SpA, neither serum biomarkers of inflammation nor clinical parameters improved upon nilotinib treatment in axial SpA. During the trial one serious adverse event occurred, which was considered unrelated to the study drug. There were no unexpected safety signals in comparison with published large scale data on nilotinib in chronic myeloid leukemia (CML). Conclusions This small proof-of-concept study supports the concept that mast cells can contribute to synovial inflammation in SpA and that tyrosine kinase inhibition targeting these cells has a biological and clinical immunomodulatory effect in peripheral but not axial SpA. These results support further clinical evaluation of nilotinib in larger clinical trials in pure peripheral SpA, as well as evaluation of other drugs targeting mast cells in SpA. References Noordenbos T, et al. Interleukin-17-positive mast cells contribute to synovial inflammation in spondylarthritis. Arthritis Rheum 2012;64:99-109. Acknowledgements We thank Novartis for the supply of the study medication for this investigator initiated and independent study. Disclosure of Interest J. Paramarta: None declared, M. Turina: None declared, T. Noordenbos: None declared, T. Heijda: None declared, I. Blijdorp: None declared, N. Yeremenko: None declared, D. Baeten Grant/research support: AbbVie, Centocor, Janssen-Cilag, MSD, Novartis, and Pfizer, Consultant for: AbbVie, BMS, Boehringer Ingelheim, Centocor, Janssen, MSD, Novartis, Pfizer, and UCB DOI 10.1136/annrheumdis-2014-eular.2823

OP0226 Human Mast Cells Engulf and Store Exogenous IL-17A

Background IL-17A plays an important role in rheumatic diseases, like rheumatoid arthritis and spondyloarthritis. Direct analysis of inflamed synovial tissue revealed an abundant presence of IL-17A-positive mast cells. Objectives As mast cells are not known to produce IL-17A in mice, we aimed to investigate the mechanism of IL-17A expression by human mast cells. Methods IL-17A, IL-17F and RORC mRNA and protein expression was assessed ex vivo and after PMA/ionomycine stimulation in primary human mast cells sorted from tonsils. Internalization of exogenous IL-17A was assessed by Western blot, imagestream, live imaging and confocal microscopy. Results Immunohistochemistry and western blot analysis confirmed the presence of IL-17A protein in primary human mast cells. In contrast to T cells, however, mast cells did not express RORC protein, the exclusive transcriptional factor controlling IL-17A expression. Accordingly, IL17A, IL17F, and RORC gene expression was readily detectable in sorted T lymphocytes but not in mast cells, even after ex vivo stimulation. Given the discrepancy between the presence of IL-17A protein and absence of its transcriptional machinery, we investigated the uptake of GFP-fused or 6xhistidin-tagged recombinant IL-17A. Imagestream and Western blot indicated that both primary mast cells and the LAD2 mast cell line engulf and store exogenous IL-17A. Live imaging and confocal microscopy revealed that internalized IL-17A is stored in endocytic vesicles and that this uptake can be blocked by inhibiting receptor-mediated endocytosis. Conclusions Human mast cells do not produce IL-17A but engulf and store exogenous IL-17A from the inflamed milieu. Molecular pathways of IL-17A uptake and eventually release are under investigation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2566

OP0297 Calprotectin (S100A8/9) as Serum Biomarker for Clinical Response in Proof-Of-Concept Trials in Axial and Peripheral Spondyloarthritis

Background Biomarkers complementing clinical evaluations may help to reduce the length and size of proof-of-concept (PoC) trials aimed to obtain quick “go/no go” decisions in the clinical development of new treatments. Objectives We aimed to identify and validate serum biomarkers to predict clinical response in spondyloarthritis (SpA) PoC trials. Methods The candidate biomarkers high sensitive-C-reactive protein (hs-CRP), interleukin-6 (IL-6), pentraxin-3 (PTX-3), alpha-2-macroglobulin (alpha-2-MG), matrix metalloproteinase-3 (MMP-3), calprotectin, and Vascular Endothelial Growth Factor (VEGF) were determined by ELISA in healthy controls (n=20) and SpA patients before and after 2 weeks of infliximab (n=18) or placebo (n=19) treatment. Clinical outcome was evaluated at week 12. Results were validated in ankylosing spondylitis (AS) with infliximab and peripheral SpA with etanercept. Results Serum levels of calprotectin, hs-CRP, PTX-3, VEGF (all P<0.001) and MMP-3 (P=0.062), but not IL-6 and alpha-2-MG, were increased in SpA versus healthy controls. Treatment with infliximab, but not placebo, significantly decreased calprotectin (P<0.001) and hs-CRP (P<0.001) levels, with a similar trend for MMP-3 (P=0.063). The Standardized Response Mean (SRM), which reflects the ability to detect changes over time, was high for calprotectin (1.26), good for hs-CRP (0.96) and moderate for MMP-3 (0.52). Calprotectin and hs-CRP, but not MMP-3, were good biomarkers of treatment response in axial SpA as evaluated in 2 separate cohorts. All 3 markers reflected response to etanercept treatment in peripheral SpA with SRMs above 0.5. Conclusions Calprotectin and hs-CRP are good serum biomarkers to predict clinical response at the group level in small-scale, short term PoC trials in SpA. Disclosure of Interest M. Turina: None declared, N. Yeremenko: None declared, J. Paramarta: None declared, L. De Rycke: None declared, D. Baeten Grant/research support: AbbVie, Centocor, Janssen-Cilag, MSD, Novartis, and Pfizer, Consultant for: AbbVie, BMS, Boehringer Ingelheim, Centocor, Janssen, MSD, Novartis, Pfizer, and UCB DOI 10.1136/annrheumdis-2014-eular.2837

A2.14 Human mast cells engulf and store exogenous IL-17A

Background The emerging role of IL-17A in immune-mediated inflammatory diseases urges characterisation of the cellular source. Numerous recent studies have described the presence of IL-17A-positive mast cells in inflamed target tissues. Since mouse studies have not indicated mast cells as IL-17A producers, we investigated the mechanism of IL-17A expression by human mast cells. Methods Quantitative PCR and Western blot analysis was performed on mast cells and CD4+ T-cells FACS-purified from human tonsils, ex vivoand after stimulation with PMA/ionomycin. Uptake of exogenous protein was analysed by microscopy, imaging-flowcytometry, live imaging and Western blot. DynaminII-inhibitor was used to block endocytosis. Results Strikingly, gene expression analysis of highly purified primary mast cells failed to detect both IL-17A, IL-17F and their transcription factor RAR-related orphan receptor C (RORC), even after stimulation. Although RORC was also absent on the protein level, IL-17A was abundantly detected, conflicting with the mRNA data. Subsequent analysis of subcellular location showed the presence of IL-17A in lysosomal storage granules and in early endosomes, suggesting a role for endocytosis. We investigated whether mast cells are capable of internalising exogenous IL-17A in vitro. Microscopy, imaging-flowcytometry and Western blot analysis indicated that LAD2 mast cell line as well as primary mast cells are able to engulf exogenous IL-17A, but not TNFa. Live imaging revealed further that internalisation of IL-17A is specific and can be halted by blocking clathrin-mediated endocytosis. Conclusion This data strongly suggest that human mast cells do not produce IL-17A, but rather engulf exogenous IL-17A from the inflamed milieu and store it in granules. Molecular mechanisms of IL-17A uptake and the contribution of this process to disease are currently under investigation.

SAT0278 Clinical and Imaging Signs of Spondyloarthritis in First Degree Relatives of HLA-B27 Positive Ankylosing Spondylitis Patients: the Pre-Spondyloarthritis (Pre-SPA) Cohort

Background Although the diagnostic delay in spondyloarthritis (SpA) has been significantly reduced over the last decade, studying the earliest disease phases remains challenging. Systematic screening of individuals at high risk, including first degree relatives (FDRs) of patients, might help to address this challenge. Objectives To assess clinical, laboratory, and imaging features of SpA in seemingly healthy FDRs of ankylosing spondylitis (AS) patients. Methods FDRs of HLA-B27 positive AS patients between an age of 18-40 years were included in Pre-SpA, a prospective inception cohort study. Clinical (including medical history, disease activity, and clinical examination), biological (HLA-B27, CRP, ESR, and calprotectin) and imaging (X-rays and MRI of the spine and sacroiliac joints (SIJ)) data were obtained. FDRs were classified according to the Assessment of Spondyloarthritis international Society for axial SpA (ASAS axSpA), ASAS peripheral SpA, the modified New York (mNY), the European Spondyloarthropathy Study Group (ESSG) and the ClASsification for Psoriatic ARthritis (CASPAR) criteria. Results We report the baseline features of the first 51 FDRs included in this ongoing prospective study. Thirty (59%) FDRs had back pain, of whom 9 (18%)had inflammatory back pain. None of the FDRs had peripheral arthritis/enthesitis or extra-articular manifestations although 39% reported a history of arthralgia and 16% had a tender joint count of 1 or more. Three (6%) FDRs had low grade sacroiliitis on X-ray, 1 (2%) FDR had cervical syndesmophytes on X-ray, and 11 (22%) FDRs showed bone marrow oedema of the SIJ on MRI and none of the FDRs had abnormalities of the spine on MRI. Thirteen out of 51 (25%) FDRs fulfilled any of the classification criteria: 9 (18%) fulfilled the ASAS axSpA criteria, 10 (20%) fulfilled the ESSG criteria, and 6 (12%) fulfilled both the ESSG and the ASAS axSpA criteria. None of the FDRs fulfilled any of the other classification criteria. FDRs fulfilling SpA criteria had more frequently back pain and inflammatory back pain, had a higher disease activity (including BASDAI), and worse function (BASFI), but there was no difference in inflammatory parameters, peripheral and extra-articular disease, and HLA-B27 status. In addition to the 13 (25%) FDRs fulfilling SpA criteria, 6 of the 38 (16%) FDRs not fulfilling any criteria had imaging abnormalities suggestive of SpA (5 on MRI and 1 on X-ray). Conclusions A substantial proportion of seemingly healthy FDRs of HLA-B27 positive AS patients had clinical and/or imaging abnormalities suggestive of SpA. Twenty-five percent of the FDRs were classified as having SpA. Further follow-up of this cohort will show which FDRs will develop clinically overt SpA over time, which initial features can predict this development, and which sequence of events is followed during disease development. Disclosure of Interest None declared

FRI0460 Differences and similarities of spondyloarthritis classification subgroups defined by the assessment of spondyloarthritis international society (ASAS)

Background Since the Assessment of SpondyloArthritis international Society (ASAS) developed new classification criteria, spondyloarthritis (SpA) is no longer divided into different phenotypic subtypes such as ankylosing spondylitis and psoriatic arthritis, but into axial and peripheral SpA. Objectives This study aimed to compare the clinical characteristics and disease activity of the different SpA subpopulations according to the new ASAS criteria. Methods 389 patients presenting on two dedicated SpA outpatient clinics fulfilling the European Spondyloarthropathy Study Group (ESSG) criteria were included in a prospective observational cohort. Baseline characteristics were collected and patient’s and physician’s global assessment of disease activity, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS), 68 swollen and tender joint count, Schober, ESR and CRP were measured every 3 months. Data were analyzed with Mann Whitney U and chi square tests. Results 314 of the 389 SpA patients (81%) fulfilled the ASAS classification criteria. Patients fulfilling the ASAS criteria had a younger age at disease onset (median 34.1 vs 38.2), were more often male (61.8 vs 36.0%) and HLA-B27 positive (65.1 vs 2.9%), and had more often sacroiliitis on imaging (53.4 vs 1.4%) than those who did not fulfil the ASAS criteria. Strikingly, most disease activity parameters were similar between the two groups and the median BASDAI (4.6 vs 5.3) and TJC (0 vs 3) was even higher in the latter group. Of the patients fulfilling the ASAS criteria, 230 (59% of the total population) fulfilled the axial and 84 (22%) the peripheral SpA criteria. ASAS peripheral SpA had an older age at disease onset (32.3 vs 38.2) and were less often HLA-B27 positive (76.2 vs 27.3%) than ASAS axial SpA. The parameters of global disease activity (patient’s and physician’s global assessment and ASDAS) were higher in axial than peripheral SpA, whereas CRP and ESR were similar. As the ASAS criteria exclude patients with active axial symptoms from the peripheral SpA group but do not exclude patients with peripheral disease from the axial SpA group, we additionally subdivided patients who fulfilled the ASAS axial SpA criteria into pure axial and combined disease (back pain plus arthritis, enthesitis or dactylitis). The combined group was intermediate between pure axial SpA and peripheral SpA in terms of clinical characteristics such as disease symptoms and age at disease onset. Most importantly, the combined group had the highest disease activity compared to the other two groups (eg. ASDAS 3.0 vs 2.6 and 2.0, respectively). Conclusions The ASAS criteria fail to classify a subgroup of 20% of the patients fulfilling the ESSG criteria and having high disease activity. Within the patients fulfilling the ASAS axial SpA criteria, we discriminate two separate groups corresponding to exclusive axial disease versus combined axial and peripheral disease. These data support a classification into axial, combined, or peripheral disease rather than just axial versus peripheral disease. The combined group could enter the classification through either the axial or peripheral SpA criteria. Disclosure of Interest None Declared

Elevated levels of CD5+ B cells in spondyloarthritis patients

We recently proposed that spondyloarthritis (SpA) is characterised by primary alterations in the innate rather than the acquired immune system. However, SpA patients develop a strong IgM antinuclear antibody profile upon tumour necrosis factor blockade. The characteristics of these autoantibodies are compatible with natural antibodies originating from ‘innate’ B cells rather than with genuine, pathogenic autoantibodies. As in mice natural autoantibodies are produced by CD5 expressing peritoneal B cells responding to innate immune signals, we aimed to quantify and …