I Blijdorp

Ectopic lymphoid neogenesis is strongly associated with activation of the IL-23 pathway in rheumatoid synovitis

IntroductionThe functional relevance of synovial ectopic lymphoid neogenesis (ELN) in rheumatoid arthritis (RA) remains unknown. As ELN correlates with the degree of tissue inflammation, we investigated whether ELN was associated with specific cytokine profiles.MethodsSynovial ELN was determined by immunohistology and long CD21 isoform (CD21L) expression. Cytokine expression was determined by multiplex enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) as well as immunohistology in synovial fluid (SF) (n = 44) and tissue (ST) (n = 108), respectively. Production of ELN-associated chemokines by fibroblast-like synoviocytes (FLS) was studied in vitro.ResultsScreening analysis of SF by multiplex ELISA showed higher protein levels of interleukin (IL)-23 (p = 0.018) and IL-17F (p = 0.028) in ELN+ versus ELN- samples. Other cytokines, including IL-17A, IL-6, and tumor necrosis factor (TNF)-α, were not different. The association between IL-23 and ELN was not biased by disease activity or other clinical features and was confirmed by higher IL-23 mRNA expression in ELN+ versus ELN- ST samples (p = 0.030), a correlation between IL-23 and CD21L expression in the same samples (r = 0.70 p < 0.0001), and a similar correlation in two independent ST sample sets (r = 0.778 p < 0.0001 and r = 0.817 p = 0.011). IL-23 p19 staining was neither restricted nor enhanced in close proximity of ectopic lymphoid follicles, and neither IL-23 nor IL-17A stimulation induced expression of the ELN-associated CC chemokine ligand, CCL21 and CXC chemokine ligand CXCL13, by FLS. Downstream of IL-23, CD21L expression was significantly associated with IL-17F, IL-21, and IL-22, but not IL-17A in two independent ST sample sets.ConclusionsSynovial ELN in RA is strongly associated with activation of the IL-23 pathway but not with IL-17A.

Human mast cells capture, store, and release bioactive, exogenous IL-17A

IL‐17A, a major proinflammatory cytokine, can be produced by a variety of leukocytes, but its exact cellular source in human inflammatory diseases remains incompletely understood. IL‐17A protein is abundantly found in mast cells in human tissues, such as inflamed synovium, but surprisingly, mechanistic murine studies failed to demonstrate IL‐17A production by mast cells. Here, we demonstrate that primary human tissue mast cells do not produce IL‐17A themselves but actively capture exogenous IL‐17A through receptor‐mediated endocytosis. The exogenous IL‐17A is stored in intracellular granules and can subsequently be released in a bioactive form. This novel mechanism confers to mast cells the capacity to steer IL‐17A‐mediated tissue inflammation by the rapid release of preformed cytokine.

The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin.

Platinum-based chemotherapy (e.g. cisplatin, carboplatin) is standard of care for many types of cancer including ovarian cancer, however, the efficacy of treatment is hampered by the development of therapy resistance. The mechanisms behind platinum resistance are not completely understood. Here, we have investigated the role of the family of p90 Ribosomal S6 kinases (RSK), important downstream mediators of ERK1/2, in the response to cisplatin chemotherapy. Strikingly, whereas treatment with cisplatin did not alter the levels of RSK1 in response to cisplatin treatment, the structurally related RSK2 protein was downregulated in an ovarian cancer cell line (A2780). Furthermore, we found that knockdown of RSK2, in contrast to knockdown of RSK1, gave rise to enhanced cisplatin sensitivity in a cisplatin sensitive as well as a cisplatin-resistant A2780 cell line. These results indicate that RSK2 is regulated in response to cisplatin treatment, and this downregulation may contribute to the cytotoxic action of cisplatin. Since RSK2 is frequently amplified in a growing number of cancers, this may have implications for the sensitivity of these tumours to platinum-based cytotoxics.

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

AB0049 Human Type 1 Innate Lymphoid Cells Accumulate in the Inflamed Synovium in Spondyloarthritis

Background Spondyloarthritis (SpA) is a major form of chronic inflammatory arthritis characterized by inflammation of axial and peripheral joints and by pathologic new bone formation leading to ankylosis. Consistent genetic, experimental, and clinical evidence indicates that IL-23/IL-17 immune axis plays a pivotal role in the pathophysiology SpA. It remains, however, unknown which IL-23 responsive cells are the major cellular source of IL-17 in SpA. Innate lymphoid cells (ILCs) are an emerging family of innate immune cells that produce various cytokines, including IL-17 and IL-22, and play critical roles in regulation of inflammation and tissue remodeling. Objectives In this study we investigated the presence and phenotype of ILCs in the peripheral blood and inflamed peripheral joints of patients with SpA. Methods Paired peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) were obtained from SpA patients with actively inflamed knee joints. ILCs (lineage negative, CD45+CD127+ CD161+) were analysed by flow cytometry. Results ILCs were present in all three compartments of patients with SpA. Analysis of ST revealed a significantly increased frequency of total ILCs in the joint compared with PB ((median (IQR) 0.37 (0.12-1.12)% of the lymphocyte population in ST versus 0.06 (0.04-0.09) % in PB, p=0.016). Immunophenotyping of ILC subsets showed a statistically significant increase in the frequency of ILC1 (CRTH2-NKp44-ckit-) in ST (37.8%; 74.43-20.47%) versus SF (7.27%; 0.6-25.1%, p=0.008) and PB (3.45%; 1.45-9.25%, p=0.004). The second most prominent ILCs in the joint were NCR-negative ILC3 (CRTH2-NKp44-ckit+), composing 33.45% (9.54-50.64%) of the total ILCs. NCR-positive ILC3 (CRTH2-NKp44+ckit+) and ILC2 (CRTH2+) populations were present in synovium at lower frequencies. Conclusions We observed an absolute and relative enrichment of both ILC1 and NCR-negative ILC3 in the inflamed ST of patient with spondyloarthritis as compared to PB and SF. As studies in other tissues such as gut and tonsil revealed that these IL-23 responsive ILC subsets can be an important source of IL-17 and /or IL-22 (1, 2), we will further investigate the cytokine production by these synovial ILCs. References Geremia et al. J Exp Med. 2011 208(6):1127-1133 Bernink et al. Nature Immunology 14(3):221-229 Disclosure of Interest None declared

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

Interleukin-17 Blockade With Secukinumab in Peripheral Spondyloarthritis Impacts Synovial Immunopathology Without Compromising Systemic Immune Responses

Secukinumab (anti–interleukin‐17A [anti–IL‐17A]) is an effective therapy for ankylosing spondylitis and psoriatic arthritis, the prototypical forms of spondyloarthritis (SpA). We undertook this study to determine whether secukinumab modulates the immunopathology of target lesions without blunting systemic immune responses, using peripheral SpA as a model.

IL-22- and GM-CSF-expressing but not IL-17A-expressing group 3 innate lymphoid cells are expanded in the inflamed spondyloarthritis joint

Clinical trials of the anti–interleukin‐17A (anti–IL‐17A) antibody secukinumab have demonstrated a crucial role of the cytokine IL‐17A in the pathogenesis of spondyloarthritis (SpA); however, its cellular source in this condition remains a matter of controversy. Group 3 innate lymphoid cells (ILC3s) have been recently identified as potent producers of proinflammatory cytokines, including IL‐17A and IL‐22, in a number of different tissues. This study was undertaken to characterize the presence and composition of ILCs, and investigate whether these cells are an important source of IL‐17A, in the synovial tissue (ST) of patients with SpA.

08.22 The role of transmembrane rather than soluble tnf in spondyloarthritis

Background TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). Here we aimed to investigate how TNF can lead to completely different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral remodelling arthritis in SpA. Materials and methods We assessed expression of TNF, TNF-R, and TACE (ADAM17) in synovial fluid, synovial tissue, and synovial fibroblasts from SpA and RA. tmTNFtg mice (TgA86)1 were clinically scored for development of peripheral and axial disease, and sacrificed at the end of the experiments for radiologic and histologic assessment. Mechanistic studies included bone marrow chimaera experiments and crossing with TNF-R1 or TNF-R2 knock-out animals. Results Arthritis was characterised by lower levels of sTNF and higher levels of tmTNF in SpA versus RA. This misbalance was related to decreased TACE activity in SpA versus RA FLS, as further confirmed by lower levels of other soluble molecules cleaved by TACE (including sTNF-RI, sTNF-RII, and sCD163) in the inflamed SpA joint. Assessing whether tmTNF has a functional role in SpA pathology, mice selectively over-expressing the transmembrane form of TNF spontaneously developed a deforming arthritis and spondylitis, starting at 4 weeks of age and reaching a 100% incidence. Histology revealed peripheral and axial synovitis, enthesitis, and osteitis, as well as inflammation of the connective tissue located at the junction of the annulus fibrosus with the vertebral bone. tmTNFtg mice did not develop extra-articular inflammation. Structural phenotyping by histology and radiology revealed mild destructive features in combination with foci of hypertrophic chondrocytes and axial and peripheral new bone formation leading to bridging of tail vertebra over time. Mechanistic experiments revealed that this SpA-like phenotype was mediated by tmTNF expression on stromal cells but not on hematopoietic cells and required the expression of TNF-R1. Conclusions Collectively, these data suggest that tmTNF expressed by stromal cells is responsible for the key pathological features of SpA. References 1.Alexopoulou L, et al. Eur J Immunol1997;27(10):2588–92.

A6.20 Ectopic lymphoid neogenesis is strongly associated with activation of the IL-23 pathway in rheumatoid synovitis

Background and objectives The functional relevance of synovial ectopic lymphoid neogenesis (ELN) in rheumatoid arthritis (RA) remains unknown. As ELN correlates with the degree of tissue inflammation we investigated whether ELN was associated with specific cytokine profiles. Materials and methods Paired synovial tissue (ST) (n = 63) and fluid (SF) (n = 44) was obtained from the inflamed knee joints of RA patients. Synovial inflammation and ELN was determined by immunohistology. CD21L was used as molecular marker of ELN. Cytokine expression was determined by ELISA and quantitative PCR in SF and ST, respectively. Results 48% of ST displayed ELN by histology. ELN+ samples had increased T and B lymphocyte infiltration (p < 0.001) and CD21L expression (p = 0.014). SF analysis showed higher expression of IL-23 (p = 0.018) and IL-17F (p = 0.028) in ELN+ versus ELN- samples, with a similar trend for IL-22 (p = 0.070). Other cytokines, including IL-17A, IL-6, TNF-a, Th1 cytokines and Th2 cytokines, were not different. In ST, IL-23 (p = 0.030) mRNA levels were increased in ELN+ samples. Moreover, CD21L expression as molecular marker of ELN correlated significantly with mRNA expression of IL-23 (r = 0.70), IL-17F (r = 0.42), IL-21 (r = 0.30) and IL-22 (r = 0.33), but not IL-17A. The strong correlation between CD21L and IL-23, IL-17F, IL-21 end IL-22 was confirmed in an independent RA ST sample set (n = 36). IFNg and IL-2, but not IL-6 and TNF-α, also showed some correlation with CD21L expression. Conclusion Synovial ELN in RA is strongly associated with increased expression of IL-23/IL-17-related cytokines. Whether patients depicting synovial ELN respond differently to therapeutic targeting of this pathway remains to be determined.