LAUSR

Background: The spleen tyrosine kinase (syk) is an intracellular protein kinase involved in signal transmission processes of immune cells and non-hematopoietic cells, such as fibroblasts. Various syk-inhibitors are therefore currently being tested in preclinical and clinical studies, e.g. for the treatment of different types of cancer as well as autoimmune diseases. In preclinical studies, the syk inhibitor fostamatinib showed a pronounced anti-inflammatory effect on synovial fibroblasts (SF) from patients with rheumatoid arthritis (RA). These cells play an important role in the pathogenesis of RA by mediating matrix destructive processes. On the other hand, clinical trials have been only moderately successful due to the side effects and the limitation regarding increase in dosage. However, further inhibitors such as RO9021 or TAK-659 have been developed. They are characterized by a more specific kinase profile and therefore side effects could potentially be less severe. Whether these inhibitors also convey an anti-inflammatory effect on RASF has not yet been investigated. Objectives: This study examines the effect of syk-inhibitors on the inflammatory response and functional behavior of activated RASF. Methods: RASF were isolated from synovial tissue of patients with known rheumatoid arthritis during joint replacement surgery. RASF were pretreated with different concentrations of RO9021 and TAK-659 and their vehicle control for 2h and additionally stimulated with IL-1β (10 ng/ml) for 17h or with oncostatin M (OSM, 100 ng/ml) for 24h. Supernatants were collected and the concentration of IL-6 and MMP3 were determinate by ELISA. The measurement of cell viability, cytotoxicity and apoptosis was performed to exclude possible cytotoxic or apoptotic effects of the syk-inhibitors. The RASF proliferation was detected by a BrdU-incorporation assay. The effect of syk-inhibitors on cell migration towards a FCS-gradient was measured by cell culture inserts. Results: Both TAK-659 and RO9021 showed a significant reduction of the release of the pro-inflammatory cytokine IL-6 and the production of MMP3 in IL-1β stimulated RASF. However TAK-659 showed a stronger effect on IL-6 reduction (48%, p<0.005) than RO9021 (25%, p<0.05) at 10µM. MMP3 decrease was also stronger with TAK-659 (92%, p<0.001) than with RO9021 (59%, p<0.001). This effect was apparently not restricted to IL-1β because both inhibitors also decreased the OSM-induced IL-6 release of RASF by 85% (p< 0.01) with TAK-659 and 30% with RO9021 at 10µM. The proliferation of RASF was also reduced by both syk-inhibitors at 5µM and higher. The highest effect could be observed with TAK-659 for 80% and with RO9021 by 50% both at 15µM. The migration of RASF was also decreased by TAK-659 at 10µM (p<0.01, n=3). The effects were not mediated by induction of apoptosis or by cytotoxicity. Conclusion: The syk-inhibitors TAK-659 und RO9021 at concentrations above 5µM reduce the inflammatory response, the migration and proliferation of RASF. If appropriate concentrations can be reached in vivo, these syk-inhibitors could offer a possibility to modulate the aggressive phenotype of RASF. Disclosure of Interests: Jeanne Pierrette Lallah Missimana: None declared, Magnus Diller: None declared, Rebecca Hasseli: None declared, Stefan Rehart: None declared, Ulf Müller-Ladner Grant/research support from: Projekt supported by an unrestricted educational grant from Celgene GmbH., Elena Neumann: None declared