THU0011 PEFICITINIB (ASP015K) INHIBITS MONOCYTE CHEMOTACTIC ACTIVITY VIA PROINFLAMMATORY CYTOKINE PRODUCTION IN RHEUMATOID ARTHRITIS FIBROBLAST-LIKE SYNOVIOCYTES
Authors: Yuzo Ikari, Takeo Isozaki, Tsuyoshi Kasama
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway has been identified as an important signaling pathway in rheumatoid arthritis (RA) of various cytokines (eg, IL-6). Janus kinase (JAK) is a cytoplasmic protein tyrosine kinase associated with various cytokine receptors. Molecules of signaling pathways such as the JAK family are thought to be promising targets for RA treatment. Peficitinib (ASP015K) is a novel JAK inhibitor in development for the treatment of RA. Peficitinib has been suggested for its effectiveness in clinical trials, but clarification of the mechanism of RA for the inflammatory pathology is still inadequate. <h3>Objectives:</h3> We clarified the effect of peficitinib on RA fibroblast-like synoviocytes (FLS). <h3>Methods:</h3> To determine whether JAK1, JAK2 and JAK3 were expressed in RA ST and FLS, immunohistochemistry was performed. In order to confirm if IL-6 and IL-6 receptor (IL-6R) activate JAK-STAT pathway in FLS, western blot analysis was performed. RA FLS were stimulated with IL-6 (100 ng/ml) and IL-6R (100 ng/ml) for 10 minutes or 30 minutes. Next, we investigated effect of peficitinib on IL-6 and IL-6R responses in RA FLS. RA FLS were stimulated with IL-6 (100 ng/ml) and IL-6R (100 ng/ml) after treated peficitinib (0.1, 1, 5μM) for 24 h. Furthermore, we performed a proliferation assay of FLS and chemotaxis assay using THP-1 (human acute monocyte leukemia cell line) and peripheral blood mononuclear cells (PBMC) to perform functional analysis by peficitinib. In the same procedure as Western blot analysis, peficitinib (5 μM) was added to FLS and stimulate with IL-6 and IL-6R. Finally, we investigated whether peficitinib suppresses the secretion of FLS inflammatory mediator using ELISA. The amounts of RANTES/CCL5, MCP-1/CCL2, MMP-3, fractalkine/CX3CL1, ENA78/CXCL5 and IL-8 in IL-6 and IL-6R stimulated peficitinib treated RA FLS conditioned medium were determined compared with IL-6 and IL-6R stimulated non treated RA FLS conditioned medium. <h3>Results:</h3> We found JAK1, JAK2 and JAK3 were expressed in RA STs and FLS. JAK1 and JAK3 were observed in RA ST lining layers. JAK2 was expressed entirely in RA ST cell nucleus. JAK1, JAK2 and JAK3 were detected in RA FLSs. It was confirmed that JAK1, JAK2 and JAK3 is expressed in FLSs of ST. Representative western blotting showing expression of phospho STAT1, phospho STAT3 and phospho STAT5 were increased 10 minutes after stimulation with IL-6 and IL-6R. Phosphorylation of STAT1, STAT3 and STAT5 in RA FLS was suppressed by concentration dependence of peficitinib. It was proved that peficitiib suppress the activation of JAK-STAT pathway. Furthermore, peficitinib treated RA FLS conditioned medium reduced THP-1 migration compared to nontreated RA FLS conditioned medium (number of THP-1 cells migrated ± SEM; 42 ± 3 and 66 ± 6 cells migrated, respectively, p&lt;0.05). Peficitinib treated RA FLS conditioned medium also reduced PBMC migration compared to nontreated RA FLS conditioned medium (number of PBMC migrated ± SEM; 36 ± 5 and 63 ± 9 cells migrated, respectively, p&lt;0.05). In addition, peficitinib treated RA FLS showed a 14 ± 2% decrease in proliferation of RA FLS compared with nontreated RA FLS. Finally, we found peficitinib suppress the secretion of inflammatory mediators in RA FLS. MCP-1/CCL2 in RA FLS supernatant was suppressed in peficitinib compared to nontreated(mean ± SEM; 160.1 ± 65.6 and 846.0 ± 107.1 pg/ml, respectively, p&lt;0.05). From these results, suppression of chemotaxis of THP-1 and PBMC was also observed through suppression of MCP-1/CCL2 in RA FLS supernatant. <h3>Conclusion:</h3> Peficitinib suppressed the JAK-STAT pathway of RA FLS and was involved in the suppress of monocyte chemotaxis and the proliferation of FLS through suppress of inflammatory cytokines. These results suggest that peficitinib acts on FLS and suppresses inflammatory pathology. <h3>Disclosure of Interests:</h3> None declared