inflammation through regulation of cytokine, chemokine and metalloprotease expression. Thus, the study of the non-canonical pathway of Wnt5a in the aggressive phenotype of FLS and the inflammatory response may provide new therapeutic targets for the RA. Objectives: To analyse the involvement of Wnt5a in the proliferation, migration, invasion and inflammatory responses of FLS from RA patients. Methods: FLS were obtained from eight RA patients. Expression of Wnt5a was suppressed by siRNA

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... ion (Dharmacon). Cellular proliferation was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Migration was analysed by the wound-healing assay using Ibidi inserts. The occupation area was determined by Image J. Invasion was tested by quantifying Giemsa stained cells on the bottom side of Matrigel coated trans-membrane (Millipore). The expression of inflammatory mediators and metalloproteases was analysed by real-time PCR after stimulation with recombinant Wnt5a protein (rWnt5a), TNF and combined treatment. Results: We analysed the effect of the absence of Wnt5a and rWnt5a on the proliferation, migration and invasion of RA FLS. FLS lacking Wnt5a showed a significant migration decrease, 27.9% in basal condition and 16.9% after TNF stimulation, when compared with FLS transfected with siRNA control. Accordingly, migration of RA FLS treated with rWnt5a was increased by 48.5% when compared with cells treated with the vehicle. No change in migration was observed in TNF-stimulated FLS. Invasive capacity was reduced, reaching 71.4% of that observed in FLS transfected with siRNA control in basal conditions. In addition, invasion in FLS treated with rWnt5a was 35.7% higher than in controls. We also analysed the effect of rWnt5a on the inflammatory response of RA FLS. We found a significantly increase of IL6, IL8, CCL2, CXCL5, MMP9 and MMP13 basal expression and the TNF-induced expression of IL6, IL8, CXCL5 and MMP3. Nevertheless, the lack of Wnt5a or the addition of rWnt5a did not modify the spontaneous or the TNF-induced proliferation. Conclusions: These results indicate that Wnt5a contributes to the aggressive phenotype of FLS by potentiating their migration and invasion, and by stimulating the inflammatory response. Acknowledgements: Supported by grant of the ISCIII/PI14/01660/RETICS Program, RD16/0012/0014/cofinanced FEDER. Abstract THU0064 -Figure 1. Expression of addhesion molecules (A, B), activation of IRAK (C), NFkB (D) signalling and apoptosis (E) in EAhy 926.