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Background Tofacitinib (TOFA) is a Janus Kinase (JAK) inhibitor approved for the treatment of rheumatoid arthritis (RA) in different countries. TOFA treatment has recently been shown to selectively inhibit the expression of several chemokines from synovial biopsies from RA patients that were treated over 28 days (1). However, TOFA did not modify the expression of various pro-inflammatory cytokines, nor histopathological synovial inflammation, including macrophage infiltration. In this regard, animal models may allow a better understanding of the consequences of JAK inhibition in chronic arthritis (CA). Our group developed a model of CA in rabbits that mimics severe human RA, thereby allowing investigation of TOFA and its consequences in synovial tissue. Objectives We have analyzed the early treatment effect of TOFA in a model of CA, aiming to evaluate early tissue changes rather than the final therapeutic effect. Methods A well-established model of antigen-induced CA was induced in 20 rabbits via four, weekly intra-articular injections of ovalbumin in previously immunized rabbits (CA group). After two intra-articular injections, 10 CA rabbits were treated with TOFA (10mg/kg/day, orally) for two weeks (CA+TOFA) and 10 healthy rabbits were used as controls. Animals were euthanised four weeks after intra-articular challenge, when we collected serum and synovial samples for different studies. Results CA animals showed reduced weight gain compared to controls, which was partially prevented by TOFA (weight gain, kg; Control: 0.8±0.05; CA: -0.09±0.06*; CA+TOFA: 0.18±0.1*#; *p Conclusions In synovial tissue with intense inflammatory activity, TOFA treatment mainly inhibited the expression of MMPs and, to a lesser extent, the expression of other pro-inflammatory mediators. These data suggest that this drug could act as an immunomodulator of the chronic inflammatory response. TOFA does not induce a fast or drastic reduction in cellular recruitment within synovial tissue, but a moderate and generalized decrease of cytokines with a stronger effect on MMPs. References Boyle DL et al. The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis. Ann Rheum Dis. 2014 [epub ahead of print] Disclosure of Interest None declared

Background Osteoarthritis (OA) is characterized by progressive destruction of articular cartilage and changes in chondrocyte phenotype. The signalling of RhoA, a small GTPase protein, has a critical role in a range of cellular functions, including cytoskeleton rearrangement, being essential in focal adhesions and cell-matrix interactions. Several studies described the involvement of RhoA signalling in the healthy chondrocyte phenotype induced by interleukin (IL)-1α, a pro-catabolic cytokine locally increased in OA. Dysregulation of this signalling pathway could play an important role in the development of OA. Objectives We aimed to study RhoA signalling pathway in human OA cartilage, and to establish the underlying mechanism involved in its regulation employing IL-1α in “in vitro” studies. Methods Human OA cartilage was isolated from the knee of OA patients during joint replacement surgery while healthy cartilage was obtained from the knee of age and sex-matched healthy donors. In vitro studies were also conducted on human OA chondrocytes in culture (HOC). HOC were stimulated with 10 ng/ml IL-1α at different periods of time. Results We observed increased protein levels of RhoA and its effector kinase ROCK1 in the cartilage of OA patients when compared to healthy cartilage. Cytoskeletal arrangement is regulated through MYPT-1 phosphorylation -a regulatory subunit of MLC-phosphatase- by different kinases, including ROCK. OA cartilage also showed an increase in both the total and the phosphorylated (Thr-853) form of 70 KDa MYPT-1, compared to healthy cartilage. In addition, we found an increased phosphorylation of the final effector MLC in OA cartilage. In order to further elucidate the mechanism underlying RhoA activation in OA cartilage, we first studied whether IL-1α was able to mimic the RhoA signalling activation in HOC in culture. IL-1α induced a significant increase in RhoA synthesis that was not linked to ROCK1 activation. We also observed an increase in the presence and phosphorylation of the 70KDa MYPT-1 form, with a simultaneous decrease in the 110 KDa MYPT-1 form. ROCK inhibition was unable to prevent MYPT-1 phosphorylation induced by IL-1α in HOC. However, we did observe lower phosphorylation levels when using an ERK1/2 inhibitor. IL-1α induced RhoA recruitment to the HOC membrane, assessed by immunofluorescence. Phalloidin staining revealed that IL-1α triggered formation of stress fibers and the loss of focal adhesion in HOC. Both stress fiber formation and focal adhesion loss were improved by the ERK1/2 inhibitor. Conclusions RhoA signalling is enhanced in the cartilage of OA patients, probably mediated by an increase in MYPT-1 phosphorylation. Stimulation of HOC with IL-1α is able to mimic these changes. However, MYPT-1 phosphorylation in HOC appeared to depend on ERK1/2, rather than on ROCK, as observed in OA cartilage. Further studies are needed to confirm the role of ROCK and ERK1/2 in RhoA activation in chondrocytes. Our results support the hypothesis that RhoA signalling pathway could be a potential therapeutic target in OA. Disclosure of Interest None declared