Background/Purpose: TNF-α is a proinflammatory cytokine in rheumatoid arthritis that exerts effect through specific receptors TNFR1/2. Though it is a primary therapeutic target, there exist many low- and non-responder groups, potentially indicating novel or redundant signaling mechanisms. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), another TNF-superfamily member, is a pleiotropic cytokine that regulates inflammatory activity, angiogenesis, cell proliferation, and tissue remodeling by binding to the fibroblast growth factor-inducible 14 (Fn-14) receptor. Though TWEAK has been characterized in other systems, the downstream effects of TWEAK and its interaction with TNF-α are not well characterized in human rheumatoid arthritis synovial fibroblasts (RASFs). This study evaluates the role of TWEAK/Fn-14 induced effector molecules in RASFs and their crosstalk with TNF-α. We also investigate the effect of Fn-14 receptor knockdown on TNF-α-mediated inflammation in human RASFs.

Methods: Human RASFs were serum-starved overnight followed by treatment with 200 ng/ml of TWEAK for 48 hours. The role of Fn-14 in TNF-α-induced inflammation was evaluated by siRNA knockdown of Fn-14 and compared to the negative control siRNA (NCsi). Human RASFs were treated with siRNA and then stimulated with TNF-α for 24 hours. Cell lysates were collected to determine the expression of the Fn-14 receptor, podoplanin, and cadherin-11 proteins using the Western blotting method. Soluble proteins MCP-1/CCL2 and RANTES/CCL5 in the conditioned media were measured by ELISA. MMP-2 and MMP-9 activities were analyzed by gelatin zymography. Invasion and migration activities were measured using a trans-well system. The experiments were performed in at least three patient cell lines and the statistical value of p< 0.05 was considered significant.

Results: TWEAK stimulation caused a 10-fold increase in MMP-2 and a 2-fold in MMP-9 activity in human RASFs. TWEAK also enhanced the release of chemokines MCP-1/CCL2 and RANTES/CCL5 in the conditioned media. Evaluation of the effect of TWEAK on functional changes showed a significant increase in the migration and invasion activity of RASFs by 15%. Knockdown of the Fn-14 receptor showed a reduction of TNF-α-induced production of chemokines- MCP1 and RANTES by 26% and 44% respectively, and of adhesion molecule- Cadherin-11 by 41% in RASFs.

Conclusion: The induction of TNF-α-related cytokines, chemokines, and migration markers by TWEAK, as well as TNF-α’s potential use of the Fn-14 receptor, suggests that the effect of TNF-α/TNFR is partially mediated by TWEAK/Fn-14 axis. Our findings in RASFs demonstrate that pharmacological inhibition of Fn-14 may provide an effective adjunct therapy to anti-TNF therapy, which may reduce the number of non-responding patients or increase efficacy for those already on an anti-TNF regimen.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tnf-%ce%b1-utilizes-the-tweak-fn-14-axis-in-human-rheumatoid-arthritis-synovial-fibroblasts/