Background/Purpose: Tumor necrosis factor-alpha (TNF)-α is a proinflammatory cytokine in rheumatoid arthritis that transduces intracellular signal transduction pathways through specific receptors, TNF-R1, and TNF-R2. The anti-TNF therapy has been a primary approach in the management of RA, yet most of the patients fall into low- and non-responder groups with unclear reasons. TNF-like weak inducer of apoptosis (TWEAK), another TNF-superfamily member, is a pleiotropic cytokine that similar to TNF-α regulates inflammatory activity, angiogenesis, cell proliferation, and tissue remodeling by binding to its receptor, fibroblast growth factor-inducible 14 (Fn-14). As TNF-α and TWEAK activate common signaling pathways, we investigated the crosstalk between TWEAK and TNF-α in human rheumatoid arthritis synovial fibroblasts (RASFs) and studied the role of Fn-14 receptor knockdown on TNF-α-mediated inflammation.

Methods: Human RASFs were serum-starved overnight followed by treatment with different concentrations of TWEAK (50, 100, 200, 400, and 800 ng/mL) and TNF-α (5, 10, 20, 40, and 80 ng/mL) alone and in combination for 24 hrs. The secretion of chemokines MCP-1/CCL2, RANTES/CCL5, and MMP-1 was analyzed using ELISA, and synergy was studied using CompuSyn Software. Fn-14 siRNA knockdown in RASFs was done followed by TNF-α stimulation for 24 hrs. Global effects of Fn-14 gene knockdown were studied using an RNA sequencing (RNA-seq) array. Differentially expressed genes (DEGs) were analyzed using GraphPad and Metascape tools. Interactions between Fn-14 and TNF-R1 receptors were studied using the immunoprecipitation (IP) method followed by Western blot analysis. The experiments were performed in at least three patient cell lines and the statistical value of p< 0.05 was considered significant.

Results: TNF-α in combination with TWEAK resulted in greater secretion of both MCP-1 and RANTES compared to each cytokine alone. The synergy was more remarkable at the low combinations of cytokines (5 ng/mL of TNF-α + 50 ng/mL of TWEAK) for MCP-1, RANTES, and MMP-1 with a combination index < 1. RNA-seq data showed that one-third of the genes were differentially expressed in RASFs when stimulated with TNF-α. Out of the 1,389 genes that were 2-fold upregulated by TNF-α, the knockdown of Fn-14 significantly suppressed 168 genes when compared to TNF-α with NCsi treatment. In addition, out of the 2,186 genes 2-fold downregulated by TNF-α, 32 genes were restored in the absence of Fn-14. Gene Ontology analysis of the RNA-seq data suggests that Fn-14-knockdown-associated genes are involved in processes like- regulation of intrinsic apoptosis, regulation of innate immunity, and sensory perception of pain. Furthermore, our IP results with TNF-R1 pull-down showed the presence of Fn-14 indicating the interaction between these two receptors, which requires further experimental investigation.

Conclusion: Our findings suggest that low-dose TNF-α synergizes with TWEAK to induce inflammation and also demonstrate the ability of TNF-α to potentially exploit the Fn-14 receptor to transduce non-canonical signaling in RASFs. This indicates that the pharmacological inhibition of Fn-14 may provide an effective adjunct therapy to anti-TNF therapy.

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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-to-induce-inflammation/