Background/Purpose: Chronic synovitis is a debilitating manifestation of juvenile idiopathic arthritis (JIA). Synovial fibroblast is a major preparator of inflammatory arthritis and has not been well studied in JIA. Thus, we aimed to study the involvement of TGF-β activated kinase 1 (TAK1) in cytokine-driven JIA synovial fibroblasts (JIASFs) activation and subsequent induction of inflammatory arthritis.

Methods: JIASFs were isolated from the synovial fluids aspirated from the knees of deidentified JIA patients. JIASFs were treated with TNF-α and IL-1β (0-120 minutes) to study the phosphorylation of TAK1 and downstream signaling pathways. To investigate the overall impact of TAK1 inhibition, RNA sequencing (RNA-seq) analysis of JIASFs following IL-1β stimulation with and without TAK1 inhibitor- 5Z-7-oxozeaenol (5Z) was conducted. JIASFs treated with NG-25 (reversible TAK1 inhibitor) and 5Z (irreversible TAK1 inhibitor) were also examined for the expression of p-TAK1, p-p38, p-ERK, and p-JNK via Western blotting. To study the involvement of TAK1 in inducing JIASF aggressive and inflammatory phenotype, the expression of COX-2, podoplanin, IL-6, IL-8, CXCL5, and CCL5 following 5Z treatment were determined dose-dependently via ELISA and Western blotting. The statistical value of p< 0.05 was considered statistically significant.

Results: RNA-seq analysis identified 2,076 differentially expressed genes significantly regulated by IL-1β (10 ng/mL; n=3, p< 0.05). A total of 502 genes, mostly involved in inflammation and aberrant cell proliferation, were upregulated by IL-1β, and 1,574 genes primarily responsible for controlled cell proliferation and homeostasis were downregulated by IL-1β. Among those 2,076 genes, 707 genes were significantly altered following 5Z (1 µM) treatment, of which 389 genes were significantly upregulated whereas 318 genes were significantly downregulated by 5Z treatment. Gene Ontology studies on RNA-seq revealed that 5Z suppressed the genes involved in cytokine signaling in the immune system, and inflammation, and restored the genes involved in the negative regulation of centriole replication, and regulation of the cell cycle process. Furthermore, the treatment with 5Z or NG-25 (0.01 to 1 µM) followed by IL-1β (10 ng/mL) stimulation revealed that 5Z significantly inhibited the p-TAK1 at 0.01 µM (p < 0.001, n = 3) and p-p38, p-JNK and p-ERK at 0.1 µM (p < 0.0001, n= 3). Whereas NG-25 significantly suppressed p-TAK1 at 1 µM (p < 0.05, n=3) and its downstream p-p38 and p-JNK at 0.1 µM and 0.5 µM, respectively. 5Z also significantly downregulated the expression of COX-2 (at 0.05 µM, p< 0.01, n=3), IL-8 and CXCL5 (at 0.01 µM, p< 0.01, n=3). The expression of podoplanin, IL-6 and CCL5 also went down following 5Z treatment (at 0.25 µM, n=3) indicating the overall potential of TAK1 inhibition to limit JIASFs ability to exacerbate arthritis.

Conclusion: Our preliminary findings delineate the critical role of TAK1 in cytokine-driven hyperplasia and invasiveness of JIASFs, and the inhibition of TAK1 in vitro promises cessation of JIASF- mediated expression of inflammatory markers.

« Back to ACR Convergence 2023

ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-tgf-%ce%b2-activated-kinase-1-in-cytokine-driven-juvenile-idiopathic-arthritis-synovial-fibroblasts-activation/