Background/Purpose: Chronic synovitis is a hallmark of juvenile idiopathic arthritis (JIA), and synovial fibroblasts are major perpetrators of inflammation and tissue destruction. Thus, we tested the efficacy of NG25, a dual TAK1 and MAP4K2 inhibitor, on IL-1β- or cytokine cocktail-activated JIA synovial fibroblasts (JIASFs) in vitro.

Methods: JIASFs were isolated from the synovial fluid aspirated from the knees of de-identified JIA patients. JIASFs (n=3-4) were pretreated with NG25 (0.01–1 µM) followed by IL-1β (10 ng/ml) stimulation for 15 minutes to study the impact on signaling pathways or 24 hours to account for inflammatory or invasive proteins or gene expression. 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 NG25 treatment was determined by ELISA or Western blotting. The inhibitory potential of NG25 against IL-1β activation was further extended to cytokine cocktail [IL-1β (1 ng/ml), TNF-α (5 ng/ml), and IFN-γ (10 ng/ml)] driven activation of JIASFs. The efficacy of NG25 was further compared with different TAK1 inhibitors (5Z-7-oxozeaenol (5Z), takinib, or HS-276). The statistical value of p< 0.05 was considered statistically significant.

Results: NG25 (0.01–1 µM) dose-dependently inhibited IL-1β-induced p-TAK1 at Thr184/187 in JIASFs with IC50 of 531 nM (n=3, p< 0.01), which resulted in downstream inhibition of p-JNK, p-p38, and p-NF-kBp65, but no effect on p-ERK. Western blot analysis showed that NG25 selectively inhibits IL-1β-induced COX-2 expression in a dose-dependent manner (n=4; p< 0.001), with no effect on the markers of cell adhesion and invasion (ICAM-1, VCAM-1, and Podoplanin). Evaluation of the conditioned media by ELISA showed a significant and selective inhibitory effect of NG25 on CXC chemokines (ENA-78 and IL-8), IL-6, and MMP-3 production (n=4; p< 0.001 at 250 nM). In a wash-out experiment where fresh low-serum media was replaced after 24 h of IL-1β +/- NG25 (1 µM) treatment, the inhibitory actions of NG25 on IL-6, IL-8, and MMP-3 production were sustained for up to 7 days in JIASFs and were superior compared to 5Z (0.25 µM), takinib (10 µM) or HS-276 (10 µM) (n=3; p< 0.01). Furthermore, NG25 inhibited the cytokine cocktail (TNFα/IL-1β/IFN-γ)-induced IL-6 (85%), IL-8 (86%), ENA-78 (100%), and MMP-3 (100%) production in JIASFs more efficiently when compared to the other TAK1 inhibitors (5Z, takinib, and HS-276) (n=3-4; p< 0.01-0.001). Similarly, NG25 was most effective in the selective inhibition of COX-2 expression, without affecting the expression of VCAM-1, Cadherin-11, or Podoplanin in cytokine cocktail-activated JIASFs in vitro (n=4; p< 0.05).

Conclusion: Our preliminary findings delineate NG25’s potential anti-inflammatory and anti-chemotactic activities in JIASFs. These findings lay a foundation for testing the efficacy of NG25 in animal models of JIA.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-dual-inhibitor-of-tak1-and-map4k2-ng25-suppresses-cytokine-driven-inflammation-in-juvenile-idiopathic-arthritis-synovial-fibroblasts/