Human il 1β

ELISA kits were used to detect the mouse IL-1β (eBioscience), TNF-α (eBioscience), IL-6 (eBioscience), and human IL-1β (Biolegend, #437004), human TNF-α (eBioscience) according to the manufacturer’s instructions.

Acute inflammatory arthritis was induced in knee joints of C57BL/6N mice as previously described^{20 (link)} by intra-articular injection with 200 µg of mBSA (Sigma A-1009), followed by daily injections on days 0–2 with 250 ng of human IL-1β (BioLegend). At time of sacrifice, knee joints were isolated for histological analysis and qPCR. Draining popliteal and inguinal lymph nodes were isolated for flow cytometry analysis. The treatment with the TGF-βRI inhibitor SB-505124 (Sigma-Aldrich) was applied locally starting two hours before the induction of arthritis, and mice were injected intra-articularly on four consecutive days with either saline, 20% DMSO as vehicle control, or 75 nmol SB-505124 (Sigma-Aldrich) in 20% DMSO with an injection volume of 6 µl per joint. Anti-IL-17A antibodies (BioXCell) were used as positive control, administered as 50 µg antibody/mouse intra-peritoneally injected every other day (n = 8 mice/group). Mice were sacrificed by cervical dislocation four days after arthritis induction, and knee joints were subsequently isolated for histological analysis.

Fasting serum samples were used for biochemical analyses, including TG, CHOL, LDL, HDL, CHOL, AST, ALT, insulin, C-peptide, free fatty acid, BUN, CRE, eGFR, HbA1c, and glucose analyses, at Union Clinical Laboratory (Taiwan, TAF No. L1447-150325/CAP No. 6979606). The cytokine profiles were determined by enzyme-linked immunosorbent assay (ELISA) kits and performed at the Department of Life Sciences (Institute of Biomedical Science, National Chung Hsing University, Taichung, Taiwan). The ELISA kits included human IL-6 (Cat# 900-K16, PeproTech, USA), human IL-10 (Cat# 900-K21, PeproTech), human IL-17A (Cat# 900-K84, PeproTech), human TNF-α (Cat# 50-114-2609, eBioscience, USA), and human IL-1β (Cat# 437005, Biolegend, USA). A glutathione peroxidase assay kit (Cat# 703102, CAYMAN, USA) and superoxide dismutase assay kit (Cat# 706002, CAYMAN) were used in this study according to the manufacturers’ standard protocol.

To activate NLRP3 inflammasome, cells (2 × 10⁵) were primed with TNF‐α (5 ng mL⁻¹; PeproTech EC Ltd. London, UK) for 1 h at 37 °C, followed by treatment with nigericin (10 μm; InvivoGen, San Diego, CA, USA) overnight. Free‐cell culture supernatants were collected, and human IL‐1β (Biolegend) levels were quantified by ELISA, following the manufacturer's instructions.