Il 1β

For evaluation of iTreg stability, cells were (1) re‐stimulated without iTreg differentiation components and (2) challenged with Th17‐polarising cytokines. For (1), cells were re‐stimulated using 500 U mL⁻¹ of IL‐2 and a 1:1 expander beads without addition of TGF‐β, ATRA and rapamycin, for 7 days at 1 × 10⁶ cells mL⁻¹ then rested with 500 U mL⁻¹ of IL‐2 for 3 days at 2 × 10⁶ cells mL⁻¹, after an initial 7‐day stimulation and 3‐day rest. On day 3 of rest, cells were used for flow cytometric analysis and in vitro suppression assay. For (2), cells were challenged for 3 days with Th17‐polarising cytokines, IL‐1β, IL‐6, IL‐21 and IL‐23 (10 ng mL⁻¹ for all, Biolegend, San Diego, CA, USA), in the presence of 25 U mL⁻¹ of IL‐2 and a 1:10 expander beads at 1 × 10⁶ cells mL^‐1, after an initial 7‐day stimulation and 3‐day rest. Cells were stimulated with 25 U mL⁻¹ of IL‐2 and a 1:10 expander beads at 1 × 10⁶ cells mL⁻¹ as unchallenged controls. On day 3 of challenge, cells were washed three times with PBS (+2% FCS). After washing, expander beads were magnetically removed. Cells were used for flow cytometric analysis, cytokine production assays and reverse transcription quantitative polymerase chain reaction.

The laboratory personnel, who performed the cytokine assay, were blinded to clinical characteristics of the 10 gout patients. Purified monocytes were stimulated for 4 hours with 10 ng/mL lipopolysaccharide (LPS) or 200 μg/mL MSU (InvivoGen, San Diego, CA, USA) in the presence of brefeldin A (BFA; BD Biosciences, San Jose, CA, USA). The cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and stained for 30 minutes at 4 degrees with antibodies to CD14, TNF-α (both from BD Biosciences), IL-1β (BioLegend, San Diego, CA, USA), IL-6 (eBiosciences, San Diego, CA, USA). Stained cells were acquired on a BD LSRFortessa (BD Biosciences) and analyzed using Flowjo software (Tree star, Ashland, OR, USA).