Murine il 1β

Ankle joints from WT or IRAK1KD mice were dissected, incubated at 37°C in DMEM containing 0.4 mg/mL collagenase IV and 10% FBS for 45 minutes, vortexed, and filtered over a 70 μm cell strainer (Falcon, Corning). Fibroblasts were resuspended in DMEM supplemented with 10% (v/v) FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells (<5 passages) were stimulated with murine IL-1β (PeproTech) in the absence or presence of JH-X-119-01 (MedChem Express) and pacritinib (in-house synthesis). After 16 hours at 37°C, CXCL1, CXCL5, and CCL2 were quantified from supernatants by ELISA (R&D Systems). Alternatively, after incubation at 37°C for the indicated times, protein expression was quantified from lysates by automated capillary-based immunoassay system (WES) as indicated above. Stimulation of synovial fibroblasts with patient synovial fluid (10% in culture medium) was performed in the absence or presence of 10 μg/mL anti-human IL-1β antibody (canakinumab) (67 (link)). Human RA synovial fibroblasts were stimulated with 3 μg/mL PolyIC, 100 ng/mL LPS, or 0.1 ng/mL human IL-1β in the absence or presence of JH-X-119-01 or pacritinib. Protein expression was quantified from lysates at the indicated time points by an automated capillary-based immunoassay system and secreted IL-8 measured by HTRF.

For experiments, colonoids were passaged using TrypLE techniques as already described but with modifications^{3 (link), 27 (link), 29 (link)}. First, Matrigel domes were disrupted by adding ice-cold PBS containing 5 mM EDTA and incubating on ice for 15 min. Colonoids were washed twice with base media, resuspended in TrypLE express (Gibco) and incubated at 37 °C with 5% CO₂ for 2 × 5 min. The colonoids were then rapidly disrupted into single cell suspensions with repeated pipetting through a p1000 tip, and an equal volume of organoid media was added. Cells were centrifuged, then resuspended in base media. The cells were counted using an automated cell counter and the appropriate number of cells (15,000 cells/well for human; 10,000 cells/well for mouse) were seeded using Matrigel as described in the colonoid isolation section in a 24-well plate. Colonoids were stimulated (days 5–7 after seeding for mice, day 10–14 for human) with base media with FliC (100 ng/ml; InvivoGen) or human IL-1β (10 ng/mL; Sigma) or murine IL-1β (10 ng/mL; PeproTech) or LPS O55:B5 (1000 ng/ml; InvivoGen) with or without recombinant IL-37 (100 to 0,1 ng/ml; R&D) or corresponding volume of base media for 4 h.