Mil 1β

Monolayers derived from colonoids (human or mice) were generated as previously described with modifications^{27 (link)–29 (link)}. First, the growth media was removed, then Matrigel domes were disrupted and colonoids were 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 gentle pipetting through a p1000 tip, and an equal volume of monolayer media (base media supplemented with 50% WRN, N2 (Invitrogen), B27 (Invitrogen), mEGF (Invitrogen) and Y-27632 (AbMole) was added. Cells were centrifuged, then resuspended in monolayer media and added dropwise to Geltrex (Gibco) coated coverslips in 24-well plates. Monolayers were incubated at 37 °C with 5% CO₂ and media changed 24 h after seeding. 72 h after seeding, confluent monolayers were stimulated with monolayer media supplemented with FliC (100 ng/ml; InvivoGen) or mIL-1β (10 ng/mL; Invitrogen) with or without recombinant IL-37 (100 ng/ml; R&D systems) or a corresponding volume of base media for 30 min.

Concentrations of mIL‐1β (Invitrogen, 88‐7013‐22) and mIL‐23 (eBioscience, 88‐7230‐22) were measured by ELISA kit according to the manufacturer's recommendations.

Bone marrow-derived DCs (BMDC) were generated by plating bone marrow cells freshly isolated from tibia and femur into 10 cm dishes and cultured with RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin and streptomycin, mGM-CSF (10 ng/ml; eBioscience) and mIL-4 (10 ng/ml; eBioscience). On day 6, floating and loosely attached cells were collected representing the BMDCs. For co-culture with CD4⁺ T cells: BMDCs (2 × 10⁴ cells/well) were seeded in 96-well round-bottomed plate and pulsed either with OVA alone (20 μg/ml) or OVA + c-di-GMP (25 μg/ml) for 18 h. After gentle washing, BMDC were cultured for 4 days with CD4 T cells (1 × 10⁵ cells/well) purified (by magnetic selection; Miltenyi Biotec) from spleen of OT-II transgenic mice. Supernatants were analyzed by ELISA. For co-culture substitution experiment: PGE2 (Sigma-Aldrich) and the cytokines mIL-23, mIL-1α, and mIL-1β (Invitrogen) were added to BMDC culture alone or to the co-culture with CD4⁺T cells. Supernatant was analyzed by ELISA and BMDCs were separated from CD4⁺ T cells by FACS sorting and total RNA analyzed by qRT-PCR.

Cardiac γδT cells were enriched from total cardiac cells using anti-PE Microbeads UltraPure (Miltenyi Biotec) after staining with PE-conjugated anti-mouse γδTCR (BD). Isolated cardiac γδT cells were transferred to 96-well Maxisorp plate (Nunc), which was coated overnight with 100 μl of 4-μg/ml anti-CD3 antibody (145-2C11; TONBO Biosciences) and cultured in the presence of 10-ng/ml mIL-1β (Peprotech) and 10-ng/ml mIL-23 (RD systems). After 48 h, the supernatants were collected and assayed for IL-17A production using LEGEND MAX^TM ELISA Kit (BioLegend) according to the manufacturer’s instructions.