Secondary bile acid isoLCA attenuates effector functions of ILC3 and Th17 cells. (A) Inverse agonist dose–response performance of the human RORγt reporter assay system. (B) Inverse agonist dose–response performance of the human RORα reporter assay system. RLU, relative light units; ML-209, RORγt antagonist; ATRA, all-trans retinoic acid (RORα inverse agonist). (C) Fate-mapped RORγt⁺ ILC3 and RORγt⁺ T cells were sorted from naïve Rorc^RFP mice. (D) Absolute number of IL-17A⁺ production by ILC3s and Th17 cells (n = 3 mice). ILC3 and Th17 cells were cultured with and without isoLCA + IL-1β for 3 days and stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 750 ng/ml ionomycin (Sigma-Aldrich) for 4–6 h in the presence of 10 μg/ml brefeldin A (Sigma-Aldrich) in complete RPMI-1640 medium (containing 10% FBS, 50 mM 2-mercaptoethanol, 1 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) to assess IL-17A production by flow cytometry. (E) Schematic overview of the experiment design. IsoLCA was administered to Rag1^−/− mice daily (oral gavage, days 0, 1, and 2) that were then euthanized on day 3. (F) Flow cytometry plots of IL-17A–producing live CD45⁺Lineage⁻RORγt⁺ ILC3s. (G) Absolute numbers of IL-17A–producing ILC3s (n = 3 mice). (H) Frequency of Il-17A–producing ILC3s (n = 3 mice). Lineage markers: CD3ε, CD3, CD5, CD8α, TCRβ, Cd11b, CD11c, CD19, B220, Ly6C, Ly6G, Ter119. Data are representative of two independent experiments and are presented as the mean ± SEM. Statistical significance was determined using an unpaired two-tailed t test (*P < 0.05). PMA, phorbol 12-myristate 13-acetate.