ILC3s are the source of increased IL-22 within the small intestine after NAIP–NLRC4 inflammasome activation in tuft cells. (A) Representative images of ileum sections from naive Gpr44^fl/+Rorγt-Cre⁺ mice showing cells positive for DCLK-1 (white), CRTH2-GFP (green), and CD4 (red); blue color indicates DAPI counterstained nuclei. Scale bar = 50 µm; representative image; n = 3 mice. (B) Flow cytometry quantification of CRTH2 (GFP)-positive cells among Lin⁻ILC3 and Lin⁺CD4 T cells. (C–H) Flow cytometry analysis of IL-22 producing ILC3s after inflammasome activation specifically in tuft cells. iNLRC4-Pou2f3-cre^ERT2 mice or littermate controls were administered two FlaTox dosages (LFn-FlaA [0.1 µg/g] and PA [0.2 µg/g]) 48 h apart and the small intestine was harvested 24 h after last Flatox injection. Cells from the lamina propria were isolated and intracellular IL-22 was measured by flow cytometry. (C) Representative flow plots of intracellular IL-22 in NKp46⁺CCR6⁻ ILC3s from Nlrc4^−/− and iNLRC4-Pou2f3-cre^ERT2 after Flatox administration. (D–H) Quantification of the frequency of IL-22⁺ cells among different subtypes of ILC3s. Each data point represents one mouse, pooled data from multiple experiments (n = 8 [B], n = 13–14 [D–H]), and bars shown as median. Mann–Whitney test was performed to determine significance. *, P < 0.05, ns = not significant.