Activation of the NAIP–NLRC4 inflammasome in tuft cells induces an antimicrobial response in small intestinal tissue. (A) Schematic representation of the crossing of iNLRC4 mice with Pou2f3-cre^ERT2 mice to yield iNLRC4-Pou2f3-cre^ERT2 mice. (B) DCLK-1 (tuft cells: green), Phalloidin (actin: yellow), propidium iodide (pyroptosing cells: red), and DAPI (nuclei: blue) in small intestines from C57BL/6 mice were treated with 0.2 µg/g of PA and 0.1 µg/g LFn-Fla for 60 min. Scale bar = 20 µm; representative image; n = 3 mice. (C) iNLRC4-Pou2f3-Cre^ERT2 and Nlrc4^−/− littermate controls were retro-orbitally injected with Flatox (LFn-FlaA [0.4 µg/g] and PA [0.8 µg/g]) and small intestines were harvested 4 h later. Epithelial cells were isolated from tissues and the percentage of tuft cells among epithelial cells was quantified by flow cytometry (n = 6–7, data combined from two experiments). (D–L) iNLRC4-Pou2f3-Cre^ERT2 and Nlrc4^−/− littermate controls were retro-orbitally injected with two FlaTox dosages (LFn-FlaA [0.1 µg/g] and PA [0.2 µg/g]) 48 h apart. (J–L) Animals were injected 30 min before FlaTox with AZD1981 or vehicle intraperitoneally. Tissues were harvested 24 h after the last injection. IL-22 protein levels within the small intestine (D and J) and colon (G) were quantified by ELISA. Downstream targets of IL-22 receptor signaling, Reg3γ (E, H, and K), and Reg3β (F, I, and L) were measured by qPCR. Data pooled from three (D–I) or two (J–L) experiments (n = 13–16 [D and G], n = 9 [E, F, H, and I], n = 6–12 [J–L]) LOD = limit of detection. Mann–Whitney test was performed to determine significance. *, P < 0.05, **, P < 0.01, ****, P < 0.0001, ns = not significant.