DKO and TKO mice exhibit impaired antibacterial ability. (A) Immunofluorescence staining of ileal crypts from WT mice. Green: UEA-1, blue: ERAdP-HA, red: NLRP6, and pink: ANXA2. Scale bar: 5 μm. (B) Left: AB-PAS staining of intestines from ERAdP–NLRP6 DKO and ERAdP–NLRP6–ANXA2 TKO mice. Scale bar: 35 μm. Right: Quantification of DCV numbers per Paneth cell of WT, DKO, and TKO mice (n = 6 mice for each group). (C) Left: Lysozyme immunohistochemistry of intestines from WT, DKO, and TKO mice. Scale bar: 35 μm. Right: Quantification of Lyz⁺ puncta per crypt of WT, DKO, and TKO mice (n = 6 mice for each group). (D) Lysozyme in supernatant of stimulated crypts from littermate WT and TKO mice after treatment of DMSO or CCh was measured by ELISA (n = 6 mice for each group). (E) Bacterial load analysis of S. Typhimurium in secreted supernatants. SI crypts of littermate WT and TKO mice were isolated and stimulated by DMSO or CCh (n = 6 mice for each group). (F and G) Bacterial load analysis of ileal contents (F) and PPs (G) from WT, DKO, TKO, and ANXA2-overexpressing mice. 3 days after S. Typhimurium infection, ileal contents and PPs were collected, and CFUs were calculated (n = 6 mice for each group). (H–K) Bacterial load analysis in livers (H), spleens (I), ileal contents (J), and PPs (K) of control, ABX, and ABX+c-di-AMP mice. At 3 days after S. Typhimurium infection, livers, spleens, ileal contents, and PPs were collected, and CFUs were calculated (n = 6 mice for each group). (L) Body weight change analysis of control, ABX, and ABX+c-di-AMP mice after infected by S. Typhimurium (n = 6 mice for each group). Data in B–L are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments.