Figure 6.
The ERAdP–NLRP6–ANXA2 axis is required for antibacterial function. (A) Upper: HE staining of duodenum, jejunum, and ileum from littermate WT, Cnep1r1ΔIEC, ERAdP–NLRP6 DKO, and ERAdP–NLRP6–ANXA2 TKO mice. Scale bar: 30 μm. Lower: Proportion of normal and abnormal Paneth cells were quantified on the basis of whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal). n = 6 mice, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (B) Immunofluorescence staining of ileums from WT, DKO, TKO, and ANXA2-overexpressing DKO mice. Red: lysozyme; green: WGA, blue: DAPI. Scale bar: 10 μm. (C and D) Bacterial load analysis of livers (C) and spleens (D) of WT, DKO, TKO, and ANXA2-overexpressing mice. 3 days after S. Typhimurium infection, livers and spleens were collected, and CFUs were calculated (n = 6 mice for each group). (E) Body weight change analysis of WT, DKO, TKO, and ANXA2-overexpressing DKO mice after infected by S. Typhimurium (n = 6 mice for each group). (F) Survival rates of WT, DKO, TKO, and ANXA2-overexpressing DKO mice after infected by S. Typhimurium (n = 12 for each group). (G) Representative pathological manifestation of intestines from WT, DKO, TKO, and DKO+oeAnxa2 mice at 7 days after S. Typhimurium infection. Scale bar: 40 μm. (H) Schematic diagram of mice treated by ABX and c-di-AMP. (I) Survival rates of control, ABX, and ABX+c-di-AMP mice after infected by S. Typhimurium (n = 12 mice for each group). (J) Representative pathological manifestation of intestines from control, ABX, and ABX+c-di-AMP mice after infected by S. Typhimurium for 7 days. Scale bar: 40 μm. (K) Relative expression of CNEP1R1, NLRP6, and ANXA2 of ileal CD samples detected by qRT-PCR (n = 20 clinical samples for each group). Fold changes were normalized to endogenous ACTB. Data are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (A and C–E), Mantel–Cox test (F and I), and Wilcoxon matched pairs signed rank test (K) (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments.

The ERAdP–NLRP6–ANXA2 axis is required for antibacterial function. (A) Upper: HE staining of duodenum, jejunum, and ileum from littermate WT, Cnep1r1ΔIEC, ERAdP–NLRP6 DKO, and ERAdP–NLRP6–ANXA2 TKO mice. Scale bar: 30 μm. Lower: Proportion of normal and abnormal Paneth cells were quantified on the basis of whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal). n = 6 mice, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (B) Immunofluorescence staining of ileums from WT, DKO, TKO, and ANXA2-overexpressing DKO mice. Red: lysozyme; green: WGA, blue: DAPI. Scale bar: 10 μm. (C and D) Bacterial load analysis of livers (C) and spleens (D) of WT, DKO, TKO, and ANXA2-overexpressing mice. 3 days after S. Typhimurium infection, livers and spleens were collected, and CFUs were calculated (n = 6 mice for each group). (E) Body weight change analysis of WT, DKO, TKO, and ANXA2-overexpressing DKO mice after infected by S. Typhimurium (n = 6 mice for each group). (F) Survival rates of WT, DKO, TKO, and ANXA2-overexpressing DKO mice after infected by S. Typhimurium (n = 12 for each group). (G) Representative pathological manifestation of intestines from WT, DKO, TKO, and DKO+oeAnxa2 mice at 7 days after S. Typhimurium infection. Scale bar: 40 μm. (H) Schematic diagram of mice treated by ABX and c-di-AMP. (I) Survival rates of control, ABX, and ABX+c-di-AMP mice after infected by S. Typhimurium (n = 12 mice for each group). (J) Representative pathological manifestation of intestines from control, ABX, and ABX+c-di-AMP mice after infected by S. Typhimurium for 7 days. Scale bar: 40 μm. (K) Relative expression of CNEP1R1, NLRP6, and ANXA2 of ileal CD samples detected by qRT-PCR (n = 20 clinical samples for each group). Fold changes were normalized to endogenous ACTB. Data are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (A and C–E), Mantel–Cox test (F and I), and Wilcoxon matched pairs signed rank test (K) (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments.

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