Figure 5. Citrobacter rodentium elicits IL-1β production by intestinal macrophages via caspase-11 inflammasome.

(a) BM-derived macrophages (BMDMs) were obtained from WT, Nlrp3^−/−, Casp11^−/− mice and stimulated with C. rodentium (C. rod) or Salmonella (Sal; MOI=25) for 1 h without antibiotics and then cultured additional 17 h in the presence of 100 μg ml⁻¹ gentamicin. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. (n=3, representative of three independent experiments). (b) WT, Nlrp3^−/− and Casp11^−/− mice were infected with C. rodentium. On day 8 post infection, LPMCs were isolated from the infected mice, and 2 × 10⁶ cells ml⁻¹ LPMCs were cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. of 3 independent experiments. *P<0.05; **P<0.01; NS, not significant by Bonferroni test. (c) Isolated LPMCs in b were cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 h. IL-22 production in CD4⁻ ILCs (Lin^-Thy-1⁺CD3^-CD4^-) and CD4⁺ ILCs (Lin^-Thy-1⁺CD3^-CD4⁺) was assessed by flow cytometry. Data are representative of four individual mice. (d) LPMCs were isolated from uninfected and C. rodentium-infected (day 8 post infection) WT and Casp11^−/− mice and cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. IL-22 in the culture supernatant was analysed by ELISA. Data are given as mean±s.e.m (n=4–6). **P<0.01; ***P<0.001; NS, not significant by Bonferroni test. (e) CD45⁺MHC-II⁺CD11b⁺CD11c⁺CD103⁻Gr-1⁻ MP1 subset was sorted from naive WT mice and C. rodentium-infected (day 8) WT and Casp11^−/− mice. 2 × 10⁵ of MP1 cells were loaded with SDS–polyacrylamide gel electrophoresis, and blotted with anti-mouse-caspase-11 antibody. As positive and negative controls, BMDMs from WT and Casp11^−/− mice with or without LPS priming (6 h) were used. The original gel images are shown in Supplementary Fig. 12.