Fig. 3. Microbiota-driven IL-1β release by intestinal macrophages regulates Csf2 production by RORγt⁺ ILC3.

(A) FACS plot showing Csf2 expression in whole intestinal lamina propria CD45⁺ cells at the indicated time points after birth. (B) Bar graph shows percentages of Csf2⁺ cells among total lamina propria cells in mice at the indicated time points after birth. Data shown are the results of three independent experiments with at least three mice per group. (C) Percentages of Csf2⁺ cells among gated colonic lamina propria NCR⁺ ILC3 and LTi cells in conventional mice or mice treated with broad-spectrum antibiotics (ABx). Stains were performed on cells cultured for 4 hours with (+) or without (−) IL-1β in the presence of Brefeldin A. Data are shown as mean ± SD of three independent experiments with at least three mice per group. (D) Percentages of Csf2⁺ cells among gated colonic lamina propria NCR⁺ILC3 and LTi cells in Il1r^−/− or C57Bl/6 mice. Data show the results of two independent experiments with three mice per group. (E) Il1b mRNA expression in FACS-purified colonic MNPs. Data shown are representative of two independent experiments with pooled cells of three mice. (F) IL-1β protein production by purified intestinal DC subsets and MPs isolated from the colonic lamina propria of C57Bl/6 and Myd88^ΔMP mice, measured by ELISA after 24 hours of culture in complete medium. Data are representative of two independent experiments with pooled cells of three mice. (G) Percentages of Csf2⁺ cells among gated colonic ILC3 in Myd88^ΔMP mice. Stains were performed in cells cultured for 4 hours with or without IL-1β in the presence of Brefeldin A. Data are shown as mean ± SD of three independent experiments with at least three mice per group. (H) Groups of mice were injected with one injection of anti-Csf1R mAb (3 mg per mouse) or control mAb. Bar graph shows percentages of remaining macrophages in colonic tissue. (I) Il1b expression in whole colonic tissue of mice treated with anti-Csf1R mAb or isotype control. Data are shown as mean ± SD of at least three independent experiments with three mice per group. (J) Percentages of Csf2⁺ cells among gated colonic lamina propria NCR⁺ ILC3 and LTi cells in mice treated with anti-Csf1R mAb or control mAb. Staining was performed on total cells cultured for 4 hours with or without IL-1β in the presence of Brefeldin A. Data are shown as mean ± SD of three independent experiments with three mice per group. (K) Csf2 production by colonic lamina propria NCR⁺ ILC3 and LTi cells in Myd88^ΔT/LTi mice measured after 4 hours of culture with or without IL-1β in the presence of Brefeldin A. Data are shown as mean ± SD of three independent experiments with at least three mice per group. Student’s t test (D, H, and I) or one-way ANOVA Bonferroni’s multiple comparison test (B, C, F, G, J, and K) were performed. Statistical significance is indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.