Figure 3.

Butyrate exerted a suppressive effect on ILC3s in PPs of the terminal ileum. (a) The levels of SCFAs in jejunum (J) and terminal ileum (TI) were determined by GC-MS. (b) The levels of butyric acid were measured in jejunal PP and terminal ileal PP from SPF mice and ABX-treatment SPF mice. (c) The level of IL-22 in lin⁻ CD45⁺CD90.2⁺ cells prepared from terminal ileal PPs of ABX-treated mice that had been stimulated in vitro with butyrate (NaB) for 17 hr and re-stimulated with PMA, ionomycin, and IL-23 was determined as described in the Methods. The numbers indicate the percentage of cells in each gated area and data representative of three independent experiments are shown. (d) Absolute numbers of RORγt-expressing cells (upper panel) and cells expressing both RORγt and IL-22 (lower panel) were counted using the experimental procedure described above. (e) The RORγt^gfp/+ cells were sorted from terminal ileal PPs prepared from ABX-treated RORc(γt)^gfp reporter mice. Levels of transcripts for the cytokines indicated were measured in sorted RORγt^gfp/+ cells treated with or without NaB (1 μM) using a PCR array. The fold change values were calculated by comparing the values with those obtained from terminal ileal PP ILCs prepared from SPF C57BL/6 mice or RORγt^gfp/+ or sorted RORγt^gfp cells without NaB treatment. (f) Changes in the frequency of ILC3 subpopulations in jejunal and terminal ileal PPs of ABX-treated mice were determined with or without the oral administration of NaB (0.1 M). The numbers indicate the percentage of cells in each gated area and data representative of three independent experiments are shown. Data represent the mean ± SE counted from three independent experiments with three mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups compared.