IL-25 sustains gut ILC2 function, and the production of IL-25 in tuft cells decreased during colitis. (A and B) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in complete IMDM containing IL-2 (10 ng/ml) and IL-7 (10 ng/ml), and treated for 16 h in the presence of IL-25 (10 ng/ml), IL-33 (10 ng/ml), or TSLP (10 ng/ml). Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s (A). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (B). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (C–G) Mid-colon tissues were harvested from mice treated with 3% DSS for 6 days and subjected to scRNA-seq analysis. The scRNA-seq data served as the control group were generated from untreated mice by our team (GSE210415). (C) tSNE visualizations of all cell types of colonic cells of mice. 13 clusters characterized by lineage-specific and cluster-enriched gene were identified by integrated analysis. Colors represent the corresponding annotated cell subtypes. (D) Dot plot showed the abundance and intensity of lineage-specific marker gene expression across all cell types of colonic cells in mice. Dclk1 (E) and Il25 (F) levels in all cell types of intestinal tissues of DSS-treated and control mice. (G) Proportions of indicated cell type among colonic tissues of DSS-treated or control mice. (H and I) Representative sections of mid-colon tissues from DSS-treated mice or control mice stained with DCLK1 by immunohistochemistry to visualize tuft cells. Top: scale bar, 500 μm; bottom: scale bar, 100 μm (H). Statistics graph showed the average number of DCLK1-positive tuft cells per field (I). Data are shown as the mean ± SD (n = 6 per group). (J and K) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each) for 5 days. Subsequently, the IL-25 was removed for 2 days. Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s (J). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (K). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (L and M) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each) for 5 days, and IL-25 was removed for 2 days, followed by restimulation with or without IL-25 (10 ng/ml) for 1 day. Analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) by flow cytometry (L). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (M). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001.