Pro-inflammatory cytokine IFN-γ evidently restrains intestinal ILC2 function and CUT&Tag-seq. (A) mRNA levels of pro-inflammatory genes, Ifng, Il1b, Il6, and Tnf, in all cell types of colonic tissues from DSS-treated and control mice were analyzed by scRNA-seq. (B and C) 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 co-incubated for 3 days with IFN-γ (50 ng/ml), IL-1β (50 ng/ml), IL-6 (50 ng/ml), or TNF-α (50 ng/ml), respectively. Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s (B). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (C). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (D and E) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were stimulated with the IFN-γ (500 ng/ml) for 3 days in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s (D). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (E). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (F–J) WT mice were administered 3% DSS in drinking water for 6 days to induce colitis, rmIL-25 (0.4 μg) was injected i.p. five doses daily from the onset of colitis induction, while anti-IFN-γ (1 mg) was injected i.p. on days −1, 2, and 5; the mice were sacrificed for analysis on day 6. Analysis of Xbp1s expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) from large intestinal LPLs by flow cytometry (F). MFI of Xbp1s in ILC2s (G). Flow cytometry analysis of GATA3 expression after gating on CD45.2⁺Lin⁻ cells in large intestinal LPLs (H). Absolute numbers of ILC2s (CD45.2⁺Lin⁻GATA3⁺) (I). Serum IFN-γ concentrations were detected by ELISA kit (J). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (K) Density plots (top) and heatmaps (bottom) of Xbp1s binding to the TSS regions (from −3 to 3 kb) in mouse colonic ILC2s. (L) Gene component analysis showed percent distribution of Xbp1s binding sites measured by CUT&Tag-seq, including promoter, 5′UTR, 3′UTR, exon, intron, and intergenic regions. (M) Binding motif analysis of Xbp1s based on CUT&Tag-seq peaks in ILC2s. *P < 0.05, **P < 0.01, ***P < 0.001.