Compromised colonic ILC2s in colitis and IRE1α-mediated branch of UPR sustain the function of gut ILC2s. (A) Proportions of indicated immune cell compartments by conducting scRNA-seq analysis with CD45⁺ immune cells isolated from the colonic biopsies of patients with UC or healthy donors (GSE125527). (B) Dot plot showed the levels of ILC2 or ILC3 signature genes in ILC2/3 population. (C) KEGG pathway enrichment analysis of DEGs in ILC2/3 cluster between healthy donors and patients with UC. (D) Dot plot showed the downregulation of genes involved in the protein processing in ER pathways in ILC2/3 cells of patients with UC in Fig. 1 C. (E and F) Flow cytometry analysis of the MFI of XBP1s in colonic ILC2s (CD45⁺Lin⁻CD127⁺CRTH2⁺) of healthy donors or patients with UC (n = 27 per group) (E). Correlation analysis between XBP1s MFI in ILC2s and UCEIS score (F). (G and H) Flow cytometry analysis of the MFI of XBP1s in colonic ILC3s (CD45⁺Lin⁻CD127⁺CRTH2⁻CD117⁺) of healthy donors or patients with UC (n = 27 per group) (G). Correlation analysis between XBP1s MFI in ILC3s and UCEIS score (H). (I and J) Human colonic LPMNCs were treated with toyocamycin (Toyo) at 25 μM for 16 h. Flow cytometry analysis of AREG expression in ILC2s after gating on CD45⁺Lin⁻CD127⁺CRTH2⁺ (I). Percentage of AREG⁺ ILC2s out of total ILC2s (CD45⁺Lin⁻CD127⁺CRTH2⁺) (J). Connected lines are samples from the same clinical samples (n = 17). P value was determined by two-tailed paired Student’s t test. (K) 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). CD45⁺ immune cells were extracted to analyze the proportions of indicated immune cell compartments. (L–P) WT mice were administered 3% DSS in drinking water for 6 days to induce colitis, followed by flow cytometry analysis of GATA3 expression after gating on CD45.2⁺Lin⁻ cells in large intestinal LPLs (L), absolute numbers of ILC2s (CD45.2⁺Lin⁻GATA3⁺) (M). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (N), percentages (O) and absolute numbers (P) of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (Q and R) Xbp1s expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) or ILC3s (CD45.2⁺Lin⁻RORγt⁺) from large intestinal LPLs of DSS-treated or control mice detected by flow cytometry (Q). MFI of Xbp1s in ILC2s or ILC3s (R). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (S and T) Sorted ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days in the presence of IL-2, IL-7, IL-25, and IL-33 (10 ng/ml each), and then treated with Toyo at the indicated concentrations for 16 h. Flow cytometry analyses of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (S). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ cells in ILC2s (T). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (U) Heatmap of ILC2-characteristic genes by performing RNA-seq analysis with Toyo (4 μM) treated for 16 h or vehicle ILC2s. *P < 0.05, **P < 0.01, ***P < 0.001. MFI, mean fluorescence intensity.