Impaired function and changed protein processing pathway in ER in colonic ILC2s during colitis. (A) Purified CD45⁺ immune cells from the intestinal biopsies of patients with UC or healthy donors were analyzed by scRNA-seq. The tSNE visualizations of CD45⁺ immune cells, with the included subsets annotated. Data are from GSE125527. (B) Dot plot showed the expression of cell-defining signature genes across all cell types of human intestinal CD45⁺ immune cells. (C) Dot plot showed the upregulation of genes involved in the protein processing in ER pathways in ILC2/3 cells of patients with UC in Fig. 1 C. (D) Flow cytometry analysis of the MFI of XBP1s in colonic ILC2s (CD45⁺Lin⁻CD127⁺CRTH2⁺) and ILC3s (CD45⁺Lin⁻CD127⁺CRTH2⁻CD117⁺) of healthy donors (n = 27). (E) Schematic of gating strategy used to identify human colonic ILCs. Gating methods for ILC2 (CD45⁺Lin⁻CD127⁺CRTH2⁺), and ILC3 (CD45⁺Lin⁻CD127⁺CRTH2⁻CD117⁺) subsets are shown. (F) 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). tSNE visualizations of all cell types of mouse colonic CD45⁺ immune cells. 14 clusters characterized by lineage-specific and cluster-enriched genes were identified by integrated analysis. Colors represent the annotated cell subtypes. (G) Dot plot showed the abundance and intensity of lineage-specific marker gene expression across all cell types of mouse colonic CD45⁺ immune cells. (H) Dot plot showed the abundance and intensity of the signature genes of ILC2s or ILC3s in all cell types of mouse colonic CD45⁺ immune cells. (I and J) Dot plot showed the levels of representative ILC2 feature genes in ILC2s (I) and ILC3 signature genes in ILC3s (J) from DSS-treated or control mice. (K) ILC2s identified in Fig. 1 K were extracted for KEGG pathway enrichment analysis. (L) Heatmap of the cytokine receptors responsible for ILC2 survival or activation by conducting SMART-seq analysis with sorted colonic ILC2s of mice treated with 3% DSS for 6 days or control mice. (M–R) WT mice were fed with 3% DSS for 6 days to induce colitis model. Experimental design (M). DAI scores (N) were monitored at the indicated time points. Representative large intestine images (O). Lengths of colons (P). Representative H&E staining of the colon. Scale bar, 500 μm (Q). Histological severity scores of colons (R). Data were compiled from two independent experiments and are shown as the mean ± SD (n = 5 per group). (S) Large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were sorted from the mice treated with 3% DSS for 6 days and control mice, followed by qRT-PCR analysis. The bar graph showed the Xbp1 mRNA splicing in ILC2s. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). *P < 0.05, ***P < 0.001. tSNE, t-distributed stochastic neighbor embedding.