Figure 3.
IL-25 and the pro-inflammatory cytokine IFN-γ affect ILC2 function by regulating Xbp1 mRNA splicing. (A) Heatmap of ILC2s activator genes and pro-inflammatory genes by conducting RNA-seq analysis with mid-colon tissues of mice treated with 3% DSS for 6 days or control mice. The data are from GSE210405, previously published by our team. (B–D) Sorted 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 16 h with IL-25 (10 ng/ml), IL-33 (10 ng/ml), or TSLP (10 ng/ml), respectively. Xbp1 mRNA splicing in ILC2s was detected using qRT-PCR (B). Xbp1s, spliced form of Xbp1. Xbp1t, total Xbp1. Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (C). MFI of Xbp1s (D) in indicated groups. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (E–H) Sorted 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. Experimental design (E). Xbp1 mRNA splicing of indicated ILC2s was detected by qRT-PCR (F). Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) was determined by flow cytometry (G), MFI of Xbp1s in ILC2s (H). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (I–L) Sorted 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. Experimental design (I). Xbp1 mRNA splicing of indicated ILC2s was detected by qRT-PCR (J). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (K). MFI of Xbp1s in ILC2s (L). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (M–O) Sorted 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. Xbp1 mRNA splicing in ILC2s was detected using qRT-PCR (M). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (N). MFI of Xbp1s in ILC2s (O). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (P–R) Sorted 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). Xbp1 mRNA splicing of ILC2s was detected using qRT-PCR (P). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (Q). MFI of Xbp1s in ILC2s (R). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (S and T) After inducing colitis in WT mice with 3% DSS in drinking water for 6 days, the concentrations of IL-25 (S) and IFN-γ (T) in colon tissues were measured by ELISA. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (U–AC) 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. Experimental design (U). DAI scores (V) were monitored at the indicated time points. Representative large intestine images (W). Lengths of colons (X). Representative H&E staining of the colon. Scale bar, 500 μm (Y). Histological severity scores of colons (Z). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in large intestinal ILC2s (CD45.2+Lin−GATA3+) (AA). Percentages (AB) and absolute numbers (AC) of Areg+, IL-5+, and IL-13+ ILC2s. Data were compiled from two independent experiments and are shown as the mean ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Refer to the image caption for details.

IL-25 and the pro-inflammatory cytokine IFN-γ affect ILC2 function by regulating Xbp1 mRNA splicing. (A) Heatmap of ILC2s activator genes and pro-inflammatory genes by conducting RNA-seq analysis with mid-colon tissues of mice treated with 3% DSS for 6 days or control mice. The data are from GSE210405, previously published by our team. (B–D) Sorted 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 16 h with IL-25 (10 ng/ml), IL-33 (10 ng/ml), or TSLP (10 ng/ml), respectively. Xbp1 mRNA splicing in ILC2s was detected using qRT-PCR (B). Xbp1s, spliced form of Xbp1. Xbp1t, total Xbp1. Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (C). MFI of Xbp1s (D) in indicated groups. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (E–H) Sorted 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. Experimental design (E). Xbp1 mRNA splicing of indicated ILC2s was detected by qRT-PCR (F). Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) was determined by flow cytometry (G), MFI of Xbp1s in ILC2s (H). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (I–L) Sorted 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. Experimental design (I). Xbp1 mRNA splicing of indicated ILC2s was detected by qRT-PCR (J). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (K). MFI of Xbp1s in ILC2s (L). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (M–O) Sorted 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. Xbp1 mRNA splicing in ILC2s was detected using qRT-PCR (M). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (N). MFI of Xbp1s in ILC2s (O). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (P–R) Sorted 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). Xbp1 mRNA splicing of ILC2s was detected using qRT-PCR (P). Analysis of Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (Q). MFI of Xbp1s in ILC2s (R). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (S and T) After inducing colitis in WT mice with 3% DSS in drinking water for 6 days, the concentrations of IL-25 (S) and IFN-γ (T) in colon tissues were measured by ELISA. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (U–AC) 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. Experimental design (U). DAI scores (V) were monitored at the indicated time points. Representative large intestine images (W). Lengths of colons (X). Representative H&E staining of the colon. Scale bar, 500 μm (Y). Histological severity scores of colons (Z). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in large intestinal ILC2s (CD45.2+Lin−GATA3+) (AA). Percentages (AB) and absolute numbers (AC) of Areg+, IL-5+, and IL-13+ ILC2s. Data were compiled from two independent experiments and are shown as the mean ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

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