Figure 4.
JAK/STAT1 signaling mediates RIPK1/RIPK3 induction of chemokines and MHC class II molecules in IECs. (A-B) Small intestinal organoids prepared from WT, Ripk3−/−, and Mlkl−/− B6 mice were treated with control (PBS) or IFN-γ (10 ng/mL) for 12 hours, with qPCR measurement of Cxcl9 and Cxcl10 expression (A); ELISA measurement of CXCL9 and CXCL10 protein levels (B). Identical concentrations for IFN-γ were used in later experiments unless otherwise stated. (C) qPCR analysis for the expression of Cxcl9 and Cxcl10 in small intestinal organoids from WT and Ripk1K45A/K45A B6 mice that were treated with control, IFN-γ for 12 hours. (D) Gene expression of antigen presentation-related genes in small intestine samples collected from WT, Ripk3−/−, and Vil-cre Ripk3fl/fl mice on day 17 after allo-HCT. (E) qPCR analysis of CIITA and H2-DMB1 mRNA levels in the small intestine from the indicated B6 recipients (BALB/c→B6) 17 days after allo-HCT. (F) Small intestinal organoids prepared from indicated B6 mice were treated with IFN-γ or control for 12 hours. CIITA and H2-DMB1 expression levels were analyzed using qPCR. (G) Small intestinal organoids prepared from indicated B6 mice were treated with IFN-γ or control for 24 hours. The MFI of MHC II was determined by flow cytometer analysis. (H-I) Small intestinal organoids prepared from WT and Stat1−/− B6 mice were treated with control or IFN-γ for 12 hours. qPCR for expression of Cxcl9, Cxcl10 (H), CIITA, and H2-DMB1 (I). (J) Small intestinal organoids from WT B6 mice were treated with 300 nM ruxolitinib (RUX) for 2 hours before IFN-γ treatment for 12 hours; gene expression was analyzed by qPCR. (K-L) Immunoblotting analysis of p-STAT1, STAT1, RIPK3, MLKL, and GAPDH in small intestine and colon from the indicated B6 recipients on day 7 after allo-HCT. Quantification of p-STAT1 or STAT1 normalized to GAPDH was shown under the band. Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD (A-C,F-J), qPCR analysis in the small intestine are shown as the mean ± SEM (E). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA (A-B,E-G,J), two-group comparisons used unpaired t tests (two-tailed) (C,F-I). ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay analysis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MFI, mean fluorescence intensity; n.s., not significant; PBS, phosphate-buffered saline; SD, standard deviation; SEM, standard error of the mean.

JAK/STAT1 signaling mediates RIPK1/RIPK3 induction of chemokines and MHC class II molecules in IECs. (A-B) Small intestinal organoids prepared from WT, Ripk3−/−, and Mlkl−/− B6 mice were treated with control (PBS) or IFN-γ (10 ng/mL) for 12 hours, with qPCR measurement of Cxcl9 and Cxcl10 expression (A); ELISA measurement of CXCL9 and CXCL10 protein levels (B). Identical concentrations for IFN-γ were used in later experiments unless otherwise stated. (C) qPCR analysis for the expression of Cxcl9 and Cxcl10 in small intestinal organoids from WT and Ripk1K45A/K45A B6 mice that were treated with control, IFN-γ for 12 hours. (D) Gene expression of antigen presentation-related genes in small intestine samples collected from WT, Ripk3−/−, and Vil-cre Ripk3fl/fl mice on day 17 after allo-HCT. (E) qPCR analysis of CIITA and H2-DMB1 mRNA levels in the small intestine from the indicated B6 recipients (BALB/c→B6) 17 days after allo-HCT. (F) Small intestinal organoids prepared from indicated B6 mice were treated with IFN-γ or control for 12 hours. CIITA and H2-DMB1 expression levels were analyzed using qPCR. (G) Small intestinal organoids prepared from indicated B6 mice were treated with IFN-γ or control for 24 hours. The MFI of MHC II was determined by flow cytometer analysis. (H-I) Small intestinal organoids prepared from WT and Stat1−/− B6 mice were treated with control or IFN-γ for 12 hours. qPCR for expression of Cxcl9, Cxcl10 (H), CIITA, and H2-DMB1 (I). (J) Small intestinal organoids from WT B6 mice were treated with 300 nM ruxolitinib (RUX) for 2 hours before IFN-γ treatment for 12 hours; gene expression was analyzed by qPCR. (K-L) Immunoblotting analysis of p-STAT1, STAT1, RIPK3, MLKL, and GAPDH in small intestine and colon from the indicated B6 recipients on day 7 after allo-HCT. Quantification of p-STAT1 or STAT1 normalized to GAPDH was shown under the band. Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD (A-C,F-J), qPCR analysis in the small intestine are shown as the mean ± SEM (E). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA (A-B,E-G,J), two-group comparisons used unpaired t tests (two-tailed) (C,F-I). ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay analysis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MFI, mean fluorescence intensity; n.s., not significant; PBS, phosphate-buffered saline; SD, standard deviation; SEM, standard error of the mean.

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