RIPK1 activates RIPK3 signaling in IECs during GVHD. (A-E) The lethally irradiated WT, Ripk1K45A/K45A, and Zbp1−/− B6 recipients (BALB/c→B6) received BALB/c TCD BM cells with or without CD4+ T cells. (A) Survival of mice after allo-HCT. (B-C) Histology analysis of small intestine and liver samples obtained from WT and Ripk1K45A/K45A B6 recipients on day 17 after allo-HCT. Representative H&E images (B) and quantification of pathology scores (C). Bar in small intestine represents 100 μm. Bar in liver represents 50 μm. Black arrows indicate areas of inflammatory cell infiltration or cell damage. (D) Levels of TNF-α and IFN-γ in serum from WT and Ripk1K45A/K45A B6 recipients on day 17 after allo-HCT. (E) ELISA analysis of CXCL9 and CXCL10 proteins in the small intestine from WT and Ripk1K45A/K45A B6 recipients on day 17 after allo-HCT. (F) Sections of 10 colon biopsies from patients with GVHD involved in the GI tract after allo-HCT, 10 control colon samples (from patients with Hirschsprung's disease) and 7 control colon samples adjacent to polyps were stained with an anti–p-RIPK1 antibody. Shown are representative images of IHC staining with anti–p-RIPK1 antibody and H&E staining from the control colon sample and 2 biopsies of patients with GVHD (left panel), and quantification of p-RIPK1 positive crypts in each sample (right panel). Bars represent 50 μm. Data shown are representative of 2 or 3 independent experiments. Data are shown as the mean ± SD (D-F), histology scores are shown as the mean ± SEM (C). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Survival comparisons were evaluated by log-rank test (A). Multiple comparisons were evaluated by one-way ANOVA (D-F), two-group comparisons used unpaired t tests (two-tailed) (C). ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay analysis; IHC, immunohistochemistry; SD, standard deviation; SEM, standard error of the mean.
