Necroptosis raises IL-6 levels in the circulation, which facilitates the death of Ripk1 ^S161E/S161E mice. (A) Mice (n = 6) were i.v. injected with 50 μg/kg TNF or 400 μg/kg TNF as indicated. At the indicated time points, serum samples were collected for ELISA analysis of IL-6. All mice were 8- to 12-wk-old male littermates (A, B, and E). (B) Representative H&E staining images of ceca from three mice of each indicated genotype 6 h after i.v. injection with 50 μg/kg of TNF. Asterisks indicate areas of edema. Scale bars, 100 μm. (C) 8- to 12-wk-old male littermate mice of the indicated genotypes were treated with TNF (50 μg/kg, i.v.) for 4 h and then EB (0.5 mg/mouse) for 20 min. Organs were collected, and the amount of EB extracted from the organs was measured by spectrophotometry. (D) Survival curves and body temperature of 8- to 12-wk-old male littermate mice of the indicated genotypes after TNF injection (50 μg/kg, i.v.). Mouse survival is presented as a Kaplan–Meier plot, and the log-rank test is performed. *P < 0.05. Data of body temperature are presented as mean ± SD. (E) Quantitative RT-PCR analysis of Il-6 mRNA levels in organs of Ripk1^+/+ and Ripk1^S161E/S161E mice (n = 6) after TNF injection (50 μg/kg, i.v.) at the indicated time points. (F) 14-wk-old male littermate Ripk1^S161E/S161E or Ripk1^S161E/S161EIl-6^−/− mice (n = 3) were reconstituted with Ripk1^S161E/S161E or Ripk1^S161E/S161EIl-6^−/− bone marrow cells and then i.v. injected with TNF (50 μg/kg). At the indicated time points, serum samples were collected for ELISA analysis of IL-6. (G and H) 8- to 12-wk-old male littermate mice of the indicated genotypes were i.v. injected with TNF (50 μg/kg). At the indicated time points, serum samples were collected for ELISA analysis of IL-6. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. *P < 0.05; **P < 0.01; ****P < 0.0001. The above experiments were independently performed twice.