Necroptosis raises IL-6 levels in the circulation, which facilitates the death of Ripk1 ^S161E/S161E mice. (A) 8- to 12-wk-old male littermate Ripk1^+/+ and Ripk1^S161E/S161E mice (n = 3) were i.v. injected with TNF (50 μg/kg). At the indicated time points, serum samples were collected for ELISA analysis of IL-1β, CCL2, IL-12/23 p40, and IFN-γ. (B) Representative immunofluorescence staining images of ceca from five 8- to 12-wk-old male littermate mice of each indicated genotype after TNF treatment for 6 h (50 μg/kg, i.v.). Endomucin, red; p-MLKL, green; E-Cad, purple. Scale bars, 50 μm. Percentages of p-MLKL⁺ cells in Endomucin⁺ cells or E-Cad⁺ cells were quantified using ImageJ. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ns, P ≥ 0.05. (C) 8- to 12-wk-old male littermate mice of the indicated genotypes were treated with TNF for 4 h (50 μg/kg, i.v.) 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) Quantitative RT-PCR analysis of Il-6 mRNA levels in organs of mice (n = 6) after TNF injection (50 μg/kg, i.v.) at the indicated time points. All mice were 8- to 12-wk-old male littermates. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. *P < 0.05; **P < 0.01. (E)RIPK1-deficient HT-29 cells were reconstituted with Flag-hRIPK1 WT or mutants (D138N, D138N + S161E, and S161N) and then treated with hTNF (30 ng/ml) + zVAD (20 μM) + SMAC mimetic (100 nM) for the indicated time points. Cell survival was measured. Data are presented as mean ± SD of triplicate. Cell lysates were analyzed by immunoblotting. The above experiments were independently performed twice. (F) A graphic model for the functions and mechanisms of RIPK1 S161 phosphorylation in TNF-induced SIRS, created in BioRender. Msf, (2025) https://BioRender.com/w9bzp6q. Source data are available for this figure: SourceData FS5.