Fig 2: Inhibition of necroptotic signalling protects against lethal sepsis. (A) Animal survival was monitored after CLP in WT mice treated with GSK (3 mg/kg) or GW (2 mg/kg). (B) Plasma IFNβ, TNF‐α and IL‐6 were tested by ELISA. (C) Representative images of H&E staining of intestine, lung and kidney in RIPK3−/− and WT mice treated with GSK (3 mg/kg) or GW (2 mg/kg) after CLP for 24 h. (D) Intestines was collected in WT mice after CLP at 0 h, 6 h, 12 h and 24 h. STING signalling and necroptosis in intestines were detected by western blot. The experiment was repeated at least three times. (E) qPCR analysis of IFNβ, TNF‐α and IL‐6 mRNA in murine intestines of WT and RIPK3−/− mice after CLP for 24 h. (F) qPCR analysis of IFNβ, TNF‐α and IL‐6 mRNA in murine intestines of WT treated with GSK (3 mg/kg) or GW (2 mg/kg) after CLP for 24 h. (G–I) Western blot was used to detect STING signalling in intestines of RIPK3−/− mice or WT treated with GSK (3 mg/kg) or GW (2 mg/kg) after CLP for 24 h. The experiment was repeated at least three times. Scale bars = 1 µm. Data were shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Enhanced necroptosis is associated with tissue damage, inflammation and STING signalling in sepsis. (A) Circulating RIPK1 in patients with no sepsis, sepsis and septic shock was analysed through enzyme‐linked immunosorbent assay (ELISA). (B) Circulating RIPK1 was compared between survivors and non‐survivors. (C) Correlation analysis between lactate and circulating RIPK1 was performed. (D) Circulating RIPK3 in patients with no sepsis, sepsis and septic shock was analysed through ELISA. (E) Circulating RIPK3 was compared between survivors and non‐survivors. (F–H) circulating lactate, PCT and CRP were recorded. Correlation analysis was performed. (I) Intestinal mucosae were collected from healthy control and intra‐abdominal infection (IAI) patients. Intestinal IHC staining for p‐RIPK3 and p‐MLKL was conducted. (J) Peripheral blood mononuclear cells (PBMC) were collected from healthy volunteers and patients with IAI. Western blot was performed to detect STING signalling and necroptotic signalling. Correlation analysis between p‐MLKL and p‐STING was conducted.

Fig 4: RIPK3 sustains activation of STING signalling by restraining the autophagy of STING. (A) Western blot analysis of STING signalling in BMDM from WT and RIPK3−/− mice after stimulation with DMXAA (50 µg/mL). The experiment was repeated at least three times. (B) HEK293T cells were transfected with various STING mutant plasmid and RIPK3 plasmid. Western blot was performed to analyse STING signalling. The experiment was repeated at least three times. (C) Immunoblot analysis of autophagy‐related protein (p62 and LC3) in WT and RIPK3 knockout HT‐29 cells stimulated with ADU (27 µM). The experiment was repeated at least three times. (D) Immunoblot analysis of STING signalling in WT and RIPK3 knockout HT‐29 cells stimulated with ADU (27 µM) and bafilomycin A1 (100 nM). Quantitative analysis of STING and LC3‐II was performed. The experiment was repeated at least three times. Data were shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: RIPK3 knockout abrogates DMXAA‐induced intestinal injury. (A) Representative images of intestinal H&E staining in WT, STING−/− and RIPK3−/− mice after DMXAA (25 mg/kg) injection for 6 h. Black arrowheads point to destruction of the intestinal mucosa. (B) qPCR analysis of interferon‐stimulated genes (ISG15 and Ifit3b) in small intestines of mice. (C) Principal component analysis (PCA) of protein in which input samples are clustered in the small intestines from WT and RIPK3−/− mice. (D) Heatmap showing the differential expressed protein upon DMXAA stimulation from WT and RIPK3−/− mice. (E) Gene Ontology (GO) showing significant pathways between WT‐Control and WT‐DMXAA. (F) GO showing significant pathways between WT‐DMXAA and RIPK3−/−‐DMXAA. The abscissa represents the enrichment factor of each GO item (richFator ≤ 1). The colour of bubbles represents the significance of enriched GO items based on Fisher's Exact Test. The bubble size indicates the number of differentially expressed proteins involved in the GO entry. Scale bars = 0.5 µm. Data were shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.