Fig 1: DAPT attenuates endothelial PANoptosis by inhibiting Notch1-DLL4 interaction.PVECs (0.5 × 106) were cocultured with sorted DLL4+ (0.5 × 106) neutrophils or DLL4– (0.5 × 106) neutrophils, then treated with DAPT or PBS. After 16 hours, cell viability was assessed using propidium iodide staining. (A) Cell death was quantified as the percentage of propidium iodide–positive cells. n = 6–15 per group. Data are representative of 3 independent experiments. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. (B) RNA was extracted, and ZBP1 expression was analyzed by RT-PCR. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 11–25 per group. *P < 0.05 vs. PBS. #P < 0.05 vs. DLL4+ neutrophils/PBS. (C–F) Western blot analysis was performed to measure protein levels of ZBP1, c-GSDMD, t-GSDMD, c-caspase-3, t-caspase-3, p-MLKL, and MLKL in PVECs. Pyroptosis, apoptosis, and necroptosis were quantified as c/t GSDMD, c/t caspase-3, and p-MLKL/MLKL. n = 4–12 per group. Data are representative of 3 independent experiments. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. *P < 0.05 vs. PBS. #P < 0.05 vs. DLL4+ neutrophils.
Fig 2: Schema of findings.DLL4+ neutrophils induce pulmonary endothelial cell PANoptosis via the Notch1-DLL4 pathway in sepsis. eCIRP induces neutrophils to express DLL4, and these DLL4+ neutrophils interact with PVECs through the Notch1-DLL4 pathway. This interaction triggers endothelial PANoptosis, driving inflammation and ALI in sepsis. NDI effectively inhibits the Notch1-DLL4 interaction, reducing endothelial PANoptosis and ALI, thereby improving survival outcomes in sepsis. The schema was created in BioRender (Murao A, 2025, https://BioRender.com/y914a2i).
Fig 3: DLL4+ neutrophils induce lung endothelial cell PANoptosis in sepsis.BMDNs isolated from wild-type mice were stimulated with eCIRP for 2 hours. FACS-sorted DLL4+ neutrophils and DLL4– neutrophils (1 × 106/100 mL/mouse) with or without CFSE label were then delivered into mice via retro-orbital injection at the time of CLP. At 20 hours, the lungs were harvested. (A and B) CFSE+ neutrophils and their viability were analyzed by flow cytometry. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 8 per group. (C) Total RNA was extracted from lungs, and ZBP1 expression was analyzed by RT-PCR. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 6 per group. (D and F–H) Western blot analysis was performed to measure protein levels of ZBP1 in total lung tissues and cleaved gasdermin D (c-GSDMD), total GSDMD (t-GSDMD), cleaved caspase-3 (c-caspase-3), total caspase-3 (t-caspase-3), phosphorylated MLKL (p-MLKL), and MLKL in lung. Pyroptosis, apoptosis, and necroptosis (PANoptosis) were quantified as c/t GSDMD, c/t caspase-3, and p-MLKL/MLKL. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 6 per group. (E) Confocal microscopy was used to assess ZBP1 expression in lungs. ZBP1 was stained red, pulmonary endothelial cells were stained green (CD31), and nuclei were stained blue (DAPI). Scale bars: 50 μm. *P < 0.05 vs. sham. #P < 0.05 vs. DLL4– neutrophils.
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