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.

Fig 4: NDI attenuates PVEC PANoptosis by inhibiting Notch1-DLL4 interaction in murine CLP model.FACS-isolated DLL4+ neutrophils (1 × 106/mL) or DLL4– neutrophils (1 × 106/mL) were retro-orbitally injected into mice at the time of CLP; then mice were treated with PBS or 10 mg/kg NDI. After 20 hours, lungs were harvested. (A) Total RNA was extracted from lung, 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 = 9–14 per group. (B and D–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 total lung tissue. Pyroptosis, apoptosis, and necroptosis 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 = 5–9 per group. (C) 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–CLP/PBS.

Fig 5: NDI attenuates ALI and inflammation and improves survival in sepsis.FACS-sorted DLL4+ neutrophils (1 × 106/mL) or DLL4– neutrophils (1 × 106/mL) were retro-orbitally injected into mice at the time of CLP. C57BL/6 male mice were then treated with PBS or 10 mg/kg NDI. After 20 hours, lungs and blood were harvested. (A–C) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were determined using specific colorimetric enzymatic assays. (D and E) Serum levels of IL-6 and TNF-α were assessed by ELISA. (F–H) Lung mRNA levels of TNF-α, IL-6, and keratinocyte chemoattractant (KC) were assessed by PT-PCR. n = 8–17 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. sham. #P < 0.05 vs. DLL4+ neutrophils–CLP/PBS. (I and J) Lung tissue sections were stained with H&E to assess lung injury severity. Lung injury score (original magnification, ×200) is shown as a bar graph. n = 5 high-power fields per group. Scale bars: 100 μm. (K and L) Lung water content [(wet weight – dry weight)/wet weight %] and Evans blue dye (EBD) (EBD/dry lung tissue) were measured to evaluate pulmonary permeability. n = 9–12 per group. 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. *P < 0.05 vs. sham. #P < 0.05 vs. DLL4+ neutrophils–CLP. C57BL/6 male mice were treated with PBS or 10 mg/kg NDI at the time of CLP. (M) Kaplan-Meier 10-day survival curve generated from PBS- and NDI-treated CLP mice. To better mimic the clinical scenario and assess the therapeutic potential of NDI, exogenous DLL4+ neutrophils were not used in the survival experiment. N = 20 mice per group. *P < 0.05 vs. vehicle (PBS).