DLL4+ neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis
Hui Jin, Saoirse Holland, Alok Jha, Gaifeng Ma, Jingsong Li, Atsushi Murao, Monowar Aziz, Ping Wang
Hui Jin, Saoirse Holland, Alok Jha, Gaifeng Ma, Jingsong Li, Atsushi Murao, Monowar Aziz, Ping Wang
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Research Article Immunology Inflammation

DLL4+ neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis

Abstract

Neutrophils play a critical role in sepsis-induced acute lung injury (ALI). Extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, promotes neutrophil heterogeneity. While delta-like ligand 4 (DLL4) expression has been studied in various cell populations, its expression in neutrophils and impact on inflammation remain unknown. Here, we discovered that eCIRP induces DLL4+ neutrophils. These neutrophils trigger PANoptosis, a novel proinflammatory form of cell death initiated by Z-DNA–binding protein-1 (ZBP1) in pulmonary vascular endothelial cells (PVECs). In sepsis, DLL4+ neutrophils increase in the blood and lungs, upregulating ZBP1, cleaved gasdermin D, cleaved caspase-3, and phosphorylated MLKL, all of which are markers of PANoptosis, exacerbating ALI. DLL4 binds to Notch1 on PVECs and activates Notch1 intracellular domain to increase ZBP1-mediated endothelial PANoptosis. We discovered what we believe to be a novel Notch1-DLL4 inhibitor (NDI), derived from Notch1 to specifically block this interaction. Our findings reveal that NDI reduced endothelial PANoptosis in vitro and in vivo, attenuated pulmonary injury induced by DLL4+ neutrophils, and decreased lung water content and permeability, indicating improved barrier function. NDI also reduced serum injury and inflammatory markers and improved survival rate in sepsis. These findings underscore the Notch1-DLL4 pathway’s critical role in DLL4+ neutrophil–mediated ALI. Targeting the Notch1-DLL4 interaction with an NDI represents a promising therapeutic strategy for sepsis-induced ALI.

Authors

Hui Jin, Saoirse Holland, Alok Jha, Gaifeng Ma, Jingsong Li, Atsushi Murao, Monowar Aziz, Ping Wang

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Figure 4

Development and evaluation of small peptide NDI attenuates PVEC PANoptosis by inhibiting Notch1-DLL4 interaction.

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Development and evaluation of small peptide NDI attenuates PVEC PANoptos...
(A) Computational modeling for the protein-protein interaction analysis between Notch1 extracellular domain and DLL4. Notch1 is shown in green, DLL4 in blue, and the interaction between Notch1 and DLL4 in purple. (B) Computational modeling was used to design Notch1-DLL4 inhibitor (NDI) derived from the Notch1 sequence that binds to DLL4. DLL4 is shown in blue, NDI in red. (C and D) BIAcore analysis was conducted to determine the binding affinity (KD value) of DLL4 to Notch1 and the inhibitory effect of NDI on this interaction. Wild-type PVECs (0.5 × 106) were cocultured with sorted DLL4+ neutrophils (0.5 × 106) or DLL4– neutrophils (0.5 × 106), then treated with either 10 μM scramble or NDI. PVECs (0.5 × 106) were first treated with Notch1-knockdown plasmid or its negative control for 24 hours. These PVECs were then cocultured with sorted DLL4+ neutrophils (0.5 × 106). (E) After 16 hours, total 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 = 8–15 per group. (F–I) 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 = 6–14 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/scramble. Con, Notch1-knockdown plasmid negative control–treated PVECs; Ko, Notch1-knockdown plasmid–treated PVECs.