Inhibition of ALOX12–12-HETE Alleviates Lung Ischemia–Reperfusion Injury by Reducing Endothelial Ferroptosis-Mediated Neutrophil Extracellular Trap Formation

Abstract

Lung ischemia–reperfusion injury (IRI) stands as the primary culprit behind primary graft dysfunction (PGD) after lung transplantation, yet viable therapeutic options are lacking. In the present study, we used a murine hilar clamp (1 h) and reperfusion (3 h) model to study IRI. The left lung tissues were harvested for metabolomics, transcriptomics, and single-cell RNA sequencing. Metabolomics of plasma from human lung transplantation recipients was also performed. Lung histology, pulmonary function, pulmonary edema, and survival analysis were measured in mice. Integrative analysis of metabolomics and transcriptomics revealed a marked up-regulation of arachidonate 12-lipoxygenase (ALOX12) and its metabolite 12-hydroxyeicosatetraenoic acid (12-HETE), which played a pivotal role in promoting ferroptosis and neutrophil extracellular trap (NET) formation during lung IRI. Additionally, single-cell RNA sequencing revealed that ferroptosis predominantly occurred in pulmonary endothelial cells. Importantly, Alox12-knockout (KO) mice exhibited a notable decrease in ferroptosis, NET formation, and tissue injury. To investigate the interplay between endothelial ferroptosis and NET formation, a hypoxia/reoxygenation (HR) cell model using 2 human endothelial cell lines was established. By incubating conditioned medium from HR cell model with neutrophils, we found that the liberation of high mobility group box 1 (HMGB1) from endothelial cells undergoing ferroptosis facilitated the formation of NETs by activating the TLR4/MYD88 pathway. Last, the administration of ML355, a targeted inhibitor of Alox12, mitigated lung IRI in both murine hilar clamp/reperfusion and rat left lung transplant models. Collectively, our study indicates ALOX12 as a promising therapeutic strategy for lung IRI.