Unexpected Protective Role of Thrombosis in Lung Injury via Endothelial Alox15

BACKGROUND: Patients with sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) commonly suffer from severe pulmonary thrombosis, but clinical trials of anticoagulant therapies in patients with sepsis and ARDS have failed. Patients with ARDS with thrombocytopenia also exhibit increased mortality, and widespread pulmonary thrombosis is often seen in patients with COVID-19 ARDS. METHODS: Different amounts of microbeads were administered intravenously to adult mice to induce various levels of pulmonary thrombosis. ALI was induced by either intraperitoneal lipopolysaccharide or cecal ligation and puncture. Endothelial cell (EC)–targeted nanoparticles were used to deliver plasmid DNA expressing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) system for EC-specific gene knockout of Alox15 (arachidonate 15-lipoxygenase) or plasmid DNA expressing Alox15 for EC-specific overexpression. Lipidomic profiling and in vivo rescue studies with the identified Alox15-regulated lipids were performed. In addition, thrombocytopenia was induced by genetic depletion of platelets using DTR Pf4Cre mice, and the effects of restoration of pulmonary thrombosis were assessed. RESULTS: We show that although severe pulmonary thrombosis or thrombocytopenia augments sepsis-induced ALI, the induction of mild pulmonary thrombosis conversely reduces EC apoptosis, ALI, and mortality via sustained expression of endothelial Alox15. Endothelial Alox15 knockout in adult mice abolished the protective impact of mild lung thrombosis. Conversely, overexpression of endothelial Alox15 inhibited the increases in ALI caused by severe pulmonary thrombosis. Treatment of the endothelial Alox15 knockout mice with 1-palmitoyl-2-oleoyl-3-arachidonoyl-rac-glycerol (C 57 H 100 O 6 ), a top candidate of the 32 Alox15-regulated lipids identified by lipidomic profiling, markedly reversed the defective phenotype, suggesting that Alox15 protects from lung injury via protective lipids. The clinical relevance of the findings was supported by the observation of reduced ALOX15-expressing ECs in lung autopsy samples of patients with ARDS. In addition, restoration of pulmonary thrombosis in thrombocytopenic mice normalized endotoxemia-induced ALI. CONCLUSIONS: We have demonstrated that moderate levels of lung thrombosis protect against sepsis-induced inflammatory lung injury via endothelial Alox15. Overexpression of endothelial Alox15 inhibits severe pulmonary thrombosis-induced increases in ALI. Thus, upregulation of ALOX15 expression or treatment with ALOX15-dependent protective lipid(s) represents a promising therapeutic strategy for treatment of ARDS, especially in subpopulations of patients with thrombocytopenia and widespread pulmonary thrombosis.