Integrated metabolomic and expression analyses identify macrophage ALOXE3 as a modulator of oxidative stress in acetaminophen-induced liver injury

Macrophage adaptation to oxidative stress may influence the progression of acetaminophen (APAP)-induced liver injury, but the underlying mechanisms remain incompletely understood. In this study, we combined hepatic transcriptomic and proteomic data with untargeted metabolomics to prioritize candidates for functional investigation. Joint pathway analysis highlighted arachidonic acid metabolism, and arachidonate lipoxygenase 3 (ALOXE3) was prioritized for functional investigation based on its concordant upregulation at the mRNA and protein levels and its previously characterized hydroperoxide isomerase activity. We then combined spatial and single-cell transcriptomics with macrophage-directed ALOXE3 manipulation to examine its distribution and function. Spatial and single-cell analyses provided cellular context for Aloxe3 expression, including its detection in macrophage-associated populations. Single-cell analysis showed higher scores for a glutathione (GSH) metabolism-related gene set in Aloxe3 -positive than in Aloxe3 -negative macrophages. In vivo, macrophage-directed ALOXE3 overexpression reduced serum ALT and AST activities and hepatic necrosis, whereas knockdown produced the opposite effects. In bone marrow-derived macrophages exposed to conditioned medium from APAP-treated hepatocytes, ALOXE3 knockdown increased 4-hydroxynonenal signals and reactive oxygen species-associated fluorescence and reduced GSH-associated fluorescence. Overexpression produced the opposite changes. These findings support a role for macrophage ALOXE3 in redox responses during APAP-induced liver injury.