Integrated Metabolomic and Bioenergetic Profiling Reveals Axl as a Key Regulator of Macrophage Homeostasis

Abstract

The TAM receptor Axl is essential for efferocytosis and inflammatory regulation, but its role in macrophage immunometabolic homeostasis remains unclear. Here, we combined phenotypic, functional, metabolomic, and bioenergetic analyses to define how Axl deficiency alters peritoneal macrophages (PMs) under steady‐state conditions. Axl−/− PMs exhibited reduced cell numbers, downregulation of resident macrophage markers, and increased Ly6C expression, indicating a shift toward a less mature, monocyte‐like phenotype. Functionally, these cells showed impaired inflammatory activation, with reduced nitrite release, diminished LPS‐induced ROS production, and lower mitochondrial ROS. Metabolomic profiling revealed broad disruption of central carbon metabolism, including reduced glycolytic intermediates, depletion of tricarboxylic acid (TCA) cycle metabolites, and decreased NAD+/NADH and NADP+/NADPH pools. Despite reduced glycolytic flux, preserved pyruvate levels suggested compensatory carbon utilization. Bioenergetic assessment demonstrated pronounced mitochondrial dysfunction, characterized by reduced maximal respiration, elevated proton leak, and limited oxidative phosphorylation flexibility. Redox balance was also impaired, with decreased GSH, GSSG, and dehydroascorbic acid and increased ophthalmic acid, consistent with elevated oxidative stress. Systemically, Axl−/− mice displayed hepatic redox imbalance and biochemical signs of liver dysfunction. Collectively, these findings identify Axl as a central regulator of macrophage identity, mitochondrial performance, and redox resilience, required for maintaining local and systemic homeostasis.