Bulk RNA Sequencing of Chimera-Derived Monocyte-Derived Macrophages Targeted for Mitochondrial ROS During Peak Experimental Autoimmune Encephalomyelitis

Reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs) are widely believed to drive tissue damage in multiple sclerosis (MS), yet the distinct roles of central nervous system (CNS)-resident versus CNS-invading MPs remain unclear. Here, we combined single-cell profiling and conditional gene targeting to map and modulate ROS production across CNS MPs in a preclinical mouse model of MS. We show that monocyte-derived macrophages (MdMs) exhibit a higher oxidative stress gene signature and produce more ROS than Mglia. Challenging previous assumptions, our findings reveal that phagocytic NADPH oxidase 2 is dispensable for neuroinflammation. In contrast, quenching mitochondrial ROS (mtROS) through mitochondria-targeted catalase (mCAT) expression in MdMs, but not in Mglia, ameliorated disease severity in acute neuroinflammation. While core phagocyte functions were unaltered in mCAT-expressing MdMs, our results demonstrate a direct neurotoxic role of mtROS. Together, we identify MdMs as the primary driver of ROS-mediated oxidative neurological tissue damage.