Multimodal Single-Cell Transcriptomic and Chromatin Accessibility Profiling Reveals Monocyte-Derived Macrophage...

Ischemic stroke promotes monocyte recruitment to the injured brain and their differentiation into monocyte-derived macrophages (MDMs). These cells contribute to debris clearance but may also exacerbate neuroinflammation. However, the heterogeneity of MDM subsets and the phenotypic transitions that shape MDM functional states during the subacute phase of stroke remain incompletely characterized. To address this, we first performed single-cell RNA sequencing (scRNA-seq) to define the transcriptional landscape of the mouse brain 48 h after transient middle cerebral artery occlusion/reperfusion compared with sham controls. Reclustering of macrophage-lineage cells identified multiple MDM subsets, including a distinct Cd68^hi/Ctsd^hi MDM subset enriched for lysosomal and lipid-processing gene expression programs. Cell trajectory inference supported a transition from early recruited MDMs toward the Cd68^hi/Ctsd^hi state, accompanied by induction of transcriptomic networks that drive MDM function to favor a clearance-competent phenotype in response to ischemic stroke. Complementary single-cell ATAC sequencing (scATAC-seq) demonstrated cell type-specific chromatin remodeling after stroke and revealed MDM subclusters with accessibility at key loci regulating lysosomal function and lipid metabolism. Together, our findings define a cellular and regulatory framework of the subacute post-stroke brain and identify a lysosome-enriched Cd68^hi/Ctsd^hi MDM trajectory, highlighting endolysosomal and lipid-processing programs during early stroke recovery.