CTSS regulates macrophage lipid metabolic reprogramming and white matter repair after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) causes secondary white matter injury, which contributes substantially to long-term neurological disability. Although macrophages accumulate in the perihematomal region and participate in tissue remodeling after ICH, the molecular programs that link macrophage responses to white matter repair remain poorly understood, and no current strategies specifically target macrophage‑mediated white matter restoration. Cathepsin S (CTSS), a lysosomal cysteine protease involved in immune regulation and tissue remodeling, is strongly induced after brain injury; however, its role in post‑ICH white matter pathology has not been defined.

A collagenase-induced mouse model of intracerebral hemorrhage (ICH) was established, followed by pharmacological inhibition of Cathepsin S (CTSS) using LY3000328. Single-cell RNA sequencing was performed on perihematomal tissues from Vehicle- and CTSS inhibitor-treated mice to explore CTSS-responsive cell populations and transcriptional programs. Macrophage lipid handling was assessed using flow cytometry, immunofluorescence, and fluorescent myelin debris-based uptake and lipid-transfer assays. A bone marrow-derived macrophage (BMDM)-oligodendrocyte precursor cell (OPC) co-culture system was used to determine whether CTSS inhibition alters macrophage-derived lipid support for OPC differentiation. The LXR agonist GW3965 was applied as a rescue intervention. White matter repair and neurological recovery were evaluated by myelin-associated protein analysis, immunofluorescence, behavioral testing, and transmission electron microscopy.

Single-cell analysis identified infiltrating macrophages, particularly adaptive lipid-associated macrophages (aLAMs), as a major CTSS-expressing population after ICH, displaying a transcriptional state related to lipid metabolism and tissue repair. CTSS inhibition attenuated this lipid-associated macrophage program and reduced ApoE, ABCA1, and ABCG1 expression. Mechanistically, CTSS inhibition reduced Npc1/Npc2 expression and disrupted lysosomal cholesterol trafficking, leading to lysosomal cholesterol retention. These changes suggest impaired Npc1/Npc2-associated cholesterol mobilization and attenuation of the LXR-associated macrophage lipid efflux program. In BMDM-OPC co-cultures, CTSS inhibition reduced the transfer of fluorescent macrophage-processed myelin-derived lipids to OPCs and limited OPC differentiation, whereas GW3965 partially restored Npc1/Npc2 expression, macrophage-derived lipid support, and OPC differentiation. Consistently, in vivo CTSS inhibition reduced MBP expression and MBP⁺ axonal wrapping, increased g-ratio values, aggravated myelin ultrastructural abnormalities, and delayed neurological recovery after ICH.

CTSS in macrophages supports white matter repair after ICH by maintaining lysosomal cholesterol trafficking and macrophage lipid efflux. CTSS inhibition limits macrophage-derived lipid support for OPCs and impairs remyelination, whereas LXR activation partially restores these reparative responses.