Macrophage-Derived, Rather Than Microglial, Cathepsin B Exacerbates Brain Injury After Ischemic Stroke

Abstract

Neuroimmune responses critically shape the progression and outcome of ischemic stroke, yet the cell type–specific roles of myeloid populations remain incompletely defined. Cathepsin B (CatB), a lysosomal cysteine protease, has been implicated in neuroinflammation, but its contribution from distinct myeloid subsets is unclear. Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and CatB-deficient mice. Bone marrow chimeras were generated to selectively delete CatB in peripheral myeloid cells or resident microglia. Infarct severity, mortality, myeloid phenotypes, and transcriptional programs were assessed using flow cytometry, immunofluorescence, and bulk and cell type–resolved RNA sequencing. CatB expression was markedly upregulated in ischemic brain myeloid cells, with higher levels in infiltrating macrophages than in microglia. Selective deletion of CatB in peripheral myeloid cells, but not in microglia, significantly reduced infarct size, mortality, and pro-inflammatory gene expression after tMCAO. Transcriptomic analyses showed suppression of inflammatory pathways and preservation of neuronal signaling in mice lacking macrophage-derived CatB. Cell type–resolved RNA sequencing revealed that CatB deficiency shifted infiltrating macrophages toward a pro-resolving, tissue-repair phenotype characterized by increased expression of M2-associated genes, including Chil3. Consistently, CatB-deficient macrophages displayed enhanced pro-resolving polarization in response to ischemic neuron–derived signals in vitro. CatB was also elevated in circulating Ly6C⁺ monocytes after stroke. These findings identify peripheral myeloid–derived CatB as a key driver of ischemic brain injury and a potential therapeutic target.