Multi-omics analysis and experimental validation reveal the IRF7-CXCL10 axis as a master regulator of microglial PCD in ischemic stroke

BACKGROUND: Microglia-driven neuroinflammation serves as a critical factor in secondary injury following ischemic stroke, yet the primary regulators governing detrimental microglial phenotypes remain unclear. As a key component of this process, the cell type-specific regulatory mechanisms of programmed cell death (PCD) are poorly understood. METHODS: We performed an integrative analysis of public single-cell and bulk transcriptomic datasets from a murine stroke model. A multi-layered computational pipeline, incorporating pseudotime trajectory, weighted co-expression network analysis (WGCNA), and gene regulatory network inference (SCENIC), was used to identify master regulators of PCD. Functional validation was conducted using in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) on primary microglia-neuron co-cultures and in vivo via a transient middle cerebral artery occlusion (tMCAO) model, employing AAV-mediated microglia-specific gene silencing, comprehensive in vitro and in vivo rescue strategies, and detailed behavioral assessments. RESULTS: Our single-cell analysis identified microglia as the central hub of PCD activity post-stroke. An unbiased, multi-layered computational pipeline converged upon Interferon Regulatory Factor 7 (IRF7) as the master transcriptional regulator of this high-PCD, pathological microglial state. We confirmed IRF7 upregulation in microglia following ischemic injury and delineated a novel downstream pathway where IRF7 directly binds the CXCL10 promoter to drive its expression, promoting microglial dysfunction and neurotoxicity. In vitro, silencing IRF7 skewed microglia toward an anti-inflammatory phenotype and protected co-cultured neurons from apoptosis. Critically, microglia-specific IRF7 knockdown in vivo significantly reduced infarct volume, suppressed neuronal death, and led to significant improvements in long-term neurological and cognitive function after stroke. Crucially, both in vitro genetic overexpression of CXCL10 and in vivo administration of recombinant CXCL10 completely abolished the neuroprotective benefits of IRF7 inhibition, establishing a definitive functional causality for the IRF7-CXCL10 axis. CONCLUSION: Our findings uncover the IRF7-CXCL10 axis as a pivotal driver of detrimental neuroinflammation in ischemic stroke and establish IRF7 as a potent therapeutic target for neuroprotection.