C/EBPβ Drives Ischemia-Induced Microglial Inflammatory Reprogramming Through Transcriptional Activation of ATF3 and Exacerbates Brain Injury.

Microglial activation contributes substantially to secondary brain injury after ischemic stroke, but the transcriptional mechanisms governing this response remain poorly defined. Here, we identified CCAAT/enhancer-binding protein β (C/EBPβ) as a key regulator of ischemia-induced microglial inflammation. RNA sequencing of microglia isolated from mice subjected to transient middle cerebral artery occlusion (tMCAO) revealed extensive transcriptional reprogramming, characterized by activation of inflammatory pathways, including chemokine, TNF, IL-17, and efferocytosis signaling, as well as cell-cycle programs, together with suppression of homeostatic processes involved in synaptic regulation and neuronal support. Network analysis identified Cebpb as a highly connected hub across multiple inflammatory pathways. C/EBPβ expression was markedly increased in peri-infarct microglia after tMCAO and in primary microglia exposed to oxygen-glucose deprivation/reoxygenation. Microglia-selective Cebpb knockdown reduced TNF-α, IL-1β, CCL2, and CD16 expression, increased IL-4, IL-13, and ARG1 expression, and attenuated ischemia-induced morphological remodeling. Consistent effects were observed in primary microglia and BV2 cells, supporting a central role for C/EBPβ in microglial pro-inflammatory activation under ischemic conditions. Mechanistically, C/EBPβ directly bound the Atf3 promoter and activated its transcription, while ATF3 partially mediated C/EBPβ-dependent Il1b induction. In vivo, microglial Cebpb knockdown reduced acute ischemic lesion volume, neuronal apoptosis in the striatum and cortex, and long-term cerebral atrophy. These histopathological improvements were accompanied by sustained recovery of neurological function, somatosensory performance, motor coordination, recognition memory, and spatial learning and memory. Collectively, these findings establish the C/EBPβ-ATF3 axis as a transcriptional pathway that promotes microglial inflammation and secondary neuronal injury after cerebral ischemia. Targeting microglial C/EBPβ may therefore limit ischemic brain damage and improve long-term neurological recovery after stroke.