Analysis of CXCR4 in microglial pyroptosis after ischemia–reperfusion

Abstract

Cerebral ischemia–reperfusion injury elicits rapid sterile inflammation in which microglia undergo marked state transitions and can contribute to secondary tissue damage. Pyroptosis, executed by gasdermin D cleavage and pore formation, has been implicated in amplification of neuroinflammation, while the chemokine receptor CXCR4 is induced after stroke and may intersect with inflammatory execution pathways. This thesis investigated the role of CXCR4 in microglial pyroptosis after ischemia–reperfusion and evaluated whether pharmacological CXCR4 blockade with AMD3100 attenuates acute pyroptotic execution. Public datasets were reanalyzed, including bulk RNA sequencing of microglia three days after 60-min middle cerebral artery occlusion (MCAO) with reperfusion and brain-wide single-cell RNA sequencing 24 h after MCAO or sham. Bulk RNA-seq identified extensive transcriptional remodeling and significant induction of CXCR4 together with prominent inflammatory, and enrichment analyses mapped CXCR4-containing programs to chemokine signaling, chemotaxis, cytoskeletal remodeling, and receptor trafficking. Single-cell analysis resolved eight microglial states indicating pronounced post-ischemic heterogeneity. Pyroptosis-related module scores were broadly reduced across most MCAO-associated states but increased in the interferon-responsive state. CXCR4-axis activity showed weak yet significant positive associations with pyroptosis composite and execution programs after ischemia–reperfusion, whereas no significant correlations were detected in sham microglia, and a network-based virtual CXCR4 knockout suggested the most coherent attenuation of pyroptosis-related regulation in the interferon-responsive microglial state. For in vitro validation, primary mouse microglia were subjected to oxygen–glucose deprivation followed by reoxygenation (OGD/RO). CXCR4 protein increased transiently and peaked at 6 h OGD with 12 h reoxygenation. Under this condition, AMD3100 treatment reduced the cleaved GSDMD N-terminal fragment without altering full-length GSDMD and was accompanied by reduced membrane-associated GSDMD patterns and partial morphological normalization. Collectively, these findings support a model in which CXCR4 contributes to microglial pyroptotic execution in a time- and state-dependent manner after ischemia–reperfusion and indicate CXCR4 antagonism as a potential strategy to dampen acute inflammatory injury.