Gasdermin D-dependent neutrophil pyroptosis exacerbates Coxsackievirus A6-induced neuroinflammation through NET formation

Coxsackievirus A6 (CVA6) is a nonenveloped, single-stranded RNA virus linked to neurological complications. Emerging evidence suggests neutrophil pyroptosis drives inflammation. However, the role of neutrophil pyroptosis in CVA6 pathogenesis remains unexplored. Ten-day-old wild-type (WT), Caspase-1 KO, and GSDMD KO mice were infected with a lethal dose of CVA6. For in vivo and in vitro studies, we used the caspase-1 inhibitor belnacasan, disulfiram, and anti-Ly6G antibody. We also generated neutrophil-specific PAD4-knockout mice (PAD4 Ne-KO) by deleting Padi4 under the S100A8 promoter. Post-infection, clinical scores, survival, and body weight were monitored. Brain tissues and bone marrow-derived neutrophils (BMDNs) were collected for analysis. Key methods included qPCR, Western blotting, histology/immunofluorescence, flow cytometry, and TEM to assess pyroptosis, inflammation, and immune cell infiltration. Findings were further validated using blood samples from HFMD patients. In this study, we investigated how the Caspase-1/GSDMD pathway mediates neutrophil extracellular trap (NET) release and drives CVA6-induced neuroinflammation. CVA6 infection increased neutrophil numbers in mouse brain and peripheral blood, along with elevated MPO-DNA—a NET marker. In BMDNs, degranulation and NET formation occurred by 24 hpi, accompanied by Caspase-1/GSDMD activation. Caspase-1 knockout prolonged survival and reduced GSDMD-N expression in brain neutrophils; pharmacological Caspase-1 inhibition decreased mature IL-1β and IL-18 in brain tissue and suppressed CVA6 replication in BMDNs. Together, in vitro and in vivo data indicate that Caspase-1/GSDMD activation and NETosis critically contribute to CVA6-induced brain injury. This was confirmed by GSDMD knockout or disulfiram-mediated GSDMD inhibition, both of which markedly reduced NET release and neuropathology. Notably, global neutrophil depletion worsened infection—suggesting a protective role—whereas neutrophil-specific PAD4 knockout improved survival. Clinically, GSDMD expression showed a significant positive correlation with NETosis markers in patient samples from CVA6-infected individuals. These findings enhance understanding of enteroviral pathogenesis, identify GSDMD as a promising therapeutic target, and provide a novel framework for developing precision interventions that reduce excessive inflammation without impairing essential host defenses. The proposed mechanism of CVA6-induced neuroinflammation: Following the invasion of the central nervous system by CVA6, neutrophil aggregation is induced, which triggers the activation of the classical pyroptosis pathway. During this process, Caspase-1 cleaves GSDMD, pro-IL-1β, and pro-IL-18 to produce active N-GSDMD, mature IL-1β, and IL-18, respectively. The pyroptosis mediated by N-GSDMD leads to the release of neutrophil extracellular traps (NETs) and is accompanied by a robust secretion of inflammatory cytokines. This cascade ultimately results in a cytokine storm and severe neuroinflammatory responses.