The C5a-C5aR1 axis facilitates blood-brain barrier disruption induced by EV-A71 infection through neutrophil activation.

Enterovirus A71 (EV-A71), a member of the genus Enterovirus within the family Picornaviridae, induces neuroinflammation; however, the underlying mechanisms remain incompletely understood. This study demonstrates that the C5a-C5aR1 axis plays a pivotal role in EV-A71-induced blood-brain barrier (BBB) disruption and neuroinflammation, primarily by regulating neutrophil migration. Using human brain specimens and a mouse model, we observed pronounced inflammatory cell infiltration in the brainstem and BBB disruption following EV-A71 infection. Immunofluorescence analysis revealed robust activation of the C5a-C5aR1 axis in fatal EV-A71 cases. Notably, C5aR1 knockout (KO) mice displayed reduced Evans blue extravasation and preserved tight junction protein expression after infection. Immunopathological examination of fatal human cases further confirmed perivascular neutrophil (CD177⁺) infiltration in the brainstem. Importantly, C5aR1 deficiency significantly attenuated neutrophil accumulation and neutrophil extracellular trap (NET) release. Given that peptidylarginine deiminase 4 (PAD4) is a key enzyme driving NET formation, we generated neutrophil-specific PAD4 knockout mice (PAD4 Ne-KO) by crossing S100A8-Cre and PAD4fl/fl lines. As anticipated, neutrophil-specific PAD4 deletion or pharmacological NET blockade substantially ameliorated BBB injury and neuroinflammation following EV-A71 infection. Overall, our findings underscore a critical role for the C5a-C5aR1-neutrophil/NETs pathway in EV-A71 encephalitis pathogenesis and support its targeting as a therapeutic strategy for critically ill patients.