Recombinant SARS-Cov-2 spike protein S1 subunit upregulates P2 × 7-NLRP3 leading to macrophage pyroptosis promoting acute lung injury.

BACKGROUND: Our previous study confirmed recombinant SARS-CoV-2 spike protein subunit S1 (S1SP) independently induces acute lung injury (ALI), but its mechanism remains unclear. This study thoroughly investigates the role of the purinergic receptor P2 × 7 and its downstream NLRP3 inflammasome in S1SP-induced lung injury. METHODS: S1SP was used to intervene in humanized K18-hACE2 mice and THP-1 cells. In vivo, mice were divided into Control, S1SP, S1SP + MCC950 (NLRP3 inhibitor), S1SP + A438079 (P2 × 7 antagonist) and their respective control groups to study the effect of S1SP on NLRP3 inflammasome in mouse lung tissues. In vitro, THP-1-derived macrophages were grouped as Control, S1SP + ATP, S1SP + ATP A438079 and A438079 control groups. qRT-PCR was used to measure NLRP3, Caspase-1 and P2 × 7 mRNA levels. Immunofluorescence and western blot detected related proteins, and ELISA measured IL-1β and IL-18 levels in bronchoalveolar lavage fluid (BALF). RESULTS: S1SP caused severe lung injury, edema, and elevated ATP in BALF. NF-κB and NLRP3 inflammasome activation led to increased TNF-α, IL-6, and Gasdermin D N-terminal fragment (GSDMD-NT) (p < 0.05). NLRP3 and P2 × 7 co-localized with macrophages, indicating their central role. MCC950 or A438079 reduced tissue damage, restored tight junction integrity, and suppressed IL-1β/IL-18 release. In vitro, S1SP + ATP co-stimulation upregulated P2 × 7 and NLRP3 activity in macrophages, exacerbating pyroptosis and cytokine release. CONCLUSION: P2 × 7 receptor mediates the occurrence of cellular pyroptosis in macrophages through the upregulation of its downstream NLRP3 inflammasome activity, contributing to the expression and release of inflammatory factors, which in turn promotes the onset and progression of S1SP-induced ALI. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12931-026-03513-9.