SARS-CoV-2 N protein induces alveolar epithelial apoptosis via NLRP3 pathway in ARDS.

Acute Respiratory Distress Syndrome (ARDS) is a severe inflammatory condition often resulting from sepsis and viral infections, including (Severe Acute Respiratory Syndrome Coronavirus 2) SARS-CoV-2. This study investigates the molecular mechanisms by which the SARS-CoV-2 nucleocapsid (N) protein influences alveolar macrophage activation, leading to alveolar epithelial cell apoptosis and exacerbating ARDS. Single-cell RNA sequencing data from ARDS patients were analyzed to identify cell subpopulations and their interactions, revealing significant macrophage-epithelial cell communication through the (NOD-like receptor family pyrin domain containing 3) NLRP3 pathway. Differential gene expression in SARS-CoV-2-infected macrophages highlighted key genes, with WGCNA pinpointing core modules. In vitro experiments demonstrated that N protein overexpression in MH-S macrophages activates the NLRP3 pathway, promoting M1 macrophage polarization and inducing apoptosis in co-cultured MLE-12 epithelial cells. Immunoprecipitation, pull-down assays, Enzyme-Linked Immunosorbent Assay (ELISA), RT-qPCR, Western blotting, and flow cytometry confirmed these findings. In vivo, ARDS mouse models induced by CLP surgery or N protein administration showed increased M1 macrophage infiltration, heightened inflammatory responses, and significant epithelial cell damage, as evidenced by H&E staining, immunofluorescence, RNA-ISH, and ELISA. These results suggest that the SARS-CoV-2 N protein activates the NLRP3 signaling pathway, driving M1 macrophage polarization and the release of pro-inflammatory factors, thereby inducing alveolar epithelial cell apoptosis and worsening ARDS. Targeting this pathway may provide new therapeutic avenues for treating ARDS associated with SARS-CoV-2.