Characterization of neutrophil activation, netosis and mast cell activation in SARS-COV-2 infection: A translational study utilizing a feline model for COVID-19

Neutrophil activation, neutrophil extracellular trap (NET) formation (NETosis), and mast cell activation are increasingly recognized as central drivers of severe immunopathology in SARS-CoV-2 infection. Despite extensive human studies, key mechanistic details remain unclear, highlighting the critical need for translational animal models to elucidate pathogenesis and identify therapeutic targets. This study characterizes neutrophil activation, NETosis, and mast cell activation utilizing a translational in vivo and in vitro feline model of COVID-19. Temporal analyses reveal early acute-phase elevations in neutrophil activation markers, including myeloperoxidase, neutrophil elastase, citrullinated histone H3, MPO-DNA complexes, and cell-free DNA. These markers strongly correlate with clinical severity and histopathological outcomes, highlighting the detrimental role of neutrophil hyperactivation during acute infection. Comparative assessment of feline responses to SARS-CoV-2 Delta and Omicron variants reveal significant variations. Delta-infected cats experience severe illness characterized by pronounced respiratory tissue damage and robust neutrophil activation, while Omicron-infected cats exhibited milder symptoms with localized tissue damage. Variant-specific cytokine profiles were noted, with Delta infections inducing intense inflammatory responses, emphasizing variant-dependent immune dynamics. In vitro experiments conducted with feline neutrophils demonstrate the involvement of both ROS-dependent and independent pathways during SARS-CoV-2 induced NETosis. While inhibiting ROS significantly decreased NET formation, residual NETosis persisted, suggesting alternative mechanisms. Silencing of S100A9 consistently decreased neutrophil activation, ROS production, and NET formation, identifying S100A9 as a pivotal regulator and potential therapeutic target. Conversely, DAPP1 knockdown produced variable results, reflecting its complex role in neutrophil signaling. Moreover, we demonstrated the prominence of acute mast cell activation during infection, particularly in lung tissues, which correlated with clinical severity. Subtle elevations in mast cell mediators in brain tissues suggested a possible contribution to neurological symptoms associated with post-COVID syndrome (long COVID). Collectively, our findings validate the feline model as a robust translational tool for investigating immune-driven mechanisms underpinning COVID-19 severity. The distinct immune responses triggered by the Delta and Omicron variants emphasize the necessity for variant-specific therapeutic strategies. Overall, this study underscores the pathological significance of neutrophil and mast cell activation in SARS-CoV-2 infection and highlights potential therapeutic targets to mitigate severe disease manifestations associated with NETosis in COVID-19.