Investigating functional NETosis pathways and siRNA mediated suppression of NETosis in feline and HL-60 neutrophil in vitro models of SARS-CoV-2 infection 2738

Abstract Description Neutrophil extracellular trap (NET) formation, or NETosis, aids pathogen clearance, while causing tissue damage when excessive, as seen in severe cases of COVID-19. This study investigates SARS-CoV-2-induced NETosis, via ROS-dependent and ROS-independent pathways in feline and human neutrophil in vitro models,further assessing the potential of small interfering RNA (siRNA) mediated gene silencing in suppressing NETosis and related complications during COVID-19. Isolated feline neutrophils and differentiated human HL-60 granulocytes were infected with SARS-CoV-2 B.1.617.2 (Delta) variant and treated with the ROS inhibitor diphenyleneiodonium. Viral load, neutrophil functionality and NETosis were quantified using ddPCR, phagocytosis assay, ROS assay, multiplex cytokine assay, RNA-Seq, MPO-DNA ELISA and dsDNA assays respectively. Statistical analyses identified DAPP1 and S100A9 as effective siRNA targets, with gene silencing assays confirming their role in reducing NETosis without impairing regular neutrophil functions. Functional assays and pathway analyses (DEGSeq2, KEGG/GO) revealed activation of both ROS-dependent and -independent NETosis, with ROS-dependent pathways showing significantly higher NETosis in both in vitro models of SARS-CoV-2 infection. Overall, our findings underscore the relevance of feline models in COVID-19 studies and suggest siRNA as a promising approach to mitigate excessive NETosis and related immune dysregulation during SARS-CoV-2 infection. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)