Development and application of a high-content biology-based assay to quantify effects of modulators of NETosis in primary human neutrophils

Abstract Neutrophils represent the first line of defense against pathogens by using multiple mechanisms, including phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). NET formation can lead to a unique form of programmed cell death in neutrophils, called NETosis. In addition to its microbicidal function, compelling evidence has linked NETosis to the pathogenesis of diseases including autoimmune diseases, atherosclerosis and thrombosis. To facilitate drug discovery programs for NETosis modulators, a high content biology-based method was developed and applied to blood derived primary human neutrophils. In summary, NETosis was triggered by PMA or BSA-Immune Complexes and the loss of membrane integrity together with spreading of NET formation beyond cell membranes as well as nuclear decondensation were captured in a live-cell setting on a CellVoyager (CV8000) platform. Furthermore, after addition of NETosis modulators (DPI and R406) to the triggered-neutrophils, a dose-dependent NETosis reduction was observed with optimal assay windows between 2.5 and 4 hours after trigger addition. Based on their distinct changes in nuclear morphology and loss of membrane integrity, this method was also able to distinguish various mechanisms of neutrophil cell death including NETosis and apoptosis. This procedure was successfully applied in medium-throughput screenings (96-well plate format), assessing dose-dependent inhibitory effects of selected large molecules, small molecules, and lipids on NETosis processes. In conclusion: We developed a high content biology-based approach that can be employed to advance drug discovery for therapeutic areas associated with NETosis. NA