Development and application of a novel high- throughput technique for screening neutrophil extracellular traps

摘要

Neutrophil extracellular traps (NETs) are antimicrobial web-like structures whose release is mediated by reactive oxygen species (ROS) and purpose is to combat infections. Unbalanced NET production and clearance is, however, associated with auto-antibody production and disease. This thesis aimed to develop a High-Content-Analysis (HCA) approach to study human NETosis and its modulation. Initially, NET-related disease-relevant conditions were studied. Individual periodontal-bacteria generated substantial NET production compared with bacterial biofilms. Calcium or magnesium ions and increases in cell density enhanced NET responses. As part of this study the use of fixation and cell adherence procedures were explored and data indicated that addition of paraformaldehyde prior to centrifugation and the absence of poly-L-lysine provided appropriate conditions for downstream cytological analysis. ‘Compartmental Analysis’ and ‘Tube Formation’ algorithms were initially assessed for HCA, however, it was determined that bespoke ‘NET Detection’ and ‘Nuclear Decondensation’ algorithms provided more accurate analysis of NETosis and peptidyl-arginine-deiminase-4 translocation. The optimised protocol employed for the high-content-screening of a 56-compound library identified 8 NETosis modulators. Further characterisation of compounds’ abilities to modulate ROS and NET production, identified roles for glutathione reductase, Src, molecular-target-of-Rapamycin and mitogen-activated-protein-kinase signalling. These pathways may provide new therapeutic targets for treatment of NET-related inflammatory disorders including periodontitis.