Medical Biology - Theses
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ItemNeutrophil extracellular trap-associated cell death - role in gout and relationship to alternate forms of cell death( 2017)Cell death has emerged as a critical process in many facets of human disease, ranging from cancer to inflammation and cardiovascular disease. One such modality, Neutrophil Extracellular Trap (NET)-related cell death, or NETosis, is a form of cell death with potential implications in a wide range of human conditions but, at present, understanding of the mechanisms of NETosis and its physiologic and pathological consequences is limited. Finding the key mechanisms underlying NETosis will illuminate roles for NETosis in human diseases and animal models of these conditions, and provide targets for intervention. This thesis examines NETosis in the context of the human inflammatory disease, gout, and explores the relationship between NETosis and the other main types of cell death - necroptosis and apoptosis. First, I developed a novel time-lapse imaging-based quantitative analysis of NETosis. The assay combines multi-well format live-cell microscopy technique with computerized analysis to obtain detailed kinetic information about the two key components of NETosis - cell death and chromatin decondensation. The technique allowed me to demonstrate that different NETosis stimuli, namely phorbol myristate acetate and monosodium urate crystals, exhibit different kinetics of cell death. Exploring the differences further, I found that crystals induce NETosis through a nicotinamide adenine dinucleotide phosphate oxidase-independent pathway that is distinct from PMA-induced NETosis. I also observed that both forms of NETosis depend on neutrophil elastase activity to cause chromatin decondensation, but not cell death. I observed NET-like structures in samples taken from gout patients both during the acute inflammation of a gout attack and from the uninflamed crystal-rich tophus tissue, a feature of chronic gout. The NETs released during MSU-induced NETosis were resistant to serum nucleases relative to PMA-induced NETs, suggesting that these DNA structures may persist within tissues, as seen in the patient samples. This nuclease resistance was at least partially attributable to the presence of increased actin in MSU-induced NETs relative to PMA-induced NETs, as identified using proteomic techniques. Given the presence of NETs in both inflammatory and uninflamed contexts, I demonstrated that the presence of NETs dampens the IL-1β responses of macrophage-like cells and reduces crystal-induced macrophage cell death. I further demonstrated that NETosis is distinct from both apoptosis and necroptosis. Unexpectedly, two MLKL inhibitors did inhibit NETosis, but this is an indirect effect, dependent on accelerating apoptosis. This finding highlighted that over time in culture, neutrophils lose the ability to release NETs when stimulated with PMA and this loss of “NET competence” is mediated by caspase activation. Using the power of a live cell imaging approach to simultaneously quantify cell death and NET release, my studies have advanced our fundamental understanding of the mechanistic differences underlying NETosis induced by different stimuli. Further, by applying this assay, I was able to establish that proteins within the apoptosis and necroptosis pathways that had been previously attributed roles in the NETosis pathway, were in fact dispensable. Consequently, I anticipate this assay and the mechanistic insights it provides will play an important part in advancing our understanding of the NETosis pathway.