Neutrophil extracellular traps - in vivo studies in zebrafish

Neutrophils are the most abundant type of leukocyte in mammals, and they are the first line of innate immune defense against invading pathogens. Upon activation, neutrophils form a cloud-like structure composed of chromatins and granule-derived proteins termed neutrophil extracellular traps (NETs) to entrap extracellular pathogens. NETosis, the process of NET release, has been studied extensively in vitro, but in vivo studies are a limited. Zebrafish have emerged as an attractive model for in vivo imaging of leukocytes due to their small size, transparency and high degree of functional similarity to mammals. These studies exploit the advantages of zebrafish for studying leukocyte biology to study NETs and NETosis. A transgenic zebrafish reporter line is generated marking the neutrophil cytoplasm with mKO2CAAX and the nucleus with histone2Bcerulean, It is used to determine the dynamics of NETs and NET-releasing neutrophils in vivo, using high-resolution microscopy. Zebrafish neutrophils were first verified to form NETs in vitro by several chemical and physical stimuli, using an improved method of purifying adult zebrafish neutrophils. Using the dual-colour NET- reporter line to image NETosis in vivo, several controversial previous observations have been re-examined, and several new aspects of NET releasing-neutrophil behaviours documented. Studies presented describe the entire sequence of NETosis by single neutrophil prior to, during and following NET release, both intravascularly and extravascularly. They display, the mobility of NETs and NET-releasing neutrophils, the velocity of NET-releasing neutrophils, NET entrapment of pathogens, NET remodelling by incoming neutrophils, and assess the persistence of NETs. In vivo observations have the potential to provide the significant insight into NETosis and the role of NETs during inflammation and infection. Additional material(s) submitted with thesis