Acute respiratory distress syndrome (ARDS) has high mortality and morbidity among Intensive Care Unit patients with a further increased number of cases due to the outbreak of the COVID-19 pandemic. Cell death of endothelial cells and excessive neutrophil extracellular traps (NET) release in the microcirculation is highlighted in the pathophysiology promoting hypercoagulation and immunothrombosis. However, the role of endothelial necroptosis and its crosslinks with neutrophil necroinflammation have been overlooked in current research.
The first part of this thesis investigated endothelial inflammation/cell death and neutrophil NETosis respectively through modulation of TNF/TNFR-1 complex signalling. For the first time, we have shown definitive evidence of TNF complex III mediated human lung microvascular endothelial cell (HLMEC) necroptosis using primary cell culture. Complex III necroptosis led to a distinct endothelial cell activation response from inflammatory phenotype with reduced expression of surface adhesion molecules, suggesting less neutrophil adhesion and interaction. Complex III necroptosis activation in neutrophils was crosslinked with NET
formation which process was abrogated with RIPK1 inhibition. NET release occurred prior to rupture of cell plasma membrane suggesting it is an early event in necroptosis activation. RIPK1/caspase mediated complex II also triggered NETosis, but likely through different
intracellular signalling.
The second part of this thesis explored the necro-inflammatory crosstalk between injured endothelium and neutrophils. Complex III necroptotic endothelium induced neutrophil NET formation through direct contact, whereas complex II apoptotic endothelium did not, suggesting neutrophil is able to distinguish activation/death states of endothelial cells and provide different immune responses. Furthermore, necroptotic endothelial cell-derived extracellular vesicles (EV) also induced NET through an indirect communication mechanism likely mediated via phospho-MLKL in its cargo. These findings indicate an in vivo translation that endothelial necroptosis suppresses neutrophil transmigration into tissue and enhances neutrophil NET release, which ultimately result in promotion of coagulation in the
microvasculature.