Neutrophil Interactions with Particle Drug Carriers and Behavior in Inflammation
Kelley, William
2020
Abstract
Neutrophils are cells of the innate immune system which make up approximately 50-70% of all circulating white blood cells. Despite their abundance, the importance of neutrophils in many contexts has only recently begun to be explored in depth due to the difficulty in maintaining neutrophils in culture. The work in this dissertation aims to investigate the role of neutrophils in inflammatory diseases and how neutrophils interact with particulate drug carriers designed to mitigate such diseases. We explored the impact of particle drug carriers on leukocyte adhesion in inflammation in vitro, finding that both targeted and non-targeted particles reduce leukocyte adhesion. This effect was linked to both particle size and concentration. Additionally, we found that under certain particle conditions, specific blocking of the endothelium via active targeting results in greater reduction in leukocyte adhesion. Intravenous particle administration resulted in a reduction in neutrophil adhesion in an in vivo model of acute mesentery inflammation and an in vivo model of acute lung inflammation, resulting primarily from particle uptake. Crucially, we found that after internalizing particles, neutrophils shed CD62L, resulting in faster rolling along the endothelium and reduced adhesion, making particle internalization the dominating mechanism for reducing neutrophil adhesion. We investigated the impact of the use of a common non-fouling coating, polyethylene glycol (PEG), on neutrophil particle phagocytosis. In contrast to the behavior of other phagocytes, we found that human neutrophils preferentially internalize PEGylated particles compared to non-PEGylated particles. This effect was linked to factors in the plasma, specifically complement; we demonstrated that PEGylated particles adsorb more complement proteins than non-PEGylated particles, resulting in increased particle uptake. We also explored whether drug-free particle effects can be used as therapeutics in inflammatory diseases, specifically in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). For this purpose, we fabricated micron-sized salicylate-based particles, termed “PolyAspirin” or “PolyA”. These particles significantly reduced neutrophil infiltration into the lungs in an LPS-induced model of ALI and in a P. Aeruginosa lung infection model of ARDS. Further, the PolyA particles reduced the release of inflammatory cytokines in the lungs in ARDS, reduced the spread of the bacteria into the bloodstream, and dramatically improved survival. These effects appear to be driven by both the presence of particles in blood circulation as well as the degradation of PolyA particles into salicylic acid. We demonstrate a novel potential for drug-free particles to be used as a therapeutic for inflammatory disease. Finally, we used in vitro and ex vivo tools to explore the role neutrophils play in antiphospholipid syndrome (APS). APS is an inflammatory autoimmune disease resulting in numerous complications including an increased risk of thrombus formation. We found that APS patient-derived neutrophils exhibit significantly increased adhesion to an unactivated endothelium, which may be in part responsible for the increased risk of thrombus. Additionally, we demonstrated that APS patient plasma can “condition” healthy donor neutrophils to exhibit enhanced adhesion. We found that this effect is driven by an increased expression of activated Mac1 on the surface of APS patient-derived neutrophils. Further, we ffound that activated Mac1 is responsible for enhanced NETosis of APS patient-derived neutrophils, which may also contribute to the increased risk of thrombosis in APS. These results suggest a novel role for neutrophils in APS pathophysiology and highlight a potential therapeutic target (activated Mac1) for the treatment of APS.Subjects
neutrophils inflammation drug delivery leukocyte adhesion
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