In Vitro Platforms for the Study and Manipulation of Neutrophil Extracellular Traps

Though only recently discovered, neutrophil extracellular traps (NETs) have rapidly attracted scientific and clinical interest as a potent weapon in the arsenal of innate immunity. These structures, fibers of decondensed nuclear material on which neutrophils localize their vast antimicrobial and proinflammatory stores, are released into sites of inflammation or injury with the presumed aim of constraining and clearing bacteria. It has also been shown, however, that NETs cause substantial harm, contributing to the pathogeneses of autoimmune diseases, cancers, and thrombotic disorders as well as inciting non-specific inflammation and collateral host damage. Thus, NETs as currently understood represent a paradox in which protection seems to be outweighed by detriment. In this light, fundamental questions have arisen surrounding the identity, function, and utility of NETs in vivo. This work describes two novel platforms rationally designed to assist in understanding and contextualizing this paradox. In the first approach, aimed at better understanding NET identity and function, a reductionist in vitro assay framework was iteratively developed to study NETs from the bottom up, beginning first with their DNA-histone fibrous substructure. Precise control of DNA-histone complexation yielded a robust, reproducible, and scalable structure that stood in stark contrast to low-yield and heterogeneous NET preparations. These structures, termed DNA-histone mesostructures (DHMs), mirrored both NET morphology and, to an extent, function. In doing so, DHMs provided a novel assay platform which elucidated the significant role of the isolated NET backbone in common NET-associated phenomena, such as bacterial trapping and immune activation. In addition, it permitted the confirmation and quantification of the role of the peptide LL-37 in altering NET degradation behavior. Beyond these structural studies, DHMs also yielded novel cell-based assays, including efforts to characterize the interaction between NET components and the immune system. Such studies elucidated the key role of DNA-histone synergism in NET-mediated immunostimulation, particularly amplified by the structural inclusion of non-methylated DNA. Additionally, they highlighted the importance of cell-structure proximity and contact in immune cell uptake and activation. In the second approach, aimed at addressing the perceived pathophysiological imbalance mediated by NETs, a nanoparticulate platform was leveraged to modify cell-derived NETs in vitro with the aim of ultimately modifying them in situ. The chosen nanoparticles, hollow nanocapsules composed of polysaccharide, were internalized into neutrophils but avoided immediate NET induction; instead, they primed neutrophils for enhanced NET production only after classical stimulation. NETs produced by nanocapsule-loaded neutrophils displayed colocalization between particles and NET fibers, thereby indicating successful modification of NETs and promise for future therapeutic engineering of these structures. Though distinct in motivation and design, these two platforms demonstrate novel approaches to understanding NETs and have revealed substantial insights about NET identity, function, and utility as described in this work. For both, the simultaneous youth and breadth of the NET field provide a profoundly large and diverse application base. Further studies leveraging both NET-mimicking in vitro assay platforms and NET-modifying nanoparticles will therefore continue to assist in the determination of both foundational and therapeutic NET biology.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/153522/1/clouttit_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/153522/2/clouttit_2.pdfDescription of clouttit_1.pdf : Restricted to UM users only.Description of clouttit_2.pdf : Restricted to UM users only