Novel nanofibrous DNA-histone structures mediate reproducible, high-throughput analyses of neutrophil extracellular traps and their effects in vitro

Abstract

Abstract Termed the “double-edged swords of innate immunity” by Kaplan and Radic, neutrophil extracellular traps (NETs) are vital contributors to innate defenses but also participate in the pathogeneses of manifold diseases including systemic lupus erythematosus, rheumatoid arthritis, thrombosis, and cancer. Despite their vast clinical impact, NETs have proven difficult to study in high quantity and isolation; indeed, over 1.9E9 neutrophils are required for full NET coverage of a 96-well plate. Our group has developed a novel NET-mimicking structure for adaptable and reproducible high-throughput analyses of NETs in vitro. Rooted in a base composition of DNA and histones interacted by slow diffusion across an aqueous two phase system, these structures exhibit the same 10–100 nm diameter fibers as their physiological counterparts and can incorporate additional NET-associated proteins and peptides. This platform therefore enables unprecedented high-throughput probes of the roles of individual NET components in health and disease. In a pilot study exploiting this opportunity, our group expanded upon the previously-reported protection of NETs from nuclease-mediated degradation by the peptide LL-37. The comparison between LL-37-containing structures and control structures without the peptide, a data point unavailable endogenously, revealed a 4.5-fold increase in structural half-life conferred by LL-37 after exposure to DNase I. Similar ongoing experiments are exploring the lupus-linked activation of plasmacytoid dendritic cells by NETs. In this way, the reported platform provides new investigative angles for NET studies while also conferring a reproducibility and statistical power unattainable with cell-based methods.