PB0878 - Physiologically Relevant NETosis Induction Reveals Distinct Triggers and Chromatin Changes

Background: NETosis is a critical part of the innate immune system by which pathogens are inactivated, degraded and removed from circulation. NETosis culminates in the extrusion of chromatin to form Neutrophil Extracellular Traps (NETs) that can span dozens of nanometers in diameter. As a result, when dysregulated, NETs can be pathophysiological, leading to thrombosis and organ damage. Furthermore, nucleosomes within chromatin are complexes containing positively charged histone proteins and negatively charged DNA. These opposing forces make nucleosomes very stable, however when decoupled can cause damage to surrounding cells. Much of our understanding of NETs is based on studies of isolated neutrophils or immortalized neutrophil-like cells, and while these models have helped understand the basic biology of NETosis, they do so in a non-physiologically relevant context and often with non-biological stimuli like Phorbol 12-myristate 13-acetate (PMA), which bypasses regulatory mechanisms.

Aims: Our goal was to assess the mechanisms underlying NETosis induction in a physiologically relevant context, specifically comparing whole blood and isolated neutrophils and induction with PMA vs. molecules associated with NETosis related diseases including LT-a, C5a and fMLP.

Methods: We measured NETosis using Sytox Green and chromatin accessibility with ATAC-seq.

Results: Interestingly, we find that while these factors are able to induce NETosis in whole blood, but not isolated neutrophils, suggesting that a critical molecule or cell type may be present in circulation that supports NETosis induction with a minimal pool of physiological factors. Furthermore, we found distinct chromatin accessibility changes preceding NET formation following PMA induction in whole blood compared to isolated neutrophils, suggesting that chromatin decondensation and extrusion signaling mechanism(s) may be different in whole blood compared to isolated neutrophils.

Conclusion(s): These findings open the possibility that using more physiologically relevant NETosis models may lead to better understanding of the regulatory mechanisms governing NETosis in vivo and the potential for new therapeutic interventions.