Atteberry, B. | Search bioRxiv

bioRxiv2025

Chromatin Changes Associated with Neutrophil Extracellular Trap (NET) Formation in Whole Blood Reflect Complex Immune Signaling

BackgroundNeutrophils are key players in innate immunity, forming neutrophil extracellular traps (NETs) to defend against infections. However, excess NET formation is implicated in inflammatory conditions such as sepsis and immunothrombosis. Studying NET formation in isolated neutrophils provides important mechanistic insights but does not reflect the complexity of immune interactions in whole blood, limiting our understanding of neutrophil responses. MethodsThis study investigates chromatin accessibility changes using Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) during phorbol 12-myristate 13-acetate (PMA) induced NET formation in whole blood. We compared chromatin accessibility patterns in neutrophils following PMA treatment in isolation and whole blood to assess the impact of other immune cells and signaling environment. ResultsWhole blood PMA stimulation elicited consistent chromatin accessibility changes across donors, demonstrating organized chromatin decondensation during NET formation. The chromatin response was characterized by increased accessibility in genomic regions enriched for immune-specific pathways, highlighting the role of immune cell interactions in NET formation. Differentially accessible regions (DARs) present following PMA induction in whole blood and isolated neutrophils showed greater association with NET-related and inflammatory transcription factors, while DARs specific to isolated neutrophils showed fewer relevant motifs. Pathway analysis indicated that whole blood responses involved more robust activation of immune-specific pathways, such as interleukin and cytokine signaling, compared to isolated neutrophils. ConclusionsOur findings underscore the importance of studying NET formation within a whole blood environment to capture the complexity of neutrophil responses and immune cell interactions. This understanding is crucial for identifying effective therapeutic targets in NET-associated inflammatory diseases.

immunology↗

bioRxiv2024

Understanding Complex Chromatin Dynamics of Primary Human Neutrophils During PMA Induced NETosis

BackgroundPrimary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases such as sepsis, systemic lupus erythematosus, rheumatoid arthritis, and others. MethodsThe study presented here investigates chromatin dynamics during NETosis by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response. ResultsWe find that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 minutes, with many continuing across the 90 minutes assessed in this study. Regulatory regions gaining accessibility are associated with activity of pathways that have been implicated in NOX-dependent NET formation. ConclusionsOur findings enhance the understanding of the chromatin changes underlying NETosis and also identify potential early-acting targets for modulating this process in inflammatory diseases.

immunology↗