Cytokine-induced chromatin accessibility in whole blood neutrophils links to sepsis transcriptional states

Background Neutrophils play an important role in the immune system by sensing environmental perturbations, including pathogens. Upon activation, neutrophils extrude their chromatin to form neutrophil extracellular traps (NETs), which trap and remove pathogens. Chromatin decondensation during NET formation is a regulated process that reflects both the inducing pathways and the cellular environment. However, most studies rely on non-physiological stimuli like phorbol 12-myristate 13-acetate (PMA), which bypass key regulatory mechanisms. As a result, how physiologically relevant inflammatory signals change neutrophil chromatin accessibility and relate to disease associated transcriptional states remains poorly understood. Methods We used the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) to profile chromatin accessibility in neutrophils stimulated in whole blood with PMA and physiologically relevant inflammatory natural factors (NFs), including TNF-α, GM-CSF, fMLP, C5a, and IL-1β, alone and in combination. Chromatin responses were compared across conditions and integrated with publicly available transcriptomic sepsis cohorts. Results NF stimulation induced stimulus specific chromatin accessibility programs distinct from PMA. Individual NFs enriched specific transcription factor (TF) motif enrichments in a stimulus dependent manner, with GM-CSF associated with STAT motifs, TNF-α with NF-κB, and C5a/fMLP with AP-1, while the combined condition showed a cooperative response including CEBP. Integration with sepsis transcriptomic datasets revealed that promoter accessibility changes under NF stimulation corresponded to transcriptional states associated with disease severity, highlighting the upstream regulatory programs linked to clinical outcomes. Conclusions These findings demonstrate that NF stimulation in whole blood reveals chromatin accessibility programs in neutrophils that correlate with disease severity in sepsis. This approach provides a framework for linking cytokine driven neutrophil regulation to heterogenous inflammatory states in sepsis and other NET-associated diseases.