Z-DNA Reveals a Conformational Dimension of Neutrophil Extracellular Trap Organization

Abstract

Neutrophil extracellular traps (NETs), with their networked extracellular DNA (eDNA), are key effectors of innate immunity which when ineffectively cleared can lead to immunopathologies. Here, we demonstrate intrinsic, host-derived left-handed noncanonical Z-DNA in NETs eDNA. Immunofluorescence imaging revealed Z-DNA forming extensive filaments tracing the non-Z-DNA backbone across diverse NETosis stimuli, while circular dichroism independently supported its presence. Temporal analysis revealed an early mitochondria-associated component with preferential ZBP1 association, followed by progressive accumulation within the eDNA network as NETs matured. Z-DNA accumulation was associated with progressive changes in NET architecture and topology, including network organization, branching, junctional complexity, filament thickness, and pore organization. Chemical perturbation of the Z:non-Z equilibrium was accompanied by NET architectural remodeling, associating DNA conformational state with higher-order scaffold organization. Z-DNA showed greater relative retention than non-Z-DNA following nuclease treatment, whereas chloroquine-mediated shifting toward a non-Z state reduced this preferential retention. Finally, Z-DNA enriched NET fractions induced greater IFN-alpha; production by human PBMCs than non-Z-DNA fractions. These findings identify Z-DNA as a dynamic component of NETs whose abundance and spatial organization are associated with NET architecture, preferential persistence after nuclease challenge, and the immunostimulatory activity of Z-DNA-enriched NET fractions.