Macrophage polarization modulates neutrophil extracellular trap dynamics and reciprocal macrophage responses

The interplay between neutrophils and macrophages is central to inflammatory regulation, yet how macrophage polarization governs the homeostasis of neutrophil extracellular traps (NETs) remains poorly understood. Here, we show that the macrophage polarizing environment is a critical determinant of NETs dynamics in human primary cell co-cultures. Using immunofluorescence, flow cytometry, kinetic extracellular DNA assays, and multiplex cytokine profiling, we demonstrate that IFN-γ/LPS (M1-polarized) conditions promote robust but transient NETs formation, accompanied by increased loss of neutrophil membrane integrity and amplified proinflammatory cytokine release. In contrast, IL-4/IL-13 (M2-polarized) conditions restrict the accumulation of NETs-associated extracellular DNA, shift neutrophil death toward an apoptotic rather than a membrane-compromised (lytic) profile, and maintain an attenuated inflammatory profile. Importantly, neutrophil co-culture reciprocally shifts macrophages toward M2-like phenotypes, an effect most pronounced in concurrently polarized (CP) macrophages and diminished once polarization has stabilized. These findings establish the polarizing environment as a bidirectional regulator of NETs homeostasis and neutrophil fate, with implications for understanding the balance between inflammatory amplification and resolution in neutrophil-rich microenvironments.