One trigger, two fates: hemolysate shapes neutrophil extracellular traps (NETs) formation in a context-dependent manner

Abstract

During systemic inflammation, neutrophil extracellular traps (NETs) capture pathogens but, in the long-term, can cause bystander damage to the vasculature. Another hallmark of sepsis in the blood is erythrocyte damage/alterations, often leading to hemolysis. Here, we report that hemolysate modulates NET formation, depending on the inducing agent. Whereas pharmacological agents, yeast components, and live Gram-positive bacteria either do not alter or somewhat enhance NET release by murine neutrophils in presence of hemolysate, it inhibits lipopolysaccharide (LPS)-induced NET formation. This inhibition depends on the number of erythrocytes undergoing hemolysis, the timing of LPS application, the neutrophil source (healthy/endotoxemic mice), and cell organization (2D/3D conditions). To verify the specificity of NET formation, Peptidyl Arginine Deiminase 4 (PAD4)-deficient mice were employed. To further elucidate the mechanisms of LPS action, separate hemolysate components were studied, namely hemoglobin, heme, and erythrocyte ghosts. Although all three contributed to NET downregulation, hemoglobin was the primary inhibitor of NETs. In line with this, the depletion of hemoglobin from the hemolysate reversed NET release, and in its presence (in hemolysate or pure), less LPS was available to neutrophils. LPS binding by hemoglobin was previously described, but it was reported to rather enhance its biological activity and amplify inflammatory signaling in some contexts. Therefore, we verified whether the process also occurs in vivo by intravital microscopy. During endotoxemia accompanied by hemolysis, both neutrophil infiltration and NET formation (relative to the cell number) were reduced. Taken together, our studies show that hemolysis diminishes Gram-negative bacteria-induced neutrophil activation and NET release.