Innate Immune Function of Neutrophil Cytoplasts Generated Post-Vital NETosis

Abstract The migration of polymorphonuclear neutrophils (PMN) into the site of infection such as lungs during pneumonia is a canonical feature of innate immunity. Formation of neutrophil-derived extracellular traps (NETs), web-like strands of varying lengths comprising DNA, histones, elastase, and myeloperoxidase, is an important determinant of PMN-mediated innate immunity. NETs form in microvessels, entrap bacteria and effete matter, and dampen PMN-mediated inflammatory injury at specific sites. However, studies have largely focused on NET release secondary to lytic NETosis and lysis of PMN. Far less is known about vital NETosis occurring in the absence of PMN rupture. As vital NETosis is characterized by generation of anuclear PMN termed cytoplasts (PMN cyto ), we addressed the function of PMN cyto as a critical determinant of PMN-mediated innate immunity. Studies were made in mice challenged with live Pseudomonas aeruginosa (PA) i.t. to induce fulminant pneumonia characterized by tissue injury in which we determined the role of generated PMN cyto population. Using Tomato Red (tDTomato) transgenic mice to mark PMN, we observed PA pneumonia induced PMN transmigration leading to PMN cyto generation in the airspace. In contrast, PMN cyto transmigration, was minimal. PMN cyto accumulating in lung tissue actively phagocytosed and killed PA. Instillation of ex vivo generated PMN cyto also prevented PA-induced inflammatory lung injury and reduced mortality as compared to control mice. We demonstrated that the salutary effects of PMN cyto required functional microchondria. Proteomic analysis revealed that PMN cyto retained bactericidal and ROS generating pathways, consistent with an intact plasma membrane. Genetic deletion of peptidyl arginine deaminase 4 (PAD4), which mediates histone citrullination and promotes NETosis, facilitates PMN cyto generation and thereby abrogated pneumonia- induced mortality. Thus, we have identified the crucial host defense function of PMN cyto generated post-vital NETosis, suggesting that PMN cyto hold promise as cell based anti-bacterial therapy in pneumonia.