Mlkl Pores Release Neutrophil Extracellular Traps in Necroptotic Neutrophils

Neutrophil extracellular traps (NETs) are networks of extracellular nuclear DNA and microbicidal proteins released from neutrophils in response to tissue damage and infection. Despite evidence of pathogenic roles for NETs in systemic lupus erythematosus, rheumatoid arthritis, diabetes, artherosclerosis and Alzheimer's disease, the major biochemical pathways controlling their formation remains poorly understood. Apoptosis does not contribute to NET formation but the role of regulated non-apoptotic cell death pathways such as necroptosis is not known. We have investigated the role of positive and negative regulators of necroptosis including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase domain-like (MLKL), receptor-interacting protein kinase-1 (RIPK1) and Caspase-8. Using immunogold electron microscopy, flow cytometry, imaging flow cytometry and fluorescence microscopy, we demonstrate that necroptosis can drive NET formation via MLKL pore formation at the cell surface. This process is caspase-independent but reactive oxygen species-dependent. Genetically-modified mouse peripheral blood and bone marrow neutrophils were used to show that Caspase-8 and RIPK1 negatively regulate NET formation driven by RIPK3 and MLKL. Mice that lack MLKL are deficient in necroptosis and NET formation, and were sensitive to methicillin-resistant Staphylococcus aureus (MRSA). Neutrophil-specific Caspase-8-deficiency also leads to increased susceptibility to MRSA due to increased rates of necroptotic neutrophil death. Killing of MRSA by necroptotic neutrophils is sensitive to DNase, and is dependent on MLKL, suggesting that necroptosis-driven NET formation contributes to the bactericidal activity of neutrophils. Human peripheral blood neutrophils also generate NETs that are sensitive to pharmacological inhibitors of necroptosis, suggesting that targeting necroptosis in general may help combat autoimmune responses to DNA. This study provides a framework to investigate the role of extracellular DNA release and cell death in the setting of infection, autoimmunity and autoinflammatory disease.