Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality

Abstract

Neutrophil extracellular traps (NETs) are chromatin structures loaded with antimicrobial molecules. They can trap and kill various bacterial, fungal and protozoal pathogens, and their release is one of the first lines of defense against pathogens. In vivo, NETs are released during a form of pathogen-induced cell death, which was recently named NETosis. Ex vivo, both dead and viable neutrophils can be stimulated to release NETs composed of either nuclear or mitochondrial chromatin, respectively. In certain pathological conditions, NETs are associated with severe tissue damage or certain auto-immune diseases. This review describes the recent progress made in the identification of the mechanisms involved in NETosis and discusses its interplay with autophagy and apoptosis.

Figure 1

Live cell imaging of subcellular events during ex vivo neutrophil apoptosis and NETosis. (a and c) Cells were incubated with Alexa488-AnnexinV (green) and the cell-impermeable DNA dye propidium iodide (red), (b and d) or with the cell impermeable DNA dye Hoechst (blue), the mitochondrial membrane potential marker TMRM (red) and the cell impermeable DNA dye, propidium iodide (green). Constitutive neutrophil apoptosis is characterized by membrane blebbing, PS exposure (green) and condensation of nuclear chromatin (blue); cells finally undergo secondary necrosis. During induced NETosis, cells display massive vacuolization and decondensation of nuclear chromatin (blue), and PS is not exposed (green) before NET formation (green). Scale bars indicate 10 μm. More information about the visualization of NETs can be found on the following link: http://www.jove.com/index/details.stp?ID=1724

Figure 2

Model for the regulation of NETosis. Bacterial DAMPs, such as LPS, are known triggers of autophagy in many cell lines, including neutrophils. However, purified LPS does not activate NADPH oxidase directly, but only sensitizes for a more powerful NADPH oxidase-derived oxidative burst induced by a subsequent trigger of NADPH oxidase. Formylated peptides are potent triggers of NADPH oxidase. Consequently, fMLP activates NADPH oxidase as well as Histone H3 citrullination. However, fMLP does not induce NETosis. The fMLP induces signaling through Akt/PI3K as well. This cascade PI3K/Akt/mTOR may inhibit autophagy and thereby prevent NETosis. Both ROS production and Histone H3 citrullination are insufficient to mediate the collapse of the nuclear membrane. Accordingly, H₂O₂ only accelerates neutrophil apoptosis. Induction of autophagy without NADPH oxidase activity, which is observed in CGD neutrophils, also results in enhanced apoptosis with no signs of NETosis. However, ROS (which are sufficient to induce consequent histone H3 citrullination) in combination with induced autophagy leads to chromatin decondensation and collapse of the nuclear membrane and prevents the activity of executioner caspases. This Figure was produced using Servier Medical Art: www.servier.com