Research ArticleCell death

The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils

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Science Signaling  04 Sep 2018:
Vol. 11, Issue 546, eaao1716
DOI: 10.1126/scisignal.aao1716

Necroptosis NET infection

Neutrophils are innate immune cells that can excrete chromatin upon activation to form neutrophil extracellular traps (NETs). Important for the control of bacterial infections and associated with autoimmunity, NET formation (NETosis) can cause neutrophil cell death. In contrast to apoptosis, necroptosis is a caspase-independent form of cell death. Using transmission electron microscopy and flow cytometry, D’Cruz et al. found that pharmacologic activation of necroptosis, but not apoptosis, stimulated neutrophil NET formation. Inhibition of RIPK1 kinase activity or loss of the necroptosis effector MLKL prevented NETosis and exacerbated methicillin-resistant Staphylococcus aureus (MRSA) infection in mice. These data identify necroptosis as a critical pathway that stimulates NETosis necessary for host defense.


Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain–like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase–independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8–dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8–dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

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