Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner

Proc Natl Acad Sci U S A. 2017 Feb 7;114(6):E961-E969. doi: 10.1073/pnas.1613305114. Epub 2017 Jan 17.


Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Keywords: Gasdermin D; MLKL; NLRP3; interleukin-1β; necroptosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism
  • Caspase 1 / genetics
  • Caspase 1 / metabolism
  • Cell Line, Tumor
  • Humans
  • Inflammasomes / metabolism*
  • Interleukin-1beta / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages / drug effects
  • Macrophages / metabolism*
  • Mice
  • Mice, Knockout
  • NLR Family, Pyrin Domain-Containing 3 Protein / genetics
  • NLR Family, Pyrin Domain-Containing 3 Protein / metabolism*
  • Necrosis
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Multimerization / drug effects
  • Receptor-Interacting Protein Serine-Threonine Kinases / genetics
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism


  • Inflammasomes
  • Interleukin-1beta
  • Lipopolysaccharides
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • MLKL protein, mouse
  • Protein Kinases
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk3 protein, mouse
  • Caspase 1