title: Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.
abstract: 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.
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Conos
authors.affiliation: Institute for Molecular Bioscience and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Kaiwen W
authors.identifier: 0000-0001-5903-3251
authors.initials: KW
authors.lastname: Chen
authors.affiliation: Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.|Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
authors.forename: Dominic
authors.identifier: 0000-0002-7045-4695
authors.initials: D
authors.lastname: De Nardo
authors.affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hara
authors.affiliation: Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.|Systems Biology & Personalised Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
authors.forename: Lachlan
authors.initials: L
authors.lastname: Whitehead
authors.affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Núñez
authors.affiliation: Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.|Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
authors.forename: Seth L
authors.initials: SL
authors.lastname: Masters
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.affiliation: Institute for Molecular Bioscience and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Kate
authors.initials: K
authors.lastname: Schroder
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: David L
authors.initials: DL
authors.lastname: Vaux
authors.affiliation: Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia; lawlor@wehi.edu.au lindqvist@wehi.edu.au vince@wehi.edu.au.|Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Lawlor
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; lawlor@wehi.edu.au lindqvist@wehi.edu.au vince@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: Lisa M
authors.identifier: 0000-0002-2421-2740
authors.initials: LM
authors.lastname: Lindqvist
authors.affiliation: Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia; lawlor@wehi.edu.au lindqvist@wehi.edu.au vince@wehi.edu.au.|Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
authors.forename: James E
authors.initials: JE
authors.lastname: Vince
mesh_terms: D000818:Animals; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000235:genetics / Q000378:metabolism*; D009336:Necrosis; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D058847:Inflammasomes; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse; D020170:Caspase 1
keywords: Gasdermin D; MLKL; NLRP3; interleukin-1β; necroptosis