Cell intrinsic MLKL activation of the NLRP3 inflammasome, not necroptotic cell lysis, induces bystander cell inflammatory signaling

S Conos, D Vaux, J Murphy, K Lawlor, L Lindqvist and J Vince

The Walter and Eliza Hall Institute of Medical Research

Necroptosis is a caspase-independent programmed cell death pathway mediated by Receptor Interacting Protein Kinase-3 (RIPK3) phosphorylation of Mixed Lineage Kinase Domain-Like (MLKL), and MLKL-dependent plasma membrane damage. The lytic nature of necroptotic death, and potential release of pro-inflammatory mediators, is thought to drive inflammation in necroptotic disease models. However, we previously documented that MLKL signaling also activates the NOD-like receptor protein 3 (NLRP3) inflammasome to trigger caspase-1 processing and maturation of the potent pro-inflammatory cytokine IL-1β. We now define the key requirements for MLKL-induced NLRP3 inflammasome activation including, i) critical MLKL domains and residues, ii) the role of MLKL oligomerisation and membrane translocation and iii) the dependence on potassium efflux. Further, we use a series of gene deficient mice and pharmacological targeting of NLRP3 to genetically and biochemically demonstrate that MLKL-killing and cellular rupture can be separated from MLKL-induced NLRP3 signalling. Finally, we show that MLKL-dependent NLRP3 inflammasome activity, not necroptotic induced cellular rupture and DAMP release, drives inflammatory signalling in healthy bystander cells. Collectively, our study emphasizes the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.