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 MLKLinduced 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 MLKLdependent diseases.aspase-dependent apoptotic cell death is required for mammalian development and the prevention of autoimmune and neoplastic diseases. Programmed cell death can also act to eliminate pathogen-infected cells, with recent studies highlighting how targeted apoptosis-inducing anticancer compounds can treat viral and intracellular bacterial infections (1, 2). On the other hand, the recently characterized caspase-independent necroptotic cell death pathway is dispensable for organism development but, like apoptosis, can be triggered to kill cells harboring pathogenic microbes (3). A number of studies have also reported how pathological activation of necroptotic signaling may contribute to diverse disease states, such as ischemia-reperfusion injury, atherosclerosis, and liver disease, presumably through cell death and the release of proinflammatory mediators (4).The execution of necroptosis is dependent on receptor interacting serine-threonine protein kinase 3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and MLKL's association with, and disruption of, plasma membrane integrity (5).In the absence of caspase activit...

Molecular mechanisms of inflammasome signaling

“…A link between the NLRP3 inflammasome and the necroptotic cell death pathway has emerged. In response to necroptotic activators, the kinase RIPK3 and the necroptotic effector MLKL are required to activate the NLRP3 inflammasome and induce IL‐1β release . In this case, MLKL oligomerizes on the cell membrane to induce pore formation, resulting in necroptosis and a reduction in the level of intracellular potassium triggering activation of the NLRP3 inflammasome .…”

“…In this case, MLKL oligomerizes on the cell membrane to induce pore formation, resulting in necroptosis and a reduction in the level of intracellular potassium triggering activation of the NLRP3 inflammasome . Of particular interest is that MLKL‐dependent secretion of IL‐1β does not rely on the pyroptosis executor gasdermin D, suggesting that IL‐1β might be released through MLKL‐induced pores instead. These studies further accentuate the complex interplay between the NLRP3 inflammasome and nonpyroptotic cell death pathways.…”

RIPK3 in cell death and inflammation: the good, the bad, and the ugly

“…The kinase activity of RIPK1 and RIPK3 is not required for this atypical non-apoptotic caspase-8 activation and in fact, treatment of cells with a RIPK3 kinase inhibitor enhances LPS-induced caspase-8 activation and thus IL-1β processing (101). Furthermore, recent studies have demonstrated an additional link between necroptosis in inflammasome activation, by showing that MLKL activation, which leads to membrane disruption, can activate the NLRP3 inflammasome in a cell-intrinsic manner by altering ion homeostasis (102, 103). This finding implies that induction of necroptosis is linked inextricably to inflammasome assembly, and that caspase-1 activation and processing of IL-1β and IL-18 likely accompany necroptotic cell death when all are present.…”

Inflammatory cell death in intestinal pathologies

“…RIPK3 is required within the hematopoietic cells to induce reparative programming through IL‐1β/IL‐23‐induced IL‐22 production . RIPK3 promotes inflammasome activation and IL‐1β release in cells of the innate immune system . Consistent with this innate cell‐intrinsic role of RIPK3, RHIM‐domain‐mediated cytokine production within the dendritic cell compartment (CD11c) and not the kinase activity (necroptosis) is required for protection against DSS‐induced damage .…”