Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.

Necroptosis is a form of programmed necrosis that is mediated by signaling complexes containing the receptor-interacting protein 3 (RIP3) and RIP1 kinases. We show that RIP3 and its interaction with the herpes simplex virus type 1 (HSV-1) protein ICP6 triggers necroptosis in infected mouse cells and limits viral propagation in mice. ICP6 interacts with RIP1/RIP3 through its RHIM domain and forms dimers/oliogmers by its C-terminal R1 domain. These binding events result in RIP1-RIP3 hetero- and RIP3-RIP3 homo-interactions and subsequent necroptosis of HSV-1-infected mouse cells. However, ICP6 RHIM cannot trigger necroptosis and even inhibits TNF-induced necroptosis in human cells. As the RHIM domain in murine cytomegalovirus protein vIRA can inhibit necroptosis in both human and mouse cells, these data suggest that both viral and host RHIM sequences determine whether the virus-host RHIM interaction is pro- or anti-necroptotic and that some viruses may evolve to escape this restriction.

Background: Receptor interacting protein 3 (RIP3)-mixed lineage kinase domain-like (MLKL) interaction is essential for necroptosis. Results: Murine RIP3 does not interact with human MLKL and vice versa due to sequence differences in and around the RIP3 phosphorylation sites. Conclusion: Different sequences in human and mouse RIP3 control the functionally conserved RIP3-MLKL interaction. Significance: This study provided new insights into the function of RIP3-MLKL interaction in necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1bd/d) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1bd/d mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.