Inhibition of caspase-1 or gasdermin-D Enable caspase-8 Activation in the Naip5/NLRC4/ASC Inflammasome

PLoS Pathog. 2017 Aug 3;13(8):e1006502. doi: 10.1371/journal.ppat.1006502. eCollection 2017 Aug.

Abstract

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / antagonists & inhibitors
  • Apoptosis Regulatory Proteins / immunology*
  • CARD Signaling Adaptor Proteins
  • Calcium-Binding Proteins
  • Caspase 1 / immunology*
  • Caspase 1 / metabolism
  • Caspase 8 / immunology*
  • Caspase 8 / metabolism
  • Disease Models, Animal
  • Enzyme Activation / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Gene Knockdown Techniques
  • Inflammasomes / immunology*
  • Inflammasomes / metabolism
  • Legionella pneumophila
  • Legionnaires' Disease / immunology*
  • Macrophages / microbiology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Neuronal Apoptosis-Inhibitory Protein
  • Real-Time Polymerase Chain Reaction

Substances

  • Apoptosis Regulatory Proteins
  • CARD Signaling Adaptor Proteins
  • Calcium-Binding Proteins
  • Gsdmd protein, mouse
  • Inflammasomes
  • Ipaf protein, mouse
  • Naip5 protein, mouse
  • Neuronal Apoptosis-Inhibitory Protein
  • Pycard protein, mouse
  • Caspase 8
  • Caspase 1

Grant support

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 2012/09363-6, 2013/08216-2 and 2014/04684-4), Conselho Nacional do Desenvolvimento Cientifico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). DSZ is a research fellow from CNPq, Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.