ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

Sci Rep. 2018 Feb 28;8(1):3788. doi: 10.1038/s41598-018-21998-3.

Abstract

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / antagonists & inhibitors
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Apoptosis*
  • CARD Signaling Adaptor Proteins / physiology*
  • CRISPR-Cas Systems
  • Calcium-Binding Proteins / antagonists & inhibitors
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Caspase 1 / physiology*
  • Caspase 8 / physiology*
  • Genome
  • Inflammasomes / metabolism*
  • Macrophages / metabolism
  • Macrophages / pathology*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout

Substances

  • Apoptosis Regulatory Proteins
  • CARD Signaling Adaptor Proteins
  • Calcium-Binding Proteins
  • Inflammasomes
  • Ipaf protein, mouse
  • Pycard protein, mouse
  • Casp8 protein, mouse
  • Caspase 8
  • Casp1 protein, mouse
  • Caspase 1