Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome

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.

Macrophage priming is dispensable for NLRP3 inflammasome activation and restriction of Leishmania amazonensis replication

“…We acknowledge that addition of LPS to study inflammasome activation in macrophages infected with Leishmania does not mimic natural infection. However, priming isolated cells prior infection is a common strategy to boost gene regulation and to synchronize inflammasome activation upon infection with many microbes including parasites, bacteria, and viruses . It is important to highlight that, in vivo, Leishmania parasites activate the inflammasome without the requirement of priming or LPS injection .…”

“…However, priming isolated cells prior infection is a common strategy to boost gene regulation and to synchronize inflammasome activation upon infection with many microbes including parasites, bacteria, and viruses. 26,[45][46][47][48] It is important to highlight that, in vivo, Leishmania parasites activate the inflammasome without the requirement of priming or LPS injection. 25,27,29,49 We believe that this occur because of tissue damage caused during infection, the release of alarmins, such as ATP, IL-1 , and high mobility group box 1 (HMGB1), and also other inflammatory mediators such as IFN-and TNF-, which can boost NF-B signaling and are extremely important in the pathogenesis of Leishmania infections.…”

The intra‐ and extracellular functions of ASC specks

“…ASC specks also recruit caspase‐8, which is activated in the absence of caspase‐1 and GSDMD, thus representing a backup mechanism to trigger apoptosis in case pyroptosis is impaired . While it is clear that ASC can nucleate caspase‐8 filaments in vitro, it is not known how the selective autocatalytic activation of caspase‐8 in case of abrogated pyroptosis is achieved on the molecular level.…”

The NLRC4 Inflammasome

“…However, unlike CASP1, CASP8 is a pro‐apoptotic caspase. In cells in which CASP1 or Gasdermin are depleted, preventing pyroptosis, Salmonella ‐induced activation of NLRC4 will result in CASP8‐dependent apoptosis …”