Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.

Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1ß (IL-1ß) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1ß and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1ß and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

Cutting edge: Cytosolic bacterial DNA activates the inflammasome via Aim2.

Pathogens are detected by pattern recognition receptors that, upon activation, orchestrate an appropriate immune response. The TLRs and the nucleotide-binding oligomerization domain-like receptors (NLRs) are prototypic pattern recognition receptors that detect extracellular and cytosolic pathogens, respectively. Listeria monocytogenes has both extracellular and cytosolic phases and is detected in the cytosol by members of the NLR family. These include two NLR members, NLRC4 and NLRP3, that, upon detection of cytosolic L. monocytogenes, induce the assembly of the inflammasome. Inflammasomes serve as platforms for the activation of the protease caspase 1, which mediates the processing and secretion of pro-IL-1beta and pro-IL-18. We previously provided evidence that L. monocytogenes is also detected by a third inflammasome. We now use biochemical and genetic approaches to demonstrate that the third detector senses bacterial DNA and identify it as Aim2, a receptor that has previously been shown to detect viral DNA.

Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome.

The mammalian innate immune system uses Toll-like receptors (TLRs) and Nod-LRRs (NLRs) to detect microbial components during infection. Often these molecules work in concert; for example, the TLRs can stimulate the production of the proforms of the cytokines IL-1beta and IL-18, whereas certain NLRs trigger their subsequent proteolytic processing via caspase 1. Gram-negative bacteria use type III secretion systems (T3SS) to deliver virulence factors to the cytosol of host cells, where they modulate cell physiology to favor the pathogen. We show here that NLRC4/Ipaf detects the basal body rod component of the T3SS apparatus (rod protein) from S. typhimurium (PrgJ), Burkholderia pseudomallei (BsaK), Escherichia coli (EprJ and EscI), Shigella flexneri (MxiI), and Pseudomonas aeruginosa (PscI). These rod proteins share a sequence motif that is essential for detection by NLRC4; a similar motif is found in flagellin that is also detected by NLRC4. S. typhimurium has two T3SS: Salmonella pathogenicity island-1 (SPI1), which encodes the rod protein PrgJ, and SPI2, which encodes the rod protein SsaI. Although PrgJ is detected by NLRC4, SsaI is not, and this evasion is required for virulence in mice. The detection of a conserved component of the T3SS apparatus enables innate immune responses to virulent bacteria through a single pathway, a strategy that is divergent from that used by plants in which multiple NB-LRR proteins are used to detect T3SS effectors or their effects on cells. Furthermore, the specific detection of the virulence machinery permits the discrimination between pathogenic and nonpathogenic bacteria.

Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection.

Listeria monocytogenes escapes from the phagosome of macrophages and replicates within the cytosolic compartment. The macrophage responds to L. monocytogenes through detection pathways located on the cell surface (TLRs) and within the cytosol (Nod-like receptors) to promote inflammatory processes aimed at clearing the pathogen. Cytosolic L. monocytogenes activates caspase 1, resulting in post-translational processing of the cytokines IL-1beta and IL-18 as well as caspase 1-dependent cell death (pyroptosis). We demonstrate that the presence of L. monocytogenes within the cytosolic compartment induces caspase 1 activation through multiple Nod-like receptors, including Ipaf and Nalp3. Flagellin expression by cytosolic L. monocytogenes was detected through Ipaf in a dose-dependent manner. Concordantly, detection of flagellin promoted bacterial clearance in a murine infection model. Finally, we provide evidence that suggests cytosolic L. monocytogenes activates caspase 1 through a third pathway, which signals through the adaptor protein ASC. Thus, L. monocytogenes activates caspase 1 in macrophages via multiple pathways, all of which detect the presence of bacteria within the cytosol.

Pseudomonas aeruginosa activates caspase 1 through Ipaf.

The innate immune system encodes cytosolic Nod-like receptors (NLRs), several of which activate caspase 1 processing and IL-1beta and IL-18 secretion. Macrophages respond to Salmonella typhimurium infection by activating caspase 1 through the NLR Ipaf. This activation is mediated by cytosolic flagellin through the activity of the virulence-associated type III secretion system (T3SS). We demonstrate here that Pseudomonas aeruginosa activates caspase 1 and induces IL-1beta secretion in infected macrophages. While live, virulent P. aeruginosa activate IL-1beta secretion through caspase 1 and Ipaf, strains that have mutations in the T3SS or in flagellin did not. Ipaf-dependent caspase 1 activation could be recapitulated by delivering P. aeruginosa flagellin to the macrophage cytosol. We examined the role of Naip5 in P. aeruginosa-induced caspase 1 activation by using A/J (Naip5-deficient) compared with C57BL/6 and BALB/c (Naip5-sufficient) macrophages and observed that A/J macrophages secrete IL-1beta in response to P. aeruginosa, S. typhimurium, and Listeria monocytogenes infection, as well as in response to cytosolic flagellin, but at slightly reduced levels. Thus, Ipaf-dependent detection of cytosolic flagellin is a conserved mechanism by which macrophages detect the presence of pathogens that use T3SS.