The innate immune system possesses potent antimicrobial activities, and successful pathogens must evade these defense mechanisms. For example, S. typhimurium
avoids multiple innate immune mechanisms, including lipocalin24
, antimicrobial peptides25
, antigen presentation26
, T cell activation26
, and as we now show, pyroptosis. An understanding of the evasion strategies used by pathogens can be used to probe the relative importance of specific immune mechanisms. In this study, we have deprived S. typhimurium
of its NLRC4 evasion strategy, and this has revealed a number of novel insights into innate immune responses to pathogens in vivo
Our first surprise was the observation that the vast majority of caspase-1-dependent clearance of flagellin expressing S. typhimurium
, L. pneumophila
and B. thailandensis
was independent of IL-1β and/or IL-18. This finding led us to investigate the cytokine-independent mechanisms by which caspase-1 could promote bacterial clearance. In the case of S. typhimurium
persistently-expressing flagellin, caspase-1-dependent control of infection occurs via pyroptosis-mediated bacterial release followed by neutrophil-dependent killing. Casp1−/−
mice are more susceptible to infection with Francisella tularensis
and the use of depleting antibodies to IL-1β and IL-18 only partially recapitulates the phenotype27
. It will be interesting to investigate the role of pyroptosis in this and other infectious model systems.
Different forms of cell death can be categorized in several ways, including programmed or accidental, caspase-dependent or -independent, and inflammatory or non-inflammatory28
. Pyroptotic cell death is programmed, caspase-1-dependent, and pro-inflammatory9
. Apoptosis is also programmed, but in contrast to pyroptosis, it is caspase 3 and 7 dependent and non-inflammatory. Apoptotic cell death results in the orderly degradation and clearance of cellular contents whereas pyroptosis, like necrosis, releases cellular contents into the extracellular environment. The role of pyroptosis in vivo
has been unclear. It was initially proposed to be a pathogenic mechanism used by bacteria to destroy host immune cells29
. However, our data suggests that pyroptosis can be a host defense mechanism used to clear intracellular pathogens.
Several other inflammasome activators in addition to NLRC4 have been shown to induce pyroptosis in vitro
and in each case, it is easy to envisage how this might promote pathogen clearance. For example, AIM2 detects cytosolic DNA released by viruses30
and by lysed L. monocytogenes
or F. tularensis31–38
, and resulting pyroptosis could prevent replication in macrophages and dendritic cells. Similarly, NLRP3 responds to intracellular bacterial pathogens, including Listeria monocytogenes39
, as well as to several viruses; again NLRP3-dependent pyroptosis of their host cells could limit their replication. NLRP3 also responds to pore-forming toxins, including those expressed by Staphylococcus aureus40, 41
, and, in this case, pyroptosis could remove the intoxicated cell.
The inflammasome is also activated by non-infectious stimuli including monosodium urate, asbestos and silica crystals. In these cases pyroptosis may cause pathology by releasing inflammatory mediators from the cell, and this would be significantly exacerbated by cyclical uptake and release of these indigestible crystals. We observe evidence for pyroptosis-induced tissue damage in Ncf1−/−mice infected with ST-FliCON. Or data suggest that in these mice ST-FliCON cause repeated cycles of pyroptosis and that this results in disruption of splenic architecture and extensive tissue necrosis. In contrast, WT mice rapidly clear ST-FliCON through pyroptosis without disruption of normal splenic architecture.
Greater numbers of ST-FliCON are recovered from Ncf1−/−mice than ST-WT. Further, we observed that the ST-FliCON bacteria move from the macrophage to neutrophil compartments. These data suggest that, in the absence of ROS, ST-FliCON actually replicate within neutrophils, which lack NLRC4 and do not undergo pyroptosis, opening the possibility that replication within ROS-deficient neutrophils is more efficient than replication in macrophages. Alternately, ST-FliCON may also be replicating in the extracellular space after pyroptotic release, while ST-WT remain relatively more restricted to the macrophage compartment.
Our data indicate that WT S. typhimurium
efficiently evade NLRC4 and caspase-1 during systemic infection, resulting in the lethal infection in WT mice. Casp1−/−
mice have modestly increased susceptibility to infection with WT S. typhimurium
suggesting that this evasion may not be complete during every stage of the infection13, 14
(Supplementary Fig. 1c, 1d
). This detection of WT S. typhimurium
does not prevent lethality, rather it delays it; WT mice still succumb to infection with WT S. typhimurium
despite the presence of functional caspase-113, 14
. Caspase-1 activation in vivo
by WT S. typhimurium
occurs via both NLRC4 and NLRP342
, and the delayed mortality is mediated predominantly by IL-18 with a smaller contribution from IL-1β14
. While the molecular mechanisms of NLRP3 detection remain to be elucidated, detection of WT S. typhimurium
by NLRC4 may arise from the delivery of residual flagellin and PrgJ rod protein present in bacteria located at or just beyond the epithelial barrier in the intestine during the early hours of the infection. Once the bacteria have replicated within host cells, more complete repression of flagellin and PrgJ expression could result in complete evasion or NLRC4 during the systemic phase of the infection.
Our results suggest that caspase-1 activation by WT S. typhimurium
is relatively weak or transitory, resulting in IL-1β and IL-18 secretion and a mild reduction in bacterial load that does not prevent mortality. In contrast, persistent and strong caspase-1 activation by ST-FliCON
during the systemic phase of infection enables a dramatic reduction in bacterial counts, resulting in an inability to cause a lethal infection. ST-
have a major reduction in their virulence, with competitive index values similar SPI2 T3SS mutant and more severe than PhoP mutant S. typhimurium43
. Thus, while IL-1β and IL-18 confer a slight delay in mortality after WT S. typhimurium
infection, WT S. typhimurium
evade pyroptosis, an innate immune effector mechanism that would otherwise confer complete protection to the host.
In summary, we report two major findings. First, we demonstrate that caspase-1 can clear intracellular pathogens independent of its two primary cytokine substrates, IL-1β and IL-18. Second, we show that caspase-1 mediates bacterial clearance via an efficient mechanism initiated by pyroptotic cell death. The relative contributions of IL-1β, IL-18, and pyroptosis to clearing a specific pathogen will likely be different between pathogens, depending on the virulence strategy used and cell types targeted. Further characterization of the role of pyroptosis in vivo may lead to novel therapies for both infectious and inflammatory diseases.