Recent studies have shown that Nalp1b, a member of the Nalp family of proteins involved in inflammasome assembly and caspase-1 activation, controls macrophage sensitivity to LT. Macrophages from caspase-1 knockout mice were not susceptible to LT, even when carrying the sensitivity-conferring Nalp1b
gene, showing that caspase-1 is required for LTmediated cell death (Boyden and Dietrich, 2006
). Previous studies investigating the potential role of caspases in macrophage death were confined to the use of caspase inhibitors, with such studies reporting either no protection from LT (Kassam et al., 2005
; Tang and Leppla, 1999
) or a reduction in LT toxicity (Kirby, 2004
; Popov et al., 2002
). Our studies with caspase inhibitors indicated that only the pan-caspase inhibitor Z-VAD-FMK was able to protect against LT, despite equivalent LT-mediated autocatalytic activation of caspase-1 in the presence of each tested inhibitor. The differences in inhibitor effects is likely due to differences in the efficiency of their inhibition of activated caspase-1, as reflected by the generation of different levels of mature IL-1β. The inhibitors' ability to prevent IL-1β maturation in response to a classic inflammasome-inducing treatment (LPS/nigericin) was similar to that observed with LT treatment (data not shown). Since we also show that LT-mediated caspase-1 activation is a very late event which requires proteasome activity and ion fluxes induced by toxin, it is possible that the more effective inhibitors are actually inhibiting a number of cellular proteases, some of which may be involved in earlier steps of the toxic process.
Compared to the cleavage of MEK proteins (beginning at 20 min), caspase-1 activation is a late LT-induced event (50-60 min), dependent on potassium efflux and proteasome activity. Following LT-induced caspase-1 activation, IL-1β and IL-18 are processed intracellularly. However, we found the release of these cytokines to be a passive late event, resulting from cell death and lysis. Moreover, the concurrent presence of equal levels of both the pro- and mature forms of these cytokines in the supernatant confirms that they are being released from dying cells and not from intact cells. Thus, the release of these cytokines is not required for macrophage death.
Caspase-1 activation alone is insufficient for LT-mediated macrophage death because nigericin-induced Nalp3 inflammasome formation did not sensitize resistant macrophages to LT. Although potassium efflux is the common trigger for formation of the LT- and nigericin-induced inflammasomes (Petrilli et al., 2007
), the LT-induced inflammasome is dependent on proteasome activity while the nigericin-induced Nalp3-inflammasome is not. Moreover, while both inflammasomes similarly activate caspase-1, the downstream effects of this activation may depend on the stimulus and the particular Nalp protein present in the inflammasome, with only the LT-induced inflammasome leading to cell death. Therefore, while the inability of LPS/nigericin to sensitize cells to LT confirms that caspase-1 activation alone is not sufficient for sensitization to LT, it is clear that LT-mediated death requires caspase-1 activation in conjunction with Nalp1b-mediated events.
The role of Nalp1b in LT-mediated macrophage killing is unclear. The actual protein components of the LT-induced inflammasome have yet to be identified, and while it has been reported that LT triggers formation of a Nalp1b inflammasome, no reports have explicitly shown that the Nalp1b inflammasome to be the sole LT-induced inflammasome. The lack of caspase-1 activation in macrophages harboring resistant Nalp1b
alleles (Boyden and Dietrich, 2006
) is used as evidence that LT specifically activates a Nalp1b-specific inflammasome in LT-sensitive cells. The absence of caspase-1 activation in resistant macrophages, however, could possibly be attributed to the parallel absence of ion fluxes as the necessary signaling event for inflammasome formation. Therefore, although Nalp1b may indeed be a required component of the LT inflammasome, additional Nalp proteins may also be activated in response to LT-induced ion fluxes. Furthermore, Nalp1b could play a role in LT-mediated cytotoxicity events upstream of LT-induced ion fluxes since expressing the sensitive Nalp1b
allele in resistant macrophages is sufficient to sensitize cells to LT-mediated killing (Boyden and Dietrich, 2006
). The crucial LT-induced early events which lead to the ion fluxes and subsequent inflammasome formation remain unknown and may include the degradation of protein(s) by the proteasome, the cleavage of yet unidentified LF substrates or downstream effects of MEK cleavage which differ between resistant and sensitive macrophages. In this model, inflammasome formation and caspase-1 activation function secondarily in LT-mediated killing as essential required sequelae of the early events that induce potassium release ().
A model of LT-induced macrophage death
Following caspase-1 activation by Nalp1b and/or other Nalp family proteins, the mechanism of the caspase-1-dependent cell death induced by LT is unknown. Unlike other proapoptotic caspases, caspase-1 is primarily associated with inflammation and rarely linked to apoptosis. Nevertheless, caspase-1 has been previously implicated in some cell death studies. Overexpression of caspase-1 in fibroblasts has been shown to induce apoptosis (Miura et al., 1993
). Other bacterial pathogens, including Salmonella
(Brennan and Cookson, 2000
; Hersh et al., 1999
; Monack et al., 2001
; van der Velden et al., 2003
), S. flexneri
(Chen et al., 1996
; Edgeworth et al., 2002
; Hilbi et al., 1998
; Hilbi et al., 1997
), Burkholderia pseudomallei
(Sun et al., 2005
), Francisella tularensis
(Mariathasan et al., 2005
), and Actinobacillus actinomycetemcomitans
(Nonaka et al., 2001
), have been reported to induce a novel form of cell death via a caspase-1-dependent mechanism. Matching our observations, a number of these studies (Hersh et al., 1999
; Sun et al., 2005
; van der Velden et al., 2003
) reported partial protection from the pathogens using caspase inhibitors and more complete protection in caspase-1-deficient macrophages. Also agreeing with our conclusion that IL-1β is not involved in LTmediated cytotoxicity, the caspase-1-dependent cell deaths associated with Salmonella
(Monack et al., 2001
), F. tularensis
(Mariathasan et al., 2005
), and S. flexneri
(Chen et al., 1996
) have been shown to be IL-1β independent by using IL-1β deficient macrophages, neutralizing antibodies to IL-1β, or IL-1β receptor antagonists, respectively.
Pyroptosis is a recently introduced term to describe the caspase-1-mediated macrophage death associated with these infections (Fink and Cookson, 2005
). The mechanism of cell death during pyroptosis is not well defined, but the pathway is characterized by the formation of plasma membrane pores, and it has been reported that in Salmonella
infection, this pore formation is dependent on caspase-1 (Fink and Cookson, 2006
). It is possible that the important events mediated by caspase-1 in these other bacterial infections have similarities to those seen with LT treatment.
In summary, the late timing of LT-mediated inflammasome formation along with the requirement of ion fluxes for its assembly suggests that caspase-1 does not initiate macrophage death. However, caspase-1 is essential to cell death by participating in a step that follows the early LT-mediated events that instigate potassium efflux. LT-induced death appears to be dependent on a unique proteasome-dependent caspase-1 activation induced specifically by Nalp1b. The particular step at which Nalp1b is required remains unknown. Future work will focus on identifying the early LT events that initiate ion fluxes, proteins that are degraded by the proteasome in response to LT treatment, cellular proteases as well as caspase-1 substrates that may be required for macrophage death and the role of Nalp1b in cell death.