The NLRP3 inflammasome is a critical nexus mediating IL-1β and IL-18 responses to pathogens and innate immune stimuli. Because inflammasome stimuli are diverse and often unrelated, discerning the mechanism of NLRP3 activation has been elusive. Recent evidence has begun to indicate mt as key players in NLRP3 inflammasome signaling (Nakahira et al., 2010
; Zhou et al., 2010
). During our analysis of CP
infection, we serendipitously uncovered evidence that mt might play an important role in activating the NLRP3 inflammasome. In this report, we have provided a mechanistic explanation for NLRP3 activation, where mt sense cellular danger that results in apoptosis, during which oxidized mtDNA is released into the cytosol and binds to NLRP3. This cascade of events resulted in activation of the NLRP3 inflammasome and caspase-1 maturation.
The identification of mt as key players in NLRP3 inflammasome induction places apoptosis at the epicenter of this important process. As both sensors and robust sources of ROS, mt rapidly respond by releasing Cyt-c and inducing apoptosis (Ott et al., 2007
). Moreover, cathepsins and ROS released during lysosomal rupture can also profoundly impact mt membrane integrity, causing membrane permeabilization and subsequent initiation of apoptosis (Boya et al., 2003
; Ferri and Kroemer, 2001
). The mt K+
cycle, inexorably linked to intracellular K+
concentration, is obligatorily central to maintaining mt volume, controlling metabolic ROS (Garlid and Paucek, 2003
) and inducing apoptosis (Park and Kim, 2002
). We have shown that, while capable of blocking IL-1β secretion in response to NLRP3 triggers, inhibiting K+
efflux using high concentrations of extracellular K+
exerted profound effects on mt, and mitigated loss of ΔΨm
in response to NLRP3 activators. Indeed, it has often been overlooked that modulating cell surface K+
channels inevitably results in altered activation of mitochondrial K+
channels. But how do mt activate the NLRP3 inflammasome?
Our data demonstrated that NLRP3 triggers, such as alum, ATP, NIG, and live CP cause mt dysfunction and cell death in macrophages. We have also shown that STS, a pro-apoptotic compound, was sufficient to act as a second signal for NLRP3 activation. Moreover, LPS-primed macrophages treated with NLRP3 activators secreted less IL-1β, but similar amounts of TNF-α in presence of CsA. Apoptosis was critical for NLRP3 inflammasome induction, because overexpression of anti-apoptotic Bcl2 attenuated IL-1β secretion by LPS-primed macrophages, and Bcl2 silencing resulted in the converse. St type III secretion mutants unable to induce apoptosis did not promote IL-1β secretion, and Bcl2 overexpression inhibited wild-type St infection-induced IL-1β secretion. Given that the physiological triggers of NLRP3 are inextricably linked to mt and apoptosis, and that a causal relationship exists between pro-apoptotic signals and NLRP3, it seems that apoptosis is an indispensable step in NLRP3 inflammasome activation. While apoptosis is often portrayed as ‘silent’ cell death, our data suggest that in the presence of proinflammatory signal 1, the apoptotic machinery activates the NLRP3 inflammasome. Thus, apoptosis can activate the NLRP3 inflammasome, but remains a silent death unless in the context of signal 1.
While the role of Bcl-2 in apoptosis is well-appreciated, it has been suggested that Bcl-2 may more directly activate the inflammasome. For example, Bcl-2 can directly bind NLRP1 and inhibit NLRP1 activation (Bruey et al., 2007
; Faustin et al., 2009
). These data raise the possibility of alternative pathways by which Bcl-2 might inhibit NLRP3 activation. However, these investigators did find that Bcl-2 does not bind NLRP3. Additionally, whether inhibition of NLRP1 activation occurs in vivo
is unclear as they stimulated Bcl2-/-
and Bcl2-overexpressing macrophages with MDP+ATP, and ATP is a well-known NLRP3 activator (Mariathasan et al., 2006b
; Sutterwala et al., 2006
). Given these data, plus the known effect of Bcl-2 on the apoptotic machinery, it is unlikely that Bcl-2 is directly involved in NLRP3 inflammasome activation.
At this point, it is not known whether both intrinsic and extrinsic apoptosis can induce the NLRP3 inflammasome together with signal 1, as all of the apoptosis inducers we tested were intrinsic. Therefore, we also investigated whether Fas ligand (FasL) could induce IL-1β secretion in LPS-primed BMDM, but did not detect IL-1β release or any reduction in mt membrane potential. However, exposure of BMDM to FasL did not induce a robust apoptotic response, suggesting that BMDM are partially insensitive to the apoptotic effects of FasL. We therefore cannot exclude a potential role for extrinsic apoptosis in NLRP3 inflammasome activation.
It is not clear how oligomeric NLRP3 inflammasome complexes sense such a wide range of cytosolic danger signals, including ATP, K+
efflux, alum, uric acid crystals, β-amyloid, and various microbial infections (Jin and Flavell, 2010
; Schroder and Tschopp, 2010
). As such, in a mechanism distinct from pattern recognition receptors, NLRP3 does not seem to sense each of these diverse ligands directly with its leucine-rich repeat (LRR) domain. Instead, it is believed that three broad physiological changes—ROS generation, K+
efflux, or lysosomal leakage—activate the NLRP3 inflammasome (Stutz et al., 2009
). Yet, it did not seem possible to reconcile these three NLRP3 activation models with one another, and so the mechanism by which the NLRP3 inflammasome was activated by diverse signals remained perplexing. Our model of oxidized mtDNA binding to NLRP3 as the activation step neatly assembles and unifies previous models of NLRP3 activation. While we found that oxidized mtDNA associated with the NLRP3 inflammasome after stimulation, our data utilizing 293 cells transfected with mtDNA suggest that mtDNA can directly bind NLRP3. However, these results do not rule out the association of the mtDNA with other members of the NLRP3 inflammasome complex.
Two recent investigations have linked autophagy and inflammasome activation with mt activity. The first report found that blocking autophagy results in accumulation of mt-driven ROS formation, which in turn activates the NLRP3 inflammasome (Zhou et al., 2010
). Our results presented here generally agree with the basic findings of the Zhou et al
. report. Specifically, those authors concluded that apoptosis was not involved in their system based on lack of LDH release after various stimuli. Moreover they were unable to detect LDH release after exposure to NIG; a proton ionophore that causes cytosolic acidification and has been shown to decrease intracellular pH and induce apoptosis in other models (Yamagata and Tannock, 1996
; Yang et al., 2008
). Indeed, we found that NIG induced nuclear condensation, and another group reported LDH release after NIG exposure (Shimada et al., 2010
). Interestingly, while Zhou et al
. did not detect LDH release after monosodium urate treatment, this compound may have off-target effects as it has been shown to inhibit neutrophil apoptosis at low concentrations, but causes LDH release at higher levels (Akahoshi et al., 1997
). Nonetheless, when considering the preponderance of data we present in this study linking apoptosis and NLRP3 activation, it seems that initiation of apoptotic events is required for proper NLRP3 activation.
Both Zhou et al
. and Nakahira et al
. found that mt-derived ROS are required for NLRP3 activation (Nakahira et al., 2010
; Zhou et al., 2010
). However, Nakahira et al
. also found that mtDNA release is critical for NLRP3 activation, and this is dependent on ROS generation. We also observed that mtDNA was released into the cytosol and that its presence was absolutely required for NLRP3 activation. However, Nakahira et al
. concluded that NLRP3 itself is required for mtDNA release, as they did not detect mtDNA in the cytosol of NLRP3 deficient macrophages. Importantly, our results suggest that transfected mtDNA can bind to NLRP3 overexpressed in 293 cells and that DNA released from mt during apoptosis binds to the NLRP3 inflammasome. Therefore, a likely explanation for the lack of cytosolic mtDNA in NLRP3 deficient macrophages in the study by Nakahira et al
. is that NLRP3 itself stabilizes mtDNA in the cytosol by binding to it. The direct binding of mtDNA to NLRP3 could therefore be the triggering mechanism for NLRP3 activation.
During apoptosis, ROS and oxidized mtDNA are generated (Esteve et al., 1999
). Our data indicate that it is this oxidized form of mtDNA that binds to and activates the NLRP3 inflammasome and that this interaction can be competitively inhibited by oxidized dG. One possibility for the inhibition by 8-OH-dG could be through the prevention of ATP binding on NLRP3 (Duncan et al., 2007
) instead of directly competing with oxidized mtDNA binding. Perhaps ATP binding and hydrolysis is required for proper mtDNA binding. However, while our data does not rule out this possibility, the end result is a lack of mtDNA binding, which is required for NLRP3 activation. Therefore, our data are in agreement with previous studies regarding the importance of mtROS, and now provide the putative mechanism for NLRP3 inflammasome activation. Additionally, while it appears that transfected oxidized mtDNA can activate both NLRP3 and AIM2, AIM2 is preferentially activated by normal DNA, and NLRP3, by oxidized DNA.
Our data clearly show that induction of apoptosis is required for NLRP3 inflammasome activation; however, we do not know at what point in the signaling cascade the two events diverge. According to our model, both the apoptosome and the NLRP3 inflammasome share common upstream activating factors derived from mt. However, it is a byproduct of apoptosis, oxidized mtDNA released into the cytosol, which seems to be the activating factor for the NLRP3 inflammasome. The divergence of the two pathways after initiation of apoptosis is evidenced by the fact that mice deficient in Apaf-1, caspase-9 or caspase-3 exhibit infertility, abnormal brain development, and lethality (Honarpour et al., 2000
; Kuida et al., 1998
; Kuida et al., 1996
) whereas no such phenotypes have been found for caspase-1, NLRP3- or ASC deficient mice. Thus, while the two pathways share a common origin, their downstream effects are different.
, we have shown that mt dysfunction leading to apoptosis occurs with, and is necessary and sufficient for, NLRP3 activation in the presence of signal 1. The generation of oxidized mtDNA in this process of apoptosis and NLRP3 inflammasome activation presents some intriguing possibilities regarding diseases such as diabetes. NLRP3 has been shown to play an important role in type II diabetes (Masters et al., 2010
) and interestingly, high concentrations of 8-OH-dG (representative of damaged DNA) have been associated with both type I and type II diabetes (Hinokio et al., 1999
; Simone et al., 2008
). One could speculate that the increased amounts of oxidized DNA generated in diabetes could be derived from the processes that induce NLRP3 activation.
A key implication of the present study is that inhibition of apoptosis by intracellular microbes serves a dual role: attenuation of IL-1β secretion and maintenance of a viable host cell for intracellular growth. Moreover, our results suggest that evolution has developed an innate immune strategy that relies on mt to determine the right time to sacrifice a jeopardized host cell for the sake of initiating a strong inflammatory cascade via IL-1β. Therefore, apoptosis is not always ‘silent’; rather, it can be a powerful voice to instruct nearby cells of imminent danger in the presence of NF-κB-activating signal 1.