Ricin is a potent ribosomal toxin considered to be a potential bioterrorist agent due to the possibility of aerosol delivery to human populations 
. Studies in animals demonstrated that delivery of ricin to the pulmonary system leads to acute lung injury and symptoms resembling acute respiratory distress syndrome 
. Previously we reported that the inflammatory and lethal effects of inhaled ricin were prevented in mice with a genetic deficiency in IL-1ß. Lung inflammation was blocked in mice that had been co-treated with IL-1 receptor antagonist (IL1RA; anakinra) or depleted of macrophages, suggesting the primacy of macrophage-derived IL-1ß in orchestrating pulmonary inflammatory responses to ricin 
. Exposure of LPS-primed BMDM to ricin in vitro
induced the processing pro-IL-1ß to mature IL-1ß in a NLRP3 dependent manner 
, raising the possibility that the toxic action of ricin on ribosomal function was responsible for activation of the NLRP3 inflammasome.
These results prompted us to examine the role that suppression of ribosomal activity plays in facilitating initiation of inflammatory signaling by IL-1ß. Indeed, the results presented here demonstrate that a panel of translation inhibitors, acting through different mechanisms on peptide initiation or elongation, promoted the conversion and release of IL-1ß by a process that required participation of the NLRP3 inflammasome (). These data led us to question whether inhibition of translation, which occurs as a result of a variety of cellular stresses, might serve as a relevant trigger for inflammasome activation in human inflammatory disease.
It has been proposed that decreased levels of intracellular potassium act to trigger release of IL-1ß as a result of activation of the NLRP3 inflammasome 
. It is well documented that reduced concentrations of intracellular potassium also fail to support protein synthesis in mammalian cells 
. Potassium ionophores such as nigericin and valinomycin and compounds that poison the membrane-associated Na+
ATPase rapidly trigger translational inhibition in cells concomitant with efflux of cellular potassium, and application of high extracellular potassium is sufficient to rescue ribosomal activity 
. Replacement of extracellular sodium with potassium suppresses the ability of nigericin and ATP to activate the NLRP3 inflammasome, supporting the notion that low intracellular potassium serves as a trigger for inflammasome activation 
. Our results demonstrating the rescue of protein translation by increased extracellular potassium in BMDM exposed to nigericin or ATP () suggested to us that impairment of ribosomal activity may explain why loss of cellular potassium activates the NLRP3 inflammasome. However, the inability of emetine, a potent and irreversible inhibitor of protein synthesis, to promote efflux of potassium () demonstrated that an intracellular environment of low potassium was not required for activation of the NLRP3 inflammasome by molecules that directly interfere with ribosomal function.
It has been concluded that MSU crystals activate the NLRP3 inflammasome 
through a process that leads to decreased concentration of cellular potassium 
. The conclusion that decreased concentration of cellular results from a leakage of potassium from cells after MSU treatment was based on the ability of high extracellular potassium (150 mM) to block processing of pro-IL-1ß to its active form 
. Exposure of BMDM to MSU results in the engulfment of the insoluble crystals within the acidic milieu of endosomes 
, resulting in solubilization of the crystals and the subsequent rapid increase in cellular volume caused by release of sodium ions. The increase in cellular volume is thought to be responsible for a drop in concentration of intracellular potassium by dilution 
. Our results showing that exposure of BMDM to MSU, but not MPU, results in the dose-dependent inhibition of protein translation and release of IL-1ß (), is consistent with the latter model and further supports the close association between inhibition of protein translation and activation of the NLRP3 inflammasome.
In view of our data showing that inhibition of translation fails to mediate loss of cellular potassium (), we were surprised that high extracellular potassium was able to block appearance of IL-1ß in the medium. Interestingly, Arlehamn et al. reported that high extracellular potassium inhibited IL-1ß release from cells after bacterial infection with P. aeruginosa
and S typhimurium
, which was dependent on the NLRC4 inflammasome, but that potassium leakage from cells could not be detected by flame photometry 
. They conjectured that a minority of cells had undergone a loss of potassium due to the nature of the pathogens, which did not infect every cell, and for this reason they could not observe measurable potassium loss from the population of cells. Alternatively, high extracellular potassium may block the release of IL-1ß by a mechanism that is independent of intracellular potassium concentration. For example, high potassium (150 mM) has been shown to suppress activation and cleavage of recombinant caspase-1 in vitro
. Petrilli et al reported that MSU-treated primed macrophages release pro-IL-1ß and procaspase-1 into the medium, and that cells exposed to high extracellular potassium release pro-IL-ß and procaspase-1 into the high potassium medium, but that proteolytic processing of these proteins failed to occur 
. These data and our data () suggest that high extracellular potassium may directly suppress the cleavage of pro-IL-1ß after externalization of the inflammasome complex by inhibiting the activation of procaspase-1. Our data suggest that cells in high potassium may suppress the activation of the NLRP3 inflammasome by two independent mechanisms: 1) by restoring intracellular potassium to normal levels in cells that have undergone leakage of potassium via pore formation (e.g. by nigericin) or stimulation of P2X7 receptors (e.g. by ATP), thereby preventing translational inhibition; and 2) by suppressing the activation of caspase-1 by an unknown mechanism.
The generation of reactive oxygen species (ROS) is commonly associated with NLRP3 inflammasome activation in response to a variety of agonists, including the mitochondrial inhibitors, rotenone and antimycin A 
. However, the conclusion that generation of ROS is responsible for inflammasome activation has been questioned 
. Inhibition of mitochondrial Complex I or Complex III following exposure of cells to rotenone or antimycin A, respectively, leads to the generation of ROS through loss of mitochondrial membrane potential 
. Rotenone and antimycin A have been shown to activate the NLRP3 inflammasome, presumably as a result of ROS production, since treatment of macrophages with Mito-Tempo, a scavenger of mitochondrial ROS, inhibited inflammasome activation 
. Uncouplers of mitochondrial function such as rotenone and antimycin A are also potent inhibitors of translation, reducing the protein synthetic rate by more than 90% at concentrations employed to generate ROS in cultured cells 
Several reports have demonstrated that ROS can inhibit mRNA translation 
. Although the mechanism by which mitochondrial inhibitors inhibit protein synthesis is incompletely understood, it has been shown recently that mitochondrial inhibitors suppress protein synthesis by inducing the rapid phosphorylation of both eIF-2alpha and the elongation factor eEF2, presumably by stimulating PERK 
. Indeed, peroxide- and hypoxia-mediated ROS have been shown to inhibit translation by increasing PERK- and PKR-mediated phosphorylation of eIF-2alpha and eEF2 
. Our data demonstrating that inhibition of translation can activate the NLRP3 inflammasome is consistent with the notion that the generation of ROS by mitochondrial dysfunction may activate the NLRP3 inflammasome by suppressing protein synthesis through stress-activated phosphorylation of eIF-2alpha and eEF2.
Inhibition of translation occurs in a variety of circumstances in nature, triggered by exposure to toxins, pathogens that co-opt host cell machinery, hypoxia, and sterile inflammatory signals released from damaged tissues. Phosphorylation of eIF-2alpha at Ser51 mediates translational inhibition in response to cellular signals 
by preventing the formation of the eIF2/GTP/Met-tRNA complex 
. Stress-induced inhibition of translation through phosphorylation of eIF-2alpha is induced by viral dsRNA through activation of protein kinase R (PKR); hypoxia through the PRK-like endoplasmic reticulum kinase (PERK); and by glucose deprivation through activation of both PKR and PERK 
. An important consequence of eIF-2alpha phosphorylation is the regulation of gene expression, as mutations that interfere with eIF-2alpha phosphorylation lead to defective expression of stress-induced genes 
. Recent evidence suggests that PKR acts as a central integrator in the inflammatory component of metabolic control by linking nutrient- and pathogen-sensing pathways in development of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies 
. Poly I:C-mediated activation of the NLRP3 inflammasome has been previously reported 
. In LPS-primed BMDM we found that poly I:C mediates eIF-2alpha phosphorylation, inhibition of protein synthesis, and the release of IL-1ß (). Phosphorylation of eIF-2alpha is required not only for attenuation of translation, but also for transcriptional induction and survival in response to endoplasmic reticulum-mediated stress (ER stress) 
. ER stress activates the NLRP3 inflammasome via a pathway that does not involve the unfolded protein response 
. Repression of translation through phosphorylation of eIF-2alpha leads to activation of NF-kappaB and the subsequent transcription of NF-kappaB-directed genes by promoting the turnover of the labile inhibitor, IkappaB alpha protein 
. Stress-induced translational inhibition by phosphorylated eIF-2alpha may contribute to inflammatory responses by simultaneously promoting the two necessary events required to produce IL-1ß: the NF-kappaB-mediated synthesis of pro-IL-1ß and the release of IL-1ß through activation of the NLRP3 inflammasome. This model could explain how stress signals that converge on eIF-2alpha could induce IL-1ß-dependent inflammatory responses.
Maintenance of the intracellular level of proteins that exhibit short half-lives, such as p53 and IkappaB, is frequently regulated by the balance between their rate of synthesis and proteasome-directed degradation 
. For example, inhibition of protein translation by stress-induced phosphorylation of eIF-2alpha leads to activation of NF-kappaB through proteasome-dependent degradation of IkappaB 
. Our experiments determined that co-treatment of BMDM with proteasome inhibitors plus ricin, cycloheximide, puromycin, pactamycin, anisomycin, MSU, or dsRNA led to a reduction or complete suppression of IL-1ß release as measured by ELISA () or immunoblot (, ). LPS-primed cells treated with poly I:C in the presence of MG-132 exhibited a reduction in IL-1ß release but did not change the phosphorylation status of eIF-2alpha (), suggesting that involvement of the proteasome in activating the inflammasome is positioned downstream of translational inhibition in these cells.
In light of our data showing a link between suppression of protein synthesis and activation of the NLPR3 inflammasome, we propose that labile protein(s) may suppress the formation of the NLRP3 inflammasome. In such a scenario, inhibition of translation, which accompanies many types of cellular stresses, would lead to a decrease in abundance of putative repressor protein(s), perhaps through proteasome-mediated degradation. In this model, blockade of proteasomal activity would extend the lifetime of the putative repressor protein(s). The validity of this model would require identification of labile protein(s) that inhibit the processing of pro-IL-1ß.
The current study demonstrates that suppression of ribosomal function by molecules acting by disparate mechanisms is sufficient to activate the NLRP3 inflammasome. These data suggest that inhibition of translation may constitute a common stimulus by which seemingly dissimilar activators promote the processing and release of IL-1ß. A decreased rate of translation may constitute a regulatory node that integrates signals from toxins, pathogens, and metabolic disturbances, thereby enhancing systemic inflammation by promoting the processing and release of IL-1ß. The suppression of IL-1ß release by proteasome inhibitors suggests that labile protein(s) may be responsible for blocking the activation of the NLRP3 inflammasome under non-stressed conditions. A decrease in translation rate may lead to reduction in cellular levels of these protein(s), thereby leading to formation of active NLRP3 inflammasomes. Further studies that focus on identification of labile inhibitors of inflammasome function are clearly necessary to test the validity of this hypothesis. A graphic depiction of the proposed mechanisms underlying NLRP3 activation by different stimuli is shown in .
Graphic depiction of proposed mechanisms by which different stimuli activate the NLRP3 inflammasome.