K+ Drops Tilt the NLRP3 Inflammasome
Authors: Thirumala-Devi Kanneganti, Mohamed Lamkanfi
Journal: Immunity (2013)

Unwarranted NLRP3 activation causes inflammatory disease. Its activation must therefore be specifically licensed. In this issue of Immunity, Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar reveal K+ efflux as the sole common denominator of an array of unrelated stimuli engaging this inflammasome. Unwarranted NLRP3 activation causes inflammatory disease. Its activation must therefore be specifically licensed. In this issue of Immunity, Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar reveal K+ efflux as the sole common denominator of an array of unrelated stimuli engaging this inflammasome. The NLRP3 inflammasome was originally identified through its association with three hereditary autoinflammatory syndromes that are characterized by urticarial skin rashes and prolonged episodes of fever. An important subset of patients suffering from the autosomal-dominant periodic fever syndromes—familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile neurological cutaneous and articular syndrome (CINCA)—turned out to harbor gain-of-function mutations in and around the central NACHT domain of NLRP3 (Lamkanfi and Dixit, 2012Lamkanfi M. Dixit V.M. Annu. Rev. Cell Dev. Biol. 2012; 28: 137-161Crossref PubMed Scopus (703) Google Scholar). These mutations are believed to force NLRP3 in a conformational state that removes the necessity for a molecular agonist to drive its activation, resulting in spontaneous assembly of the inflammasome, activation of the cysteine protease caspase-1, and secretion of the inflammasome substrates interleukin-1β (IL-1β) and IL-18. Subsequent reports—to a significant extent based on the analysis of Nlrp3-deficient mice—further established a causal relationship between unwarranted NLRP3 signaling and the ethiology of Crohn's disease, colitis, atherosclerosis, contact hypersensitivity, metabolic syndrome, and Alzheimer's disease; whereas other studies revealed its critical role in host defense against a diverse set of bacterial, viral, and fungal pathogens (Lamkanfi and Dixit, 2012Lamkanfi M. Dixit V.M. Annu. Rev. Cell Dev. Biol. 2012; 28: 137-161Crossref PubMed Scopus (703) Google Scholar). In cultured macrophages and dendritic cells, the NLRP3 inflammasome responds to endogenous danger-associated molecular patterns (DAMPs) such as ATP and hyaluronan. In addition, monosodium urate (MSU) crystals, amyloid-β fibrils and exogenous crystalline particles including alum, silica, asbestos, and nanomaterials all stimulate NLRP3 activation. Immune cells also respond to the action of pore-forming toxins such as maitotoxin, α-hemolysin, gramicidin, and the ionophore nigericin with NLRP3 inflammasome activation. Given its broad impact on diverse homeostatic systems, Nlrp3 inflammasome activation is stringently controlled (Figure 1). Basal Nlrp3 expression levels in naive macrophages and dendritic cells are low, and NF-κB-driven upregulation of Nlrp3 expression is the first hurdle to be taken to ready the NLRP3 inflammasome for activation (Lamkanfi and Dixit, 2012Lamkanfi M. Dixit V.M. Annu. Rev. Cell Dev. Biol. 2012; 28: 137-161Crossref PubMed Scopus (703) Google Scholar). Recent reports refer to a second posttranscriptional checkpoint at which deubiquitination licenses NLRP3 for activation (Juliana et al., 2012Juliana C. Fernandes-Alnemri T. Kang S. Farias A. Qin F. Alnemri E.S. J. Biol. Chem. 2012; 287: 36617-36622Crossref PubMed Scopus (503) Google Scholar, Py et al., 2013Py B.F. Kim M.S. Vakifahmetoglu-Norberg H. Yuan J. Mol. Cell. 2013; 49: 331-338Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). However, Nlrp3 inflammasome activation in TLR-activated cells only will occur upon exposure to a second stimulus from the list above comprising ATP, nigericin, and silica. Given the wide array of pathophysiological conditions and chemically unrelated stimuli activating the NLRP3 inflammasome, it is commonly thought that NLRP3 does not physically interact with these molecular entities directly but rather guards a host-derived secondary messenger, the homeostatic concentration of which is commonly perturbed by NLRP3-activating stimuli. Multiple such secondary messengers have been put forward in recent years, including K+ efflux, lysosomal destabilization, membrane permeabilization, mitochondrial damage, and release of oxidized DNA, the production of reactive oxygen species (ROS), Ca2+ influx, and cell swelling (Lamkanfi and Dixit, 2012Lamkanfi M. Dixit V.M. Annu. Rev. Cell Dev. Biol. 2012; 28: 137-161Crossref PubMed Scopus (703) Google Scholar). Although each of these mechanisms was proposed based on the analysis of the cellular responses elicited by a number of the NLRP3-activating stimuli above, most models have not been evaluated for larger sets of the ever-growing list of NLRP3-activating molecules. Based on a more comprehensive analysis of the cellular events triggered by an extended panel of NLRP3 stimuli that belong to distinct biochemical categories, Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar in this issue of Immunity concluded most of the proposed secondary messengers as being dispensable for NLRP3 activation because they were not shared by all tested NLRP3-activating triggers. In this regard, oxidative mitochondrial damage and ROS production were deemed not to be critical because NLRP3 activation was not observed in response to uncoupling of the mitochondrial respiratory chain with rotenone, oligomycin and antimycin A, nor upon inhibition of autophagy or exogenous ROS generation with H2O2 (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). Moreover, the change in metabolic rate observed with gramicidin, nigericin, and ATP temporally lagged NLRP3 activation. In addition, the authors show that the metabolic effect of gramicidin, nigericin, and ATP was rescued by the Na+-K+-ATPase inhibitor ouabain without affecting their potency to induce NLRP3 inflammasome activation, although interpretation of the latter experiment is somewhat complicated by the fact that ouabain autonomously induces NLRP3-dependent IL-1β release (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar, Walev et al., 1995Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (237) Google Scholar). Finally, concentrations of ROS scavengers that were sufficient to inhibit the cellular redox state appeared incapable of inhibiting NLRP3 inflammasome activation. These results are supported by recent observations suggesting that higher concentrations of these ROS scavengers might interfere with NF-κB-mediated transcriptional priming rather than posttranslational activation of the NLRP3 inflammasome (Bauernfeind et al., 2011Bauernfeind F. Bartok E. Rieger A. Franchi L. Núñez G. Hornung V. J. Immunol. 2011; 187: 613-617Crossref PubMed Scopus (459) Google Scholar). Instead of the above models, Munoz-Planillo et al. identified K+ efflux as the sole common denominator induced by all classes of tested NLRP3-activating stimuli (Figure 1). Indeed, cellular K+ depletion by the H+-K+ ionophore nigericin, staphylococcal α-hemolysin, gramicidin, and the purinergic P2X7 receptor has long been recognized and was previously linked to posttranslational maturation and secretion of bioactive IL-1β from LPS-primed macrophages (Walev et al., 1995Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (237) Google Scholar). The realization that these molecules all induced IL-1β maturation through the NLRP3 inflammasome set the stage for demonstrating that NLRP3 activation by molecules without apparent membrane-permeating properties—such as crystalline MSU—also was inhibited by high extracellular K+ concentrations (Pétrilli et al., 2007Pétrilli V. Papin S. Dostert C. Mayor A. Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 1583-1589Crossref PubMed Scopus (1074) Google Scholar). Munoz-Planillo et al. further established that phagosomal uptake of crystalline and particulate molecules such as silica, alum, and calcium pyrophosphate dihydrate (CPPD) crystals by LPS-primed macrophages was accompanied by a significant drop in the intracellular K+ concentration that preceded NLRP3-dependent IL-1β secretion. Notably, the phagocytosis inhibitors cytochalasin B and latrunculin B impaired both K+ efflux and NLRP3 inflammasome activation by crystalline substances, suggesting that cellular uptake is required for both events to occur. In this regard, activation of NLRP3 by media containing high extracellular concentrations of Ca2+ and phosphate was attributed to the formation of insoluble calcium phosphate salts (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar), which were previously linked to K+ efflux and NLRP3 inflammasome activation (Jin et al., 2011Jin C. Frayssinet P. Pelker R. Cwirka D. Hu B. Vignery A. Eisenbarth S.C. Flavell R.A. Proc. Natl. Acad. Sci. USA. 2011; 108: 14867-14872Crossref PubMed Scopus (177) Google Scholar). Moreover, preventing lysosomal and endosomal acidification with Bafilomycin A blocked NLRP3 activation and K+ efflux by crystalline agents and the lysosomotropic peptide derivative LL-OMe, but not by nigericin and ATP (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). This suggests that endosomal acidification might be specifically required for inducing a drop in intracellular K+ concentration and NLRP3 inflammasome activation by crystalline and lysosomotropic, but not soluble agents. In this regard, it is interesting to note that Munoz-Planillo et al. monitored K+ efflux indirectly by measuring changes in the intracellular concentration of this cation. Although drops in intracellular K+ levels usually are caused by its extracellular release through membrane-associated pores and channels, it could also reflect its dilution by cellular volume changes resultant from water influx driven by osmosis. Notably, a recent report suggested a model in which NLRP3 activation by ingested MSU crystals would result from the release of high amounts of Na+ under the acidic conditions found in maturing endosomes and lysosomes. Cytosolic release of crystal-derived Na+ from damaged organelles in turn would trigger an influx of water and an accompanying cellular volume increase that drops the intracellular K+ concentration below the threshold licensing NLRP3 activation (Schorn et al., 2011Schorn C. Frey B. Lauber K. Janko C. Strysio M. Keppeler H. Gaipl U.S. Voll R.E. Springer E. Munoz L.E. et al.J. Biol. Chem. 2011; 286: 35-41Crossref PubMed Scopus (129) Google Scholar). In support of this model, experimental removal of sodium from the crystals prevented secretion of IL-1β—but not IL-6—from macrophages ingesting Na+-free crystals (Schorn et al., 2011Schorn C. Frey B. Lauber K. Janko C. Strysio M. Keppeler H. Gaipl U.S. Voll R.E. Springer E. Munoz L.E. et al.J. Biol. Chem. 2011; 286: 35-41Crossref PubMed Scopus (129) Google Scholar), although this treatment could as well cause lattice changes in the crystal that decreased its intrinsic capacity to activate NLRP3. Although they did not test MSU crystals, Munoz-Planillo et al. showed that other crystalline NLRP3 stimuli permeabilized the membrane for ethidium and taurine rather early, suggesting that extracellular K+ efflux accounts—at least partially—for the observed decrease in intracellular K+ levels induced by MSU. In addition, they showed that K+-free buffers decreased intracellular K+ levels and triggered NLRP3 inflammasome activation in the absence of osmosis (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar), suggesting cell swelling to at least be dispensable for NLRP3 activation by noncrystalline compounds. The observation that unlike the NLRP3 inflammasome, activation of the AIM2 inflammasome by transfected double-stranded DNA was not accompanied by a decrease in intracellular K+ concentrations firmly establishes a regulatory role for K+ in modulating NLRP3 activation thresholds at the level of NLRP3 itself or upstream in the pathway. In support of this conclusion, LPS-treatment of macrophages expressing the MWS-associated constitutively active NLRP3R258W mutant triggered caspase-1 activation and IL-1β release in the absence of K+ efflux, and high K+ concentrations failed to prevent these events (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martinez-Colon G. Smith B.L. Rajendiran T.M. Nunez G. Immunity. 2013; 38 (this issue): 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). This suggests that elevated intracellular K+ concentrations might hamper wild-type NLRP3 to undergo the conformational changes that are induced by the MWS-associated mutation. Alternatively, K+ might inhibit a factor upstream of NLRP3 that signals such conformational changes. Thus, collectively these results suggest that K+ efflux might reduce the threshold for NLRP3 activation. As such, the NLRP3 inflammasome might resemble the apoptosome in that both are cytosolic caspase-activating protein platforms (caspase-1 versus caspase-9, respectively), the assembly and activation of which is inhibited by physiological K+ concentrations (Cain et al., 2001Cain K. Langlais C. Sun X.M. Brown D.G. Cohen G.M. J. Biol. Chem. 2001; 276: 41985-41990Crossref PubMed Scopus (177) Google Scholar). Once assembled, both complexes are rather insensitive to ionic perturbations. The role of K+ efflux in this model entails the removal of a checkpoint safeguarding the cell against unwarranted complex assembly caused by the inadvertent release of small amounts of their respective ligands (unknown versus cytochrome c, respectively). However, because K+ efflux also has been implicated in activation of the NLRP1b inflammasome by anthrax lethal toxin (Pétrilli et al., 2007Pétrilli V. Papin S. Dostert C. Mayor A. Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 1583-1589Crossref PubMed Scopus (1074) Google Scholar), further work is needed to determine whether it represents the long-sought-after secondary messenger that is specifically sensed by the NLRP3 inflammasome. K+ Efflux Is the Common Trigger of NLRP3 Inflammasome Activation by Bacterial Toxins and Particulate MatterMuñoz-Planillo et al.ImmunityJune 27, 2013In BriefThe NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP, and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore, but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K+ and Na+. Full-Text PDF Open Archive