J Clin Immunol (2010) 30:628–631 DOI 10.1007/s10875-010-9440-3
Molecular Mechanism of NLRP3 Inflammasome Activation Chengcheng Jin & Richard A. Flavell
Received: 2 June 2010 / Accepted: 6 June 2010 / Published online: 30 June 2010 # Springer Science+Business Media, LLC 2010
Abstract The inflammasome is an intracellular multimolecular complex that controls caspase-1 activity in the innate immune system. NLRP3, a member of the NLR family of cytosolic pattern recognition receptors, along with the adaptor protein ASC, mediates caspase-1 activation via assembly of the inflammasome in response to various pathogen-derived factors as well as danger-associated molecules. The active NLRP3 inflammasome drives innate immune response towards invading pathogens and cellular damage, and regulates adaptive immune response. Here, we review identified agonists of the NLRP3 inflammasome and the molecular mechanism by which they induce NLRP3 inflammasome activation. Three signaling pathways involving potassium efflux, generation of reactive oxygen species, and cathepsin B release are discussed. Keywords NLRP3 inflammasome . agonist . potassium efflux . reactive oxygen species . cathepsin B The NOD-like receptors (NLRs) are a large family of intracellular pattern recognition receptors which are comprised of 22 members in human and 34 members in mice [1, 2]. As an important player in the innate immune system,
C. Jin Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, TAC S-560, New Haven, CT 06520, USA R. A. Flavell (*) Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, TAC S-569, New Haven, CT 06520, USA e-mail:
[email protected]
they are able to recognize a variety of pathogen-associated molecular patterns, as well as host-derived danger signals (danger-associated molecular patterns), and mediate immune response to defend pathogen infection and endogenous damage. Several members of this family such as NLRP1, NLRP3, and NLRC4 have been shown to assemble into large multiprotein complexes named inflammasomes to control caspase-1 activity. Inflammasome-dependent caspase-1 activation may lead to the maturation and secretion of proinflammatory cytokines IL-1β and IL-18, and may also drive pyroptosis [3] or mediate unconventional protein secretion [4]. The most thoroughly characterized inflammasome is the NLRP3 inflammasome, which consists of the NLR family member NLRP3, the adaptor protein ASC and the effector protein caspase-1. Thus far, a broad range of exogenous and endogenous stimuli have been demonstrated to activate the NLRP3 inflammasome. These include infecting microorganisms such as Sendai virus, Influenza virus, adenovirus, the fungi Saccharomyces cerevisiae and Candida albicans, as well as several bacteria like Staphylococcus aureus, Listeria monocytogenes, and Shigella flexneri [5–8]. In certain cases, the specific microbial components or products that trigger the NLRP3 inflammasome activation have been identified, such as bacterial RNA [9], hemozoin crystals produced by malaria-causing parasites [10, 11] and a number of bacterial pore-forming toxins (for instance, nigericin from Streptomyces hygroscopicus, maitotoxin from Gambierdiscus toxicus, aerolysin from Aeromonas hydrophila and listeriolysin O from L. monocytogenes [8, 12]). The NLRP3 inflammasome can also be activated in response to a diversity of host-derived factors indicative of stress and injury, including extracellular ATP and hyaluronan released from injured cells [8, 13], amyloid-β fibrils constituting the Alzheimer’s disease brain plaque [14], elevated plasma glucose occurring in metabolic disorders
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[15], as well as monosodium urate (MSU) and calcium pyrophosphate dehydrate crystals, the causative agents of gout and pseudogout, respectively [16]. In addition, a number of environmental insults such as silica, asbestos and the adjuvant aluminum hydroxide (alum) have been shown to induce inflammasome activation [17–20]. Exposure to UVB irradiation [21] or chemical irritants inducing contact hypersensitivity [22, 23] was also found to trigger a NLRP3inflammasome-dependent response in skin keratinocytes. Much research interest has focused on the molecular mechanism of inflammasome assembly and activation. Previous studies suggest that NLRP3 is sequestered by SGT1 and HSP90 in an auto-inhibitory but responsive conformation in the steady state [24]. Upon challenge, NLRP3 senses the ligand via its C-terminal leucine-rich repeat domain, and undergoes an ATP-dependent selfoligomerization mediated by its intermediate NACHT domain. Then, homotypic interaction between the N-terminal PYD domains of NLRP3 and ASC, and subsequently between the CARD domains of ASC and procaspase-1 will recruit procaspase-1 to the high molecular weight complex, leading to its autocleavage and activation [2]. However, it is still unclear how those various stimuli are recognized by NLRP3 and trigger the inflammasome activation. Given the divergence of their structures and biochemical properties, those agonists are unlikely to bind NLRP3 directly, and indeed no such interaction has been observed in previous research. Instead, it is proposed that a common cellular event may be elicited by different stimuli and serve as the activating signal for the NLRP3 inflammasome. Three models are generally supported in current literature [1, 25]. First, numerous studies have suggested that potassium (K+) efflux is a necessary signal upstream of NLRP3 activation. When K+ efflux is prevented experimentally by drugs or a high concentration of K+ in the cell culture media, activation of the NLRP3 inflammasome is abolished in response to almost all known activators [10, 11, 17, 19, 20, 26]. It is known that extracellular ATP engages the ATP-gated cation channel P2X7R whereas bacterial toxins cause membrane pore formation to trigger K+ efflux [8, 27]. However, it is yet to be determined how K+ efflux is induced by other inflammasome activators, and whether it depends on the activation of specific ion channels or a nonselective increase in ion permeability due to membrane damage. On the other hand, K+ efflux may not be sufficient to activate the NLRP3 inflammasome. ATP-induced inflammasome response was found to be abrogated when Na+ in the cell culture medium was iso-osmotically substituted with Li+ or choline [28], or when Cl− was replaced by SCN− or I− [29], indicating that a general change in the intracellular ionic milieu might be involved in inflammasome activation.
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In the second model, the generation of reactive oxygen species (ROS) is considered to be critical for the inflammasome activation. In support of this, ROS blockade via chemical scavengers of ROS, pharmacological inhibitors of NADPH oxidase or siRNA-mediated knockdown of the p22phox subunit of NADPH oxidase suppressed NLRP3 activation in response to a wide range of stimuli, including ATP [30], C. albicans [11] and various crystals (MSU, asbestos, silica, and hemozoin [10, 17, 19]). Moreover, Zhou et al. found that thioredoxin-interacting protein can directly bind to NLRP3 in a ROS-responsive manner, further elucidating the link between ROS production and inflammasome activation [15]. In spite of these lines of evidence, several aspects of this model still await clarification. First, the cellular source of ROS has yet to be identified. ROS can be derived from a variety of physiological processes such as cytochrome P450 oxidase uncoupling, mitochondrial respiration, and activation of xanthine oxidase, peroxisome oxidases or NADPH oxidases [31]. Chemical inhibitors of mitochondrial complex I and complex II did not affect asbestosinduced inflammasome activation [17], arguing against the mitochondrial origin of ROS. Multiple NADPH oxidases with different subunit composition have been characterized with specific subcellular localization and differential coupling to external stimuli [31]. Among them, Nox2 is the major NADPH oxidase known to be assembled and activated upon phagocytosis in macrophages and neutrophils, and was previously speculated to be responsible for the generation of ROS in the NLRP3 activation pathway [17]. Nonetheless, ATP and crystals-induced inflammasome activation was found to rely on p22phox, a common subunit shared by several different NADPH oxidases [17], but not gp91phox, the specific subunit of Nox2 [18], suggesting the implication of some other NADPH oxidase in this process. Second, the manner by which ROS modulates NLRP3 activity is not understood. Increased ROS production in superoxide dismutase-1 (SOD-1)-deficient macrophages was observed to suppress caspase-1 activation, since superoxide directly inhibited caspase-1 activity by modifying its redox-sensitive cysteine residues [32]; it is unclear whether this represents a negative-feedback mechanism to control the caspase-1 activity conferred by ROS-induced-dependent NLRP3 inflammasome activation. A third model has been proposed for the crystalline and particulate activators of the inflammasome, wherein uptake of these substances causes destabilization of the acidic lysosomal compartment and release of cathepsin B, which is somehow sensed by NLRP3 and triggers inflammasome activation. In agreement with this model, the cathepsin B inhibitor CA-074Me or drugs disrupting the phagocytic machinery could impair the NLRP3 inflammasome activation induced by alum, silica, MSU, fibrillar amyloid-β and
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malaria hemazoin [10, 14, 18–20]. Additionally, this cathepsin-B-dependent activation model may not apply exclusively to the particulate activators of inflammasome, since other NLRP3 agonists such as the K+ ionophore nigericin and the antiviral compound R837 can also induce cathepsin B release, and lead to a caspase-1 activation sensitive to CA-074Me treatment [33, 34]. However, opposing this model, cathepsin B-deficient macrophages exhibited a normal inflammasome-dependent IL-1β production in response to hemozoin, MSU or alum [10]. This discrepant result raises the question about the offtarget effects of CA-074Me, as implied by another study on the NLRP1 inflammasome [35]. Since phagosome rupture is associated with the release of numerous other enzymes and CA-074Me might inhibit some related proteases, it is likely that an unidentified target of CA-074Me, rather than cathepsin B, acts as the essential signal to trigger the NLRP3 inflammasome. Currently no direct ligand-receptor interaction between cathepsin B and NLRP3 has been reported. In general, the three signaling pathways discussed above may not be mutually exclusive. Although accumulated evidence have converged on these models, the exact mechanism by which K+ efflux, ROS production or cathepsin B release triggers inflammasome activation remains to be characterized in detail. It is likely that a change in the intracellular ionic milieu or redox state may induce a specific conformation of NLRP3 that enables inflammasome assembly. Alternatively, these upstream signals may regulate the release, modification, or recognition of a ligand that directly binds to NLRP3 [36]. Given the important roles of the NLRP3 inflammasome in orchestrating multiple innate and adaptive immune responses in infection and autoinflammatory disorders [2], future studies are required to delineate the signaling pathways of inflammasome activation, and they will provide potential drug targets to treat a number of heritable and acquired diseases in which the activity of NLRP3 inflammasome is dysregulated.
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