Mechanism of NLRP3 inflammasome activation

Abstract

Inflammasomes continue to generate interest in an increasing number of disciplines owing to their unique ability to integrate a myriad of signals from pathogen- and damage-associated molecular patterns into a proinflammatory response. This potent caspase-1-dependent process is capable of activating the innate immune system, initiating pyroptosis (an inflammatory form of programmed cell death), and shaping adaptive immunity. The NLRP3 inflammasome is the most thoroughly studied of the inflammasome complexes that have been described thus far, perhaps owing to its disparate assortment of agonists. This review highlights our current understanding of the mechanisms of both priming and activation of the NLRP3 inflammasome.

Keywords: NLRP3; calcium; caspase-1; inflammasome; mitochondria.

Figure 1

Schematic of NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Human NLRP1 can interact with ASC and caspase-1 via an N-terminal PYD and also bind caspase-5 to the complex via the C-terminal CARD. Muramyl dipeptide (MDP), Bacillus anthracis lethal toxin, and Toxoplasma gondii can induce the activation of the NLRP1 inflammasome. Mouse Nlrp1b does not possess a functional N-terminal PYD, hence caspase-1 may interact with its C-terminal CARD. NLRP3 interacts with ASC through an N-terminal PYD domain, which then recruits caspase-1. Mitochondrial DNA (mtDNA) and cardiolipin have been postulated to bind to NLRP3 and induce its activation. The NLRC4 inflammasome is activated by the intermediary molecules NAIP1, NAIP2, and NAIP5/6, which have been shown to bind to the type three secretion system (T3SS) needle and rod proteins and bacterial flagellin, respectively. It remains unclear how ASC interacts with the NLRC4 inflammasome complex. The AIM2 inflammasome can directly bind double-stranded DNA (dsDNA) via its HIN200 domain. AIM2 recruits ASC and caspase-1 through its N-terminal PYD domain. CARD, caspase recruitment domain; FIIND, domain with function to find; NACHT, nucleotide-binding and oligomerization domain; PYD, pyrin domain; LRR, leucine-rich repeats; BIR, baculovirus IAP repeat domain; HIN200; HIN-200 domain.

Figure 2

Signals mediating NLRP3 inflammasome priming. Upon engagement, pattern recognition receptors (PRR), such as TLR4 and NOD2, or cytokine receptors, such as TNFR and IL-1R, activate NF-κB, leading to the transcription and translation of NLRP3 and pro-IL-1β. Dissociation of HSP90 and SGT1 from NLRP3 is required for NLRP3 inflammasome activation. Additionally, NLRP3 undergoes deubiquitylation by the JAMM domain–containing Zn²⁺ metalloprotease deubiquitinating enzyme BRCC3, which is crucial for subsequent NLRP3 inflammasome activation. Upon activation of the NLRP3 inflammasome (see Fig. 4), active caspase-1 can process pro-IL-1β (and pro-IL-18) into their mature secreted forms.

Figure 3

Inhibition of NLRP3 inflammasome activation. Type I IFNs acting through IFNAR inhibit the transcription of pro-IL-1β through the upregulation of the anti-inflammatory cytokine IL-10. Type I IFNs and IFN-γ inhibit NLRP3 through the production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS), resulting in nitrosylation of NLRP3. Interaction of mature or memory T cells with macrophages via CD40–CD40L results in inhibition of the NLRP3 inflammasome. Elevation of cellular cAMP levels also results in the inhibition of NLRP3. The microRNA mir-223 regulates the amount of NLRP3 mRNA and, consequently, NLRP3 expression.

Figure 4

Regulation of NLRP3 inflammasome activation in response to ion fluxes and mitochondrial dysfunction. K⁺ efflux is required for NLRP3 inflammasome activation and is achieved either directly by bacterial pore-forming toxins, such as nigericin, or indirectly via receptors such as the purinergic receptor for ATP, P2X7R. During the regulatory volume decrease response to cell swelling, Ca²⁺ fluxes can be regulated by the transient receptor potential receptors TRPM7 and TRPV2. Ca²⁺ influx can also be mediated via the ROS-sensitive TRPM2. G protein–coupled receptors CASR and GPRC6A are activated by sensing extracellular Ca²⁺ and trigger phospholipase C (PLC), generating inositol-1,4,5-triphosphate (InsP₃) and triggering the release of Ca²⁺ from the endoplasmic reticulum (ER). Ca²⁺ acts on the mitochondria via voltage-dependent anion channels (VDAC) and the mitochondrial Ca²⁺ uniporter (MCU), resulting in mitochondrial dysfunction. Decrease in mitochondrial membrane potential (Δψm), generation of mitochondrial ROS, decrease in ATP and NAD generation, and, possibly, mitochondrial outer membrane permeabilization (MOMP) are associated with NLRP3 inflammasome activation. Mitochondrial dysfunction and a reduction in NAD⁺ result in microtubule-driven apposition of ASC on the mitochondria to NLRP3 on the ER. Mitochondrial DNA (mtDNA) and the inner mitochondrial membrane lipid cardiolipin have been shown to bind to NLRP3 and induce its activation. Association between NLRP3 and mitochondrial antiviral signaling protein (MAVS) and NLRP3 and mitofusin 2 (Mfn2) have also been implicated in effective NLRP3 inflammasome activation.

Figure 5

Comparison of apoptotic pathways and NLRP3 inflammasome activation. The left panel represents the extrinsic pathway of apoptosis. Death receptors trigger the movement of pro-caspase-8 to the mitochondria, where its activation is dependent upon the translocation of cardiolipin from the inner to the outer mitochondrial membrane. This translocation follows the generation of ROS and is accompanied by the oxidation of cardiolipin by cytochrome c (cyt c), resulting in the loss of cardiolipin–cyt c association, with cyt c remaining in the intermembrane space. Active caspase-8 cleaves Bid to tBid, which in turn triggers mitochondrial outer membrane permeabilization (MOMP). Cyt c diffuses into the cytosol through the MOMP, where it binds to APAF-1, resulting in assembly of the apoptosome and inducing cell death. The right panel depicts a proposed model for NLRP3 inflammasome activation. NLRP3 agonists cause mitochondrial dysfunction, resulting in the generation of mitochondrial ROS. It is postulated that increases in mitochondrial Ca²⁺ occur through voltage-dependent anion channels (VDAC) and the mitochondrial Ca²⁺ uniporter (MCU). This results in a transient loss of mitochondrial membrane potential (Δψm). It is unclear if the mitochondrial dysfunction induced by NLRP3 agonists proceeds to MOMP. Cardiolipin, miofusin 2 (Mfn2), and mitochondrial antiviral signaling protein (MAVS) have been implicated in the association of NLRP3 with the mitochondria and are required for efficient inflammasome activation.