Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation.

As an important sensor in the innate immune system, NLRP3 detects exogenous pathogenic invasions and endogenous cellular damage and responds by forming the NLRP3 inflammasome, a supramolecular complex that activates caspase-1. The three major components of the NLRP3 inflammasome are NLRP3, which captures the danger signals and recruits downstream molecules; caspase-1, which elicits maturation of the cytokines IL-1ß and IL-18 and processing of gasdermin D to mediate cytokine release and pyroptosis; and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which functions as a bridge connecting NLRP3 and caspase-1. In this article, we review the structural information that has been obtained on the NLRP3 inflammasome and its components or subcomplexes, with special focus on the inactive NLRP3 cage, the active NLRP3-NEK7 (NIMA-related kinase 7)-ASC inflammasome disk, and the PYD-PYD and CARD-CARD homotypic filamentous scaffolds of the inflammasome. We further implicate structure-derived mechanisms for the assembly and activation of the NLRP3 inflammasome.

"Boosting" tumor immunity in elderly mice by hyperactivating dendritic cells.

Immunosenescence poses a significant challenge to tumor immunotherapy in elderly individuals. In this issue of Cell, Zhivaki et al. elucidate that dendritic cells "hyperactivated" by specific adjuvants elicit TH1-skewed CD4+ T cell responses in a manner contingent on the NLRP3 inflammasome, which can eliminate tumors in aged mice.

A 360° view of the inflammasome: Mechanisms of activation, cell death, and diseases.

Inflammasomes are critical sentinels of the innate immune system that respond to threats to the host through recognition of distinct molecules, known as pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), or disruptions of cellular homeostasis, referred to as homeostasis-altering molecular processes (HAMPs) or effector-triggered immunity (ETI). Several distinct proteins nucleate inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRC4/NAIP, AIM2, pyrin, and caspases-4/-5/-11. This diverse array of sensors strengthens the inflammasome response through redundancy and plasticity. Here, we present an overview of these pathways, outlining the mechanisms of inflammasome formation, subcellular regulation, and pyroptosis, and discuss the wide-reaching effects of inflammasomes in human disease.

Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.

NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.

The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Distinct changes in endosomal composition promote NLRP3 inflammasome activation.

Inflammasome complexes are pivotal in the innate immune response. The NLR family pyrin domain containing protein 3 (NLRP3) inflammasome is activated in response to a broad variety of cellular stressors. However, a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill defined. Here, we demonstrate that NLRP3 inflammasome activators primarily converge on disruption of endoplasmic reticulum-endosome membrane contact sites (EECS). This defect causes endosomal accumulation of phosphatidylinositol 4-phosphate (PI4P) and a consequent impairment of endosome-to-trans-Golgi network trafficking (ETT), necessary steps for endosomal recruitment of NLRP3 and subsequent inflammasome activation. Lowering endosomal PI4P levels prevents endosomal association of NLRP3 and inhibits inflammasome activation. Disruption of EECS or ETT is sufficient to enhance endosomal PI4P levels, to recruit NLRP3 to endosomes and to potentiate NLRP3 inflammasome activation. Mice with defects in ETT in the myeloid compartment are more susceptible to lipopolysaccharide-induced sepsis. Our study thus identifies a distinct cellular mechanism leading to endosomal NLRP3 recruitment and inflammasome activation.

Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

Epithelial Nlrp10 inflammasome mediates protection against intestinal autoinflammation.

Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1ß and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.

Mitochondrial damage activates the NLRP10 inflammasome.

Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome.

Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation.

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

NLRP3 licenses NLRP11 for inflammasome activation in human macrophages.

Intracellular sensing of stress and danger signals initiates inflammatory innate immune responses by triggering inflammasome assembly, caspase-1 activation and pyroptotic cell death as well as the release of interleukin 1ß (IL-1ß), IL-18 and danger signals. NLRP3 broadly senses infectious patterns and sterile danger signals, resulting in the tightly coordinated and regulated assembly of the NLRP3 inflammasome, but the precise mechanisms are incompletely understood. Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages. NLRP11 interacted with NLRP3 and ASC, and deletion of NLRP11 specifically prevented NLRP3 inflammasome activation by preventing inflammasome assembly, NLRP3 and ASC polymerization, caspase-1 activation, pyroptosis and cytokine release but did not affect other inflammasomes. Restored expression of NLRP11, but not NLRP11 lacking the PYRIN domain (PYD), restored inflammasome activation. NLRP11 was also necessary for inflammasome responses driven by NLRP3 mutations that cause cryopyrin-associated periodic syndrome (CAPS). Because NLRP11 is not expressed in mice, our observations emphasize the specific complexity of inflammasome regulation in humans.

NLRP3 inflammasome in cancer and metabolic diseases.

The NLRP3 inflammasome is a multimeric cytosolic protein complex that assembles in response to cellular perturbations. This assembly leads to the activation of caspase-1, which promotes maturation and release of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18, as well as inflammatory cell death (pyroptosis). The inflammatory cytokines contribute to the development of systemic low-grade inflammation, and aberrant NLRP3 activation can drive a chronic inflammatory state in the body to modulate the pathogenesis of inflammation-associated diseases. Therefore, targeting NLRP3 or other signaling molecules downstream, such as caspase-1, IL-1ß or IL-18, has the potential for great therapeutic benefit. However, NLRP3 inflammasome-mediated inflammatory cytokines play dual roles in mediating human disease. While they are detrimental in the pathogenesis of inflammatory and metabolic diseases, they have a beneficial role in numerous infectious diseases and some cancers. Therefore, fine tuning of NLRP3 inflammasome activity is essential for maintaining proper cellular homeostasis and health. In this Review, we will cover the mechanisms of NLRP3 inflammasome activation and its divergent roles in the pathogenesis of inflammation-associated diseases such as cancer, atherosclerosis, diabetes and obesity, highlighting the therapeutic potential of targeting this pathway.

Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

The orphan receptor Nur77 binds cytoplasmic LPS to activate the non-canonical NLRP3 inflammasome.

Intracellular sensing of lipopolysaccharide (LPS) by murine caspase-11 or human caspase-4 initiates a protease cascade, termed the non-canonical inflammasome, that results in gasdermin D (GSDMD) processing and subsequent NLRP3 inflammasome activation. In an effort aimed at identifying additional sensors for intracellular LPS by biochemical screening, we identified the nuclear orphan receptor Nur77 as an LPS-binding protein in macrophage lysates. Nr4a1-/- macrophages exhibited impaired activation of the NLRP3 inflammasome, but not caspase-11, in response to LPS. Biochemical mapping revealed that Nur77 bound LPS directly through a domain in its C terminus. Yeast two-hybrid assays identified NLRP3 as a binding partner for Nur77. The association between Nur77 and NLRP3 required the presence of LPS and dsDNA. The source of dsDNA was the mitochondria, requiring the formation of gasdermin-D pores. In vivo, Nur77 deficiency ameliorated host response to endotoxins. Thus, Nur77 functions as an intracellular LPS sensor, binding mitochondrial DNA and LPS to activate the non-canonical NLRP3 inflammasome.

The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3.

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing.