The discovery of NLRP3 and its function in cryopyrin-associated periodic syndromes and innate immunity

Summary

From studies of individual families to global collaborative efforts, the NLRP3 inflammasome is now recognized to be a key regulator of innate immunity. Activated by a panoply of pathogen-associated and endogenous triggers, NLRP3 serves as an intracellular sensor that drives carefully coordinated assembly of the inflammasome, and downstream inflammation mediated by IL-1 and IL-18. Initially discovered as the cause of the autoinflammatory spectrum of cryopyrin-associated periodic syndrome (CAPS), NLRP3 is now also known to play a role in more common diseases including cardiovascular disease, gout, and liver disease. We have seen cohesion in results from clinical studies in CAPS patients, ex vivo studies of human cells and murine cells, and in vivo murine models leading to our understanding of the downstream pathways, cytokine secretion, and cell death pathways that has solidified the role of autoinflammation in the pathogenesis of human disease. Recent advances in our understanding of the structure of the inflammasome have provided ways for us to visualize normal and mutant protein function and pharmacologic inhibition. The subsequent development of targeted therapies successfully used in the treatment of patients with CAPS completes the bench to bedside translational loop which has defined the study of this unique protein.

1 |. INTRODUCTION

By the mid-20th century, three categories of diseases were known to be caused by the immune system: immunodeficiency, allergy, and autoimmunity. Prior to the 1900s, studies of the immune system largely focused on host–microbe interactions and immune deficiencies. These studies built on the work of Edward Jenner, Louis Pasteur, Emil von Behring, and Shibasabura Kitasato,^1,2 but were fundamentally attempts at understanding immune responses that were described as early as Hippocrates. In the 1900s, experimental descriptions of autoimmunity, allergy, and anaphylaxis not only expanded knowledge of diseases involving the immune system, but also led to a paradigm shift acknowledging that the immune system was capable of self-damage.^3,4 Despite this hard-won knowledge, rare patients and families with systemic inflammatory disorders were recognized that violated this established dogma, as they failed to fit into the established categories of immune system diseases. These patients were characterized by non-infectious episodes of inflammation, without high-titer autoantibodies or antigen-specific T cells. In the late 20th century, these disorders were termed autoinflammatory syndromes to distinguish them from autoimmune disorders and were molecularly characterized by the identification of the genes whose defects caused four landmark hereditary periodic fever syndromes: TNFRSF1A in TNFR1-associated periodic syndromes (TRAPS), MEFV in familial Mediterranean fever (FMF), MVK in Hyper-IgD syndrome, and NLRP3 in cryopyrin-associated periodic syndrome (CAPS).^5–8 This review will cover the progress made in our understanding of the role of NLRP3 in human disease beginning with families experiencing unusual inflammatory symptoms and extending to over a quarter of a century of translational approaches using genetics, molecular biology, cellular and mouse immunology, structural analysis and modeling, and targeted pharmacology.

2 |. THE CLINICAL FEATURES OF CAPS AS AN AUTOINFLAMMATORY DISEASE SPECTRUM

CAPS, as it is now recognized, encompasses three previously identified syndromes: familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID), which is also called chronic infantile neurologic cutaneous articular (CINCA) syndrome (Figure 1, Table 1). Although each of these syndromes has distinct characteristics, patients with FCAS/MWS and MWS/NOMID overlap phenotypes have been reported, which suggested these disorders existed on a spectrum of inflammatory disease even before the NLRP3 gene was identified.^9–11

FIGURE 1.

Clinical manifestations of NLRP3 spectrum disease. CAPS, cryopyrin-associated periodic syndrome; FCAS, familial cold autoinflammatory syndrome; ICP, intracranial pressure; MWS, Muckle–Wells syndrome; NOMID/CINCA, neonatal-onset multisystem inflammatory disease/chronic infantile neurologic cutaneous articular syndrome. Created with Biorender.com.

TABLE 1.

Clinical characteristics of CAPS spectrum disease (modified from Welzel & Kuemmerle-Deschner¹⁷⁶).

	Mild phenotype (FCAS)	Moderate phenotype (MWS)	Severe phenotype (CINCA/NOMID)	Atypical CAPS170,171
Disease onset	<6 months–adulthood	Early childhood–adulthood	Perinatal	Early childhood–adulthood
Family history	Often positive	Often positive	Often negative (sporadic de novo mutations)	Often positive
Inflammatory flares	Cold induced	Yes, may have continuous disease symptoms	Continuous disease symptoms	Yes
Duration of inflammatory flares	30 min–72 h	1–3 Days ± subclinical	Persistent inflammation	Variable
Cold trigger	Yes	Possible	Rare	Rare
Dermatological manifestations	Cold-induced neutrophilic urticaria	Neutrophilic urticaria	Neutrophilic urticaria	Less frequent
Fever	6–24 h after cold exposure possible	Particularly in childhood	Yes	No
Ocular manifestation	Conjunctivitis	Conjunctivitis, episcleritis, optic disc edema/papilledema	Conjunctivitis, episcleritis, optic disc edema/papilledema	Conjunctivitis, episcleritis, papilledema
Musculoskeletal manifestations	Myalgia, arthralgia	Myalgia, arthralgia, oligoarthritis	Myalgia, arthralgia, polyarthritis. Epiphyseal bony overgrowth, limb-length discrepancies, contractures	Myalgia, arthralgia
Hearing loss	No	Yes	Yes	Yes
Central nervous system manifestations	Headache	Headache, intermittent aseptic meningitis	Headache, chronic aseptic meningitis, increased intracranial pressure, brain atrophy	Headache, interm
Amyloidosis	Rare	Yes	Yes	No

2.1 |. Familial cold autoinflammatory syndrome (FCAS)

FCAS was described in 1940 by Kile and Rusk who studied a unique family with affected members, equally male and female across five generations, exhibiting recurrent episodes of urticarial-like rash, limb pain, and fever following generalized cold exposure.¹² The proband described a lifelong history of burning, rather than pruritic, erythema beginning 30 min after going outside in cold damp weather, followed by fever to 101–103 °F, and joint stiffness lasting 6–8 h following mild cold exposure or 24 h following more extended cold exposures. Symptoms could be induced by immersion of her arm in cold water and were resistant to therapies for allergic urticaria commonly used at the time. Additional families with similar phenotypes and elevations in acute phase reactants were reported over the next 6 decades, variably described as cold hypersensitivity, familial cold urticaria (FCU), and finally FCAS^13,14 in an attempt to differentiate this chronic, inherited, systemic inflammatory disorder from the more common acquired cold urticaria.¹⁵

2.2 |. Muckle–Wells syndrome

While the cold-induced urticaria uniquely defined the family described by Kile and Rusk, additional descriptions of families with apparent autosomal dominantly inherited urticarial disorders began to appear in the literature. In 1962, Muckle and Wells reported a family with similar features of non-infectious episodes of urticaria-like rash, limb pain, and fever with rigors, which they described as “aguey bouts” which lasted approximately 36 h.¹⁶ In contrast to FCAS patients, this family developed progressive bilateral sensorineural hearing loss beginning in childhood, and end stage renal disease secondary to AA amyloidosis beginning in adulthood, with laboratory evaluations showing anemia, increased serum immunoglobulins and elevated inflammatory markers.¹⁶ Similar to FCAS patients, these patients failed to respond to therapy with antihistamines or daily steroids. Muckle and Wells noted the similarities of recurrent skin rash and amyloidosis between MWS patients and FMF patients,¹⁷ in what may be one of the first attempts to phenotypically link autoinflammatory syndromes.

2.3 |. CINCA/NOMID

Chronic infantile neurologic cutaneous articular (CINCA) syndrome was first described in 1980 by Prieur who reported three unrelated children presenting in the neonatal period with a chronic urticarial-like rash, neutrophilia and significant involvement of the large joints. Described as “bread crumb appearance” on radiograph, the inflammatory joint disease involved the epiphyses and patella and resulted in physical deformation and disability.¹⁸ Extensive neurologic disease including seizures secondary to chronic sterile meningitis increased intracranial pressure and developmental delay were also described.¹⁸ Progressive deafness and visual impairment further added to the morbidity for this disorder. Initially termed chronic meningo-cutaneo-articular syndrome in children, other patients with similar phenotypes were subsequently described, and the disorder was referred to as either CINCA or NOMID.^19–21

3 |. IDENTIFICATION, CLONING, AND CHARACTERIZATION OF THE NLRP3 GENE AS THE CAUSE OF CAPS

A fortuitous meeting with families that described a lifelong history of cold-induced rash, fever and arthralgias, became the turning point for the field of inflammasome biology. We used DNA from a total of 69 individuals from five families, including the initial proband described in 1940, and used positional cloning to link a defined region on chromosome 1q43-q44 to FCAS,¹¹ the same area previously linked to MWS.²² The team then screened predicted exons and flanking intronic sequences in this region, identifying missense mutations in FCAS and MWS patients in a gene initially labeled CIAS1 (for cold-induced autoinflammatory syndrome 1).⁶ Using the nascent Human Genome Project, we identified seven exons by homology and two additional exons by sequencing of RT–PCR products. This method also revealed extensive alternative splicing in the 3′ end of the gene, the relevance of which would not come to light for several years. Northern blot analysis demonstrated that transcripts of this newly discovered gene were present at a low level in peripheral blood leukocytes and had little or no expression in other tissues. This was consistent with the expression patterns seen in MEFV, establishing a link between these recurrent inflammatory disorders on a molecular level. The name of the disorder was changed to FCAS to highlight the genetic and phenotypic similarities.⁶ A year later, CIAS1 (later termed NLRP3) mutations were also identified as the cause of NOMID.^23,24 Since then, research focused on the molecular basis for these rare diseases has been the work of many scientists around the world in addition to our laboratory.

3.1 |. Genotype and phenotype of a monogenic NLRP3 associated disease

Since the initial description of the NLRP3 gene in 2001, over 250 variants have been described worldwide.^25–28 There appears to be significant genotype–phenotype correlation in that certain mutations are often associated with specific disease spectrum severity, although different mutations affecting the same amino acid can cause different diseases along the CAPS spectrum (Figure 2).⁹ One complication in the literature is that two different methionines have been used as the initiating amino acid. The NLRP3 sequence from the originally used methionine starts with the amino acid sequence MASTR (e.g., NP_004886.2 and NP_001230062.1). The NLRP3 sequence has also been redescribed as beginning with an upstream in-frame methionine so that the protein starts with the sequence MKMASTR (e.g., NP_004886.3); however, this upstream methionine is not universally conserved in mammals. This ambiguity means that the same mutation in different publications can be given a position that differs by 2, for example p.L359W and p.L361W refer to the same mutation. In this review, we exclusively use the original nomenclature; hence, we will name the above mutation p.L359W.

FIGURE 2.

NLRP3 mutations in CAPS and undefined autoinflammatory disease. Amino acid positions mutated in NLRP3-mediated autoinflammatory disease with the associated amino acid substitution placed according to the resulting phenotype. Black dots indicate the number of distinct missense mutations associated with each position. Regions A–G are local NLRP3 regions containing multiple missense mutations often affecting the same amino acid. The Δ−309 at G307 corresponds to the p.G307_F309del mutation. Data from the Infevers database.²⁵

The majority of patients in the CAPS spectrum have autosomal dominant NLRP3 missense mutations; however, there are mutation-negative patients with classic CAPS symptoms and pathology. At least some of these patients can be explained by the presence of somatic mutations that were not detected by standard sequencing, with some patients ultimately having mutations in NLRP3, and a few other patients with CAPS phenotype were determined to have NLRC4 mutations.^29–33 We have also identified a promoter variant in one NLRP3 mutation-negative patient associated with increased NLRP3 expression,³⁴ providing a potential novel disease mechanism. An extensive pathway-targeted candidate gene approach¹² and further linkage studies have not yet identified additional disease genes.

3.2 |. Low-penetrance mutations

Our initial description of NLRP3 mutations in FCAS included a variant (p.V198M) that was not initially observed in a panel of healthy controls.⁶ However, additional sequencing revealed that this variant and two other variants (p.R488K and p.Q703K) that had been initially observed in CAPS phenotypes were also identified in healthy controls as well as patients with atypical clinical presentations. The higher frequency of these variants in different populations and the genetic data is not supportive of classifying them as pathogenic variants; however, there are clinical and functional data to suggest that they have an intermediate phenotype between pathogenic CAPS and healthy controls.³⁵ The phenotypic differences between the classic CAPS mutations and low-penetrance mutations provide insight into the unique role of NLRP3 in modulating the immune response. Subsequently, detailed evaluations uniting molecular experiments and structural biology have been instrumental in our understanding of how NLRP3 responds to an array of diverse stimuli and directs formation of the cytoplasmic multiprotein platform known as the inflammasome.

4 |. INFLAMMASOMES: NOMENCLATURE, STRUCTURE, AND FUNCTION

The NLRP3 protein, originally named cryopyrin for the N-terminal pyrin domain and link to cold-induced symptoms,⁶ belongs to the nucleotide-binding domain and leucine-rich repeat (LRR) containing (NLR) family of proteins. This family is characterized by diverse N-terminal domain(s), a central nucleotide-binding NACHT (for Neuronal Apoptosis inhibitor protein CIITA, HET-E and TP1) domain,³⁶ and a C-terminal Leucine-Rich Repeat (LRR) domain (Figure 3). NLR proteins are classified into five subfamilies on the basis of the N-terminal domain:³⁷ (1) NLRA proteins like CIITA have an Acidic transactivating domain, (2) NLRB proteins like NAIP have a Baculovirus Inhibitor of apoptosis protein Repeat (BIR) domain, (3) NLRC proteins like NOD1 have a CARD (CAspase Recruitment Domain), (4) NLRP proteins like NLRP3 have a PYD (Pyrin domain; Figure 3), and (5) NLRX proteins like NLRX1 lack significant N-terminal homologies. The human genome encodes 22 NLR proteins, many of which are associated with diseases driven by inflammation.³⁸

FIGURE 3.

NLRP3 gene structure and CAPS mutations. Protein domains shown at top are the pyrin domain (PYD), the NACHT domain, and the leucine-rich repeat (LRR) domain. The NACHT domain is further subdivided into the FISNA (fish-specific NACHT associated) domain, nucleotide-binding domain (NBD), HD1, the winged-helix domain (WH), and HD2. Positions of mutations in different diseases are shown as hashes in the middle. Exon structure is shown at the bottom, with the non-coding exon 1 omitted from the diagram. Regions commonly mutagenized in autoinflammatory disease (regions A–G) are highlighted in red.

4.1 |. Similarities and differences among innate immune modulators of the inflammasome

Infection by pathogens and other environmental cues give rise to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) that induce the innate immune system. These PAMPs and DAMPs are thought to trigger the assembly of NLR proteins into large molecular weight complexes, termed inflammasomes after a pivotal study by Jorg Tshopp’s group describing a lipopolysaccharide (LPS)-inducible assembly of NLRP1 with apoptosis related speck-like protein containing CARD (ASC), caspase-1, and caspase-5.³⁹ Inflammasome formation has been verified for multiple NLR proteins, including NLRC4, NLRP1, NLRP3, and NLRP7.^39–44 Inflammasome formation by NLRP3 appears to be driven by two signals: The first signal is “priming,” and the second signal is “activation”. Signal 1 leads to NF-κB-driven expression of genes encoding NLR proteins and proinflammatory cytokines, such as IL-1β and IL-18, as well as deubiquitination of the LRR domain by BRCC3,^45–47 the recognition of LPS by Toll-like receptor (TLR) proteins, and binding of proinflammatory cytokines to their cognate receptors. Once expressed, inactive NLRP3 appears to be stabilized in the cytosol through interactions with the SCF ubiquitin ligase subunit SGT1 and heat shock protein 90 (HSP90),⁴⁸ similar to the interaction for pathogen-sensing NLR proteins in plants.⁴⁹ Signal 2 includes a large variety of triggers for NLR protein assembly into inflammasomes and the maturation and release of the proinflammatory cytokines.

Assembled inflammasomes form 10- and 11-subunit disks comprised of the NACHT and LRR domains (for NLRP3, see Figure 4).^41–43 The N-terminal pyrin domains of NLRP inflammasomes can form 5-symmetric helical filaments alone,^42,50 and electron microscopy suggests that inflammasome assembly generates a 10-or 11-subunit NLRP pyrin domain proto-filament capable of nucleating a filament made up of the pyrin domains of the ASC adapter protein.⁵¹ ASC is comprised of two domains, an N-terminal pyrin domain and a C-terminal CARD domain, which recruits pro-caspase 1 through a homotypic interaction between the CARD domains of ASC and pro-caspase 1 (Figure 4).^51–53 Activation of pro-caspase 1 molecules is likely achieved through increased local concentration mediated by ASC binding followed by trans-cleavage and dimerization of the mature caspase 1 subunits. Mature caspase 1 then cleaves the pro-inflammatory cytokines, leading to their release into the extracellular matrix for signaling, and cleaves and activates gasdermin D (GSDMD). The inflammasome-triggered formation of ASC filaments serves as a signal amplification mechanism to drive the innate immune response.⁵⁴

FIGURE 4.

A unified model for the formation of the active NLRP3 inflammasome. Predicted NLRP3 assembly states and inflammasome intermediates (top) are correlated with likely cellular localization of these assemblies (green highlighted regions, bottom).

The PAMPs and DAMPs that activate some NLR proteins have been identified; however, the mechanism of activation remains unclear for most of these proteins. NLRC4 responds to bacterial flagellins and the inner rod components of bacterial type III secretion systems; NLRP1 responds to dsRNA and dsDNA, associated with viral infections, and NLRP7 responds to microbial lipopeptides.^40,55,56 The precise mechanism by which these molecular patterns activate inflammasome formation remains poorly understood for most NLR proteins. NLRP1 assembly appears to be triggered by proteolytic cleavage of the N-terminus that is induced by a variety of factors, including viral proteases, viral replication, and ribotoxic stress, rather than by direct interaction with PAMPs and DAMPs.⁵⁷ In contrast, NLRP6 may form inflammasomes through a liquid–liquid phase separation after directly interacting with viral RNA molecules or lipoteichoic acid during infection by Gram-positive bacteria.⁵⁸ Similarly, NLRC4 utilizes an accessory subunit, one of the NLR family apoptosis inhibitory proteins (NAIPs, a single gene in humans but present in 6 copies in mice) that provides ligand specificity^56,59–61 and promotes inflammasome formation by nucleating a disk containing 1 NAIP protein and 10 NLRC4 proteins.⁴³ It has recently been suggested that NLRP3 also forms a non-canonical inflammasome that also contains NLRC4, the RNA helicase DDX17, ASC, and caspase-1 in response to the presence of short interspersed nuclear element (SINE) RNA molecules.⁶²

4.2 |. NLRP3 as a unique innate immune sensor of cellular stress

Remarkably, the NLRP3 inflammasome is unique in that it is responsive to a wide range of activating agents, including uric acid crystals, alum, silica, cholesterol crystals, extracellular adenosine triphosphate (ATP), hyaluronan, amyloid-β fibrils, mitochondrial reactive oxygen species (ROS) generation, and lowered intracellular potassium concentrations such as by nigericin or gramicidin treatment.^63–66 It seems unlikely that NLRP3 can directly detect each of these DAMPs, and a unifying feature for these agents has been suggested to be increased potassium efflux from the cell.^67,68 Consistent with this, inhibition of potassium efflux by the mammalian ketone metabolite β-hydroxybutyrate suppresses NLRP3 activity.⁶⁹

Reduced potassium levels appear to be an indirect activator of NLRP3 assembly.⁷⁰ Several of the treatments shown to activate NLRP3, including extracellular ATP and potassium efflux triggered by nigericin or gramicidin, lead to the disassembly of the trans-Golgi network. NLRP3 is recruited to vesicles in this dispersed trans-Golgi network (dTGN) through the charged interaction between the phosphatidylinositol-4-phosphate lipid and the linker between the NLRP3 pyrin and NACHT domains and a region of the fish-specific NACHT associated (FISNA) subdomain (Figure 3).^71,72 It has been proposed that the inactive “cage” form of NLRP3 could be the state at which NLRP3 binds to the dTGN vesicles⁷¹; however, constitutively activated CAPS mutant proteins are also capable of spontaneously forming membrane-bound NLRP3 puncta,⁷⁰ suggesting that active forms can also bind to membranes. Remarkably, two potassium-efflux independent NLRP3 activators, imiquimod and CL097, also promote the formation of dTGN vesicles and NLRP3 conformational changes similar to potassium efflux^70,72; however, it is currently unclear why the requirement for potassium efflux for NLRP3 activation varies among different treatments that disrupt the trans-Golgi network.

Even after full or partial assembly of NLRP3 on dTGN vesicles, several lines of evidence indicate that NLRP3 activity is further controlled through subcellular localization. First, NLRP3 activation in mice requires binding of the serine/threonine protein kinase NEK7 (centrosomal NIMA-related kinase 7) by the NLRP3 LRR domain (Figures 4 and 5).^73–75 Importantly, in humans, this activation appears to be independent of the NEK7 kinase activity.⁷⁵ While NEK7 binding would be predicted to disrupt the inactive NLRP3 “cage” structures (Figure 4),^50,71,76 NEK7 is primarily localized at the microtubule organizing center (MTOC), and activated NLRP3 is moved by microtubule retrograde transport from the trans-Golgi network to the MTOC.^77,78 This transport is responsible for the formation of ASC specks, which are large helical fibrils that typically form at one site per cell and are convenient experimental markers for NLRP3 activation, as they can be visualized by fluorescence microscopy.⁷⁹ The requirements for the NLRP3 retrograde transport are reminiscent of the retrograde transport of aggresomes, which are large molecular aggregates that are resistant to proteosome degradation and are degraded by autophagy at the MTOC.^80,81 A relocalization requirement likely explains why partial, but not complete, defects in inflammasome activity are caused by defects in the dynein adapter histone deacetylase 6 (HDAC6), HDAC6 inhibitors, defects in microtubule-affinity reducing kinase 4 (MARK4), dynein disruption, and loss of the aggresome component vimentin.^77,82–91 Cargo recruited for transport by HDAC6 is typically ubiquitinated,⁹² and extensive studies have indicated that NLRP3 inflammasome components are regulated both positively and negatively by ubiquitination.⁹³ A relocalization requirement also explains why the microtubule depolymerizing agent colchicine is useful in treating symptoms of gout, which is an NLRP3-mediated disease; colchicine blocks both ASC speck formation and IL-1β release driven by monosodium urea crystals.^94–96

FIGURE 5.

NLRP3 LRR domain interactions. (A and B) Leucine-rich repeats encoded by different exons are depicted in alternating red and yellow. An insertion within the LRR domain that binds the concave face of the LRR domain in the inactive NLRP3 decameric “cage” (A) but is unstructured in the active NLRP3-NEK7 disk (B) is colored in blue. (C) LRR domain-mediated interactions (red and blue) make up the interactions in the inactive decameric “cage” assembly. (D) LRR domain-mediated interactions (blue) in the active disk assembly are with NEK7; the NLRP3 disk is only held together by interactions between the NACHT domains (red), primarily involving the FISNA (FIS) subdomain.

It is currently unclear what assembly state NLRP3 is in during transport to the MTOC; however, a small molecule inhibitor of NLRP3, MCC950/CRID3, which likely prevents the inactive-to-active conformational change in NLRP3 subunits and subsequent inflammasome disc assembly, blocks ASC speck formation,⁹⁷ suggesting that, at minimum, the active conformation of NLRP3 is required. Similarly, the NLRP3-ASC interaction appears to be required. In the presence of ASC, activated NLRP3 is localized to a single speck; however, in the absence of ASC, activated NLRP3 is distributed in dTGN-associated puncta.^70,86 This suggests that ASC is required for retrograde transport or for retention of NLRP3 at the MTOC, possibly through recruitment of ubiquitinated ASC by HDAC6. ASC is known to be modified by linear ubiquitin chains via the linear ubiquitin chain assembly complex (LUBEC), and two LUBEC subunits, HOIL-1 and SHARPIN, promote inflammasome activity.^98,99 ASC is also subject to modification by K63-linked ubiquitin chains via the TRAF3 and Peli1 E3 ubiquitin ligases.^56,99–101 Remarkably, TRAF3, Peli1, and HOIL-1 were shown to promote ASC speck formation;^56,99,100 however, it is not yet clear if these proteins act at the step of retrograde transport.

The complex choreography required for the formation of active NLRP3 inflammasomes at the MTOC may have arisen due to the ability of NLRP3 to respond to a wide variety of inflammatory activators. Because the proximal signal for activation, likely disruption of the trans-Golgi network, is only indirectly tied to infection, preventing inappropriate activation and being able to rapidly silence the inflammasome to return to a basal state are potentially more crucial for NLRP3-mediated inflammatory responses than for inflammasomes triggered by direct interaction with PAMPs/DAMPs. Remarkably, both NLRP3 and pyrin inflammasomes localize to the MTOC, whereas absent in melanoma 2 (AIM2) inflammasomes do not.⁷⁷ Activation by MTOC recruitment and binding of the MTOC-resident protein NEK7 localizes activated NLRP3 inflammasomes where they could be rapidly degraded by autophagy. Consistent with this view, autophagosome formation is induced by many NLRP3 stimuli, autophagosomes partially colocalize with activated NLRP3 inflammasomes, loss of autophagosome function increases inflammasome activity, and K63-linked ubiquitin chain modification leads to autophagosome recruitment of ASC.¹⁰²

5 |. ALTERNATIVE SPLICING OF THE NLRP3 MRNA

We originally observed extensive alternative splicing of the 3′ end of the NLRP3 gene when sequencing RT-PCR products during the initial cloning suggesting yet another mechanism that could regulate NLRP3 inflammasome function.⁶ More recently, we collaborated with Eike Latz to leverage high throughput Illumina sequencing to characterize NLRP3 mRNAs from human monocyte-derived macrophages to more closely analyze splice variants.¹⁰³ Exons 5–9, which encode most of the LRR domain, have a remarkable structure. Each of these exons are 171 bp in length, precisely encode two leucine-rich repeats of 28 and 29 amino acids, and are in the same reading frame (Figure 5A). Each leucine-rich repeat is comprised of a sheet-turn-helix fold where the conserved leucine positions form a hydrophobic core that packs laterally with adjacent repeats to generate a structure that can be described as an arc or a solenoid. The effect of these features means that loss of any one or any combination of these exons will result in an NLRP3 protein with a shortened LRR domain that is predicted to still fold. This patten is shared by other NLR proteins but is distinct from TLR proteins in which the entire LRR domain-coding region exists in a single exon. Alternative splicing of NLRP3, and potentially other NLR proteins, is reminiscent of the adaptive immune system of jawless fish that rely upon lymphocyte receptors whose structure is varied through somatic recombination between short LRR-encoding exons.¹⁰⁴

The NLRP3 LRR domain is known to interact with a variety of proteins, including SGT1, NEK7, and other NLRP3 molecules (Figure 5C,D). The most common NLRP3 splice form, NLRP3^Δexon5 (previously termed NLRP3^Δexon4), would be predicted to disrupt key interactions in the NLRP3 inactive cage structures as well as observed interactions with NEK7. NLRP3^Δexon5,7 was also observed less commonly in this study,¹⁰³ and we as well as Martinon and colleagues¹⁰⁵ have observed yet other splice variants. Loss of exon 5 disrupted NEK7 binding and prevented both ASC speck formation and release of IL-1β in response to multiple NLRP3 activators, including nigericin.¹⁰³ Similarly, expression of constructs lacking exons 5, 6, 8, or 10 (but not lacking exons 7 or 9) prevented nigericin activation of the inflammasome, suppressed auto-activation by the CAPS mutation p.R260W, reduced affinity for SGT1, and failed to trigger ASC polymerization.¹⁰⁵ The inactive variants were unable to act as dominant negative mutants,¹⁰⁵ consistent with the fact that they are present at modest levels in normal cells.¹⁰³

Although these splice variants could arise by chance, the relative inefficiency of the splice site acceptors at exon 5 (and exon 7) that promote the more common splice variants¹⁰³ suggest that these splice variants might have a biological role. One possibility is that these splice variants might promote inflammasome activation in response to other PAMP/DAMP signals or in combination with activating proteins other than NEK7. Remarkably, one nonsense mutation, p.R554X, results in a complete loss of the LRR domain and is associated with an atypical cold-induced autoinflammatory syndrome;¹⁰⁶ however, this mutation is unusual as truncations of the LRR domain did not induce an inflammatory response in mice but could support one in response to nigericin.¹⁰⁷ Another possibility is that these splice variants are normally inactive but generate stable proteins that fine-tune the inflammatory response.

This complex, multi-level regulation may be required to modulate the formation of NLRP3 inflammasomes and NLRP3-mediated immune responses, which may ultimately prevent chronic inflammation and tissue damage. The identification of patients with CAPS, however, highlights the risks of uncontrolled NLRP3-mediated inflammation and provides additional insights into NLPR3 folding, activation, and key models to link these molecular models with human disease.

6 |. CAPS MUTATIONS CAUSE A GAIN-OF-FUNCTION PHENOTYPE

CAPS mutations in NLRP3 are autosomal dominant and result in increased inflammasome activity, including caspase-1 activation, IL-1β cleavage and release, and inflammatory cell death, which has been observed in in vitro studies using cell lines transfected with plasmids carrying mutant NLRP3 or patient cells.^108–111 These studies also demonstrate that CAPS mutations lead to spontaneous formation of NLRP3-ASC specks, and their release into the supernatant causes phagocytosis and perpetuation of inflammasome-mediated inflammation.^112,113 Similarly, peripheral blood mononuclear cells isolated from patients with CAPS show increased inflammasome activity with IL-1β and IL-18 secretion compared to healthy human controls at baseline or with minimal stimulation such as low dose LPS.^114,115

6.1 |. Cold and NLRP3 in FCAS

The cold specificity of NLRP3 activation in FCAS led to the development of ex vivo, in vitro and murine models to further study the role of NLRP3 and the CAPS phenotype (Table 2). Ex vivo, adherent monocytes derived from the peripheral blood of patients with FCAS demonstrate significant release of IL-1β with as short as 1 h of incubation at 32°C, suggesting the presence of pre-transcribed and pre-translated pro-IL-1β in the presence of mutations in NLRP3. Prolonged incubation at this reduced temperature leads to release of IL-1β, IL-6, and TNF-alpha, consistent with perpetuation of an inflammatory cascade. Cold-induced IL-1β release could be inhibited by caspase-1 inhibition, confirming a role for inflammasome activation in cold-related symptoms. In addition, anakinra failed to prevent early IL-1β release but significantly reduced the late-phase transcription and release of all cytokines, consistent with a pro-IL-1 autoinflammatory feedback loop.¹¹⁶ This ex vivo system of “intrinsic” NLRP3 activation has become a key model for pre-clinical studies of therapeutics targeting the NLRP3 inflammasome.

TABLE 2.

Knockin and knockout murine models (modified from Hoffman¹⁷⁷).

Human phenotype	Human mutation	Mouse mutation	Design	Mouse phenotype	References
FCAS	L353P	L351P	Conditional knockin	Perinatal lethality, ex vivo cold sensitivity	114 JAX:017970
MWS	A352V	A350V	Conditional knockin	Growth defect, rash, early death as juvenile	114 JAX:017969
MWS	R260W	R258W	Constitutive knockin	Rash	123
NOMID	D303N	D301N	Conditional knockin	Growth defect, rash, early death, osteoporosis	132 JAX:017971
NOMID	D303N	n/a	Syntenic knockin	Adult-onset arthritis, osteoporosis	178
NOMID	N477K	N475K	Conditional knockin	Rash, growth defect, early death, amyloidosis	133
n/a	n/a	Deletion exons 1–2	Constitutive knockout	Fertile, healthy	126,127
n/a	n/a	Insertion into intron 2	Constitutive knockout	Fertile, healthy	95
n/a	n/a	LacZ Neo replacement of exons 4–9	Constitutive knockout	Fertile, healthy	129
n/a	n/a	LacZ Neo replacement of exons 1–9	Constitutive knockout	Fertile, healthy	129
n/a	n/a	Neo replacement of exons 1–9	Constitutive knockout	Fertile, healthy	179 JAX:021302
n/a	n/a	Neo targeted deletion of exon 4	Conditional knockout	Fertile, healthy	130

Abbreviations: FCAS, Familial cold autoinflammatory syndrome; MWS, Muckle Wells syndrome; n/a, not applicable.

More recently, Karasawa and colleagues investigated the long-standing question of how cold induces NLRP3 activation in FCAS.¹¹⁷ Using transfected cell lines, the authors showed that cells expressing FCAS-associated mutations p.L353P and p.Y563N formed cryo-sensitive aggregates that act as a scaffold for inflammasome activation.¹¹⁷ Aggregate formation was dependent on caspase-1 mediated calcium influx, rather than potassium efflux, suggesting a possible divergence in inflammasome activation between wild-type and mutant NLRP3, which has implications for therapy. Additional studies have implicated the involvement of heat shock protein 70 (HSP70) in the mechanism of cold induction observed in FCAS-associated mutant NLRP3.¹¹⁸

6.2 |. How do CAPS mutations cause NLRP3 activation?

Most CAPS mutations affect exon 4 of NLRP3 (previously called exon 3 prior to the identification of a short non-coding 5′ exon¹⁰³), which encodes the NACHT domain (Figure 3). Analysis of disease-causing NLRP3 missense mutations in the Infevers database²⁵ reveals seven protein regions (regions A–G) that are affected by many missense mutations in CAPS and other autoinflammatory diseases (Figure 2). In many cases, multiple missense mutations have been identified for the same residue in these regions.

The recent availability of active and inactive NLRP3 structures provides an opportunity to decipher the effects of these disease-causing mutations.^{42,50,76,119,120} Remarkably, the NACHT domains in the human and mouse NLRP3 “cage” assemblies do not contact each other.^50,71,76 None of the mutations in the NACHT domain nor those in other NLRP3 domains affect the protein–protein contacts in the inactive assembly. Similarly, the protein–protein interfaces in the active NLRP3 disk conformation are not affected by most of the mutations, excepting p.D211N and p.H213R (NACHT-NACHT interface) and p.E567G/K/Q, p.R777C, and p.R918Q (LRR-NEK7 interface).⁴² Thus, most of the mutations likely do not stabilize or destabilize the protein–protein interactions in either the active or inactive conformations.

The NLRP3 protein undergoes a large closed-to-open conformational change when transitioning between the inactive and active states (Figure 6A,B). This motion can be described as a rigid body hinge motion between the FISNA-N BD-HD1 N-terminal subdomains of the NACHT domain and the WH-HD2-LRR portions of the protein. Strikingly, the commonly mutated regions A, B, and C form a single surface on the NBD, consistent with the predictions we made 15 years ago,⁹ and interact in the inactive conformation with the commonly mutated regions F (WH subdomain) and G (HD2 subdomain) (Figure 6C). The interface delineated by the regions A–C, F and G in the inactive conformation is where the NLRP3 inhibitor MCC950/CRID3 binds.^50,71,121 This inhibitor is thought to function by stabilizing the inactive conformation by binding a pocket that is only present in the inactive conformation. This interface is lost in the active conformation, and many of the residues in regions A–C, F and G become more solvent exposed (Figure 6D). Similarly, the commonly mutated regions D and E are part of an interface of the HD1 domain with the HD2 and LRR domains that is present in the inactive conformation but undergoes substantial alteration in the active conformation (Figure 6C). Thus, a shared feature of each of the regions commonly mutated in autoinflammatory diseases (Figure 2) is that they lie at domain-domain interfaces that are lost or substantially changed in the conversion of inactive NLRP3 to active NLRP3 (Figure 6C,D).

FIGURE 6.

Many CAPS mutations lie at intradomain interfaces in the inactive conformation. (A) Distance difference matrix calculated by determining the differences between all Cα-Cα distances for the active NLRP3 disk conformation (PDB id 7pzc;¹⁷⁴) and the inactive NLRP3 “cage” conformation (PDB id 8ej4;⁴²). Similar results were obtained using other structures with the inactive NLRP3 conformation. Regions that do not move relative to each other have low (light colored) differences whereas regions that do have high (red colored) differences. The FISNA-NBD-HD1 and WH-HD2-LRR regions of NLRP3 behave as two rigid bodies whose relative motion can be described as a hinge. (B) Superposition of the active and inactive conformations using the FISNA-NBD-HD1 portion of the molecule, which shows the relative motion of the WH-HD2-LRR rigid body; structures of intermediate conformations were generated using ChimeraX.¹⁷⁵ (C and D) Residues commonly mutated in autoinflammatory disease (regions A–G, see Figures 2 and 3) are shown as spheres in the inactive (C) and active (D) NLRP3 conformations. (E) Local environmental change (LEC) scores calculated for the conformational change between the inactive NLRP3 “cage” conformation and the active NLRP3 disk conformation with a radius cutoff of 5 Å. High scores indicate large changes to the local environment of the residue, and small scores indicate little or no change. Solid black circles are residues that are mutated in CAPS and undefined autoinflammatory disease. Open gray circles are other residues. The commonly mutated regions A–G are indicated by horizontal bars. (F) CAPS mutations that lie at interdomain boundaries in the inactive conformation (top) likely function to destabilize the inactive conformation and drive the NLRP3 conformation equilibrium to the active conformation (bottom).

To systematically explore all of the identified mutations and not just those at regions A–G, we identified domain–domain interface residues whose environment changed between the active and inactive conformation, using a local environmental change (LEC) score that we devised. This score is calculated by finding all atoms within a 5 Å cutoff in both conformations and calculating the average absolute difference between the distances in both conformations. As expected, the commonly mutated regions A–G were robustly identified by this analysis as well as some of the other mutated residues (Figure 6E). This analysis, however, indicated that there were some mutations associated with gain-of-function disease whose local environment was not substantially altered in the inactive-to-active conformational change (Figure 6E).

These data suggest the hypothesis that many of the mutations cause disease by disrupting the domain-domain interactions that stabilize the inactive state (Figure 6F). A shift in the equilibrium of the NLRP3 conformation to the active state predicts that the mutations would reduce or eliminate the need for a second activating signal such as potassium efflux. This hypothesis is consistent with multiple experimental results. First, CAPS mutations have reduced crosslinking to the MCC950/CRID3 inhibitor, which binds the inactive conformation.¹²² Second, in the absence of stimulation, the p.R260W, pA352V, and p.L353P mutant proteins form dispersed puncta that are similar to those formed by wild-type NLRP3 in the presence of stimulation.⁷⁰ Third, the p.R260W mutation did not require potassium efflux for activation,¹²³ but retained the requirement for NEK7 binding,⁷³ while other mutations such as p.L353P appear to be more dependent on calcium influx.¹¹⁷ Fourth, more missense mutations are known to cause severe disease than mild disease, which could be consistent with a mechanism involving disruption of the inactive conformation, instead of stabilization of the active conformation if this bias is not due to the increased likelihood of patients with severe phenotypes to undergo more extensive genetic analysis. In general, mutations giving rise to different disease severity (FCAS vs. MWS vs. NOMID) are not distinguished by their position on the NLRP3 protein; disease severity may be a consequence of how strongly individual mutations affect the NLRP3 conformational equilibria and affect the sensitivity to intracellular ion flux.

Some disease-causing mutations do not affect residues that directly lie at the interfaces that change during the conformational change (Figure 6E). These mutations may drive a gain-of-function phenotype through one of several mechanisms. Some of these mutations might destabilize the inactive state indirectly by affecting the local protein structure, which may include adjacent interface residues. Other mutations might locally disrupt the structure and recruit protein chaperones; the NLRP3-chaperone interactions have the potential to promote the active, open conformation due to steric collisions with NLRP3 domains in the inactive, closed conformation. Finally, the small number of mutations that lie at the protein–protein interfaces in the active conformation could function by directly stabilizing the active, signaling state.

7 |. CONSERVATION OF NLRP3 ACROSS SPECIES

The NLRP3 gene sequence is strongly conserved among primate and non-primate mammals, particularly in the pyrin and NACHT domains, which was used to PCR amplify and clone portions of NLRP3 from multiple mammalian species.¹²⁴ Analysis of the ratio of the rates of non-synonymous mutations (d_N) to synonymous mutations (d_S) within each branch in the NLRP3 phylogenic tree is less than 1.¹²⁴ This result indicates that NLRP3 is subject to purifying selection during mammalian evolution, which is consistent with its central role in mediating innate immune responses to a wide variety of signals. An important observation of this early work, which has borne out by the subsequent sequencing of mammalian genomes from essentially every order of Mammalia, is that disease-causing mutations tend to affect highly conserved residues (Figure 7). Importantly, human and mouse NLRP3 share both extensive sequence conservation and patterns of expression.¹²⁴ These similarities make the mouse a useful species to study NLRP3.

FIGURE 7.

Conservation of NLRP3 across species. Alignment of NLRP3 residues mutated in inflammatory diseases are highlighted in yellow if identical to the human sequence or red if identical to a disease-causing mutation. Sequences were sampled from species in different mammalian orders (Homo sapiens, human, Primates; Mus musculus, mouse, Rodentia; Dasypus novemcinctus, armadillo, Xenarthra; Tupaia chinensis, tree shrew, Scandentia; Galeopterus variegatus, colugos, Dermoptera; Bos taurus, cow, Cetartiodactyla; Erinaceaus europaens, hedgehog, Eulipotyphia; Loxodonta africana, elephant, Proboscidea; Equus caballus, horse, Perissodactyla; Rhinolophus ferrumequinum, horseshoe bat, Chiroptera; Canis lupis familiarus, dog, Carnivora; Orycteropus afer afer, aardvark, Tubulidentata; Oryctolagus cuniculus, rabbit, Lagomorpha). Mutation data from the Infevers database.²⁵

7.1 |. The mouse is an excellent model to study NLRP3 and CAPS

Mice have been used for the study of immunity for decades, initially for in vivo experiments that cannot be performed in humans, and later to determine specific gene/protein function using recombinant technology allowing for the study of specific gene knockouts. However, since the beginning, disagreements have arisen between human and mouse immunologists as significant differences exist, particularly in lymphocyte biology, but also in innate immune responses.¹²⁵ Despite these differences in immune function and phenotype, mice remain extremely useful as models for human immune diseases, and this holds true for the study of NLRP3 and CAPS, with some caveats.

7.2 |. Deletion of Nlrp3 in mice

In 2006, several landmark papers were published by various groups^95,126,127 using Nlrp3 knockout mice. These studies revealed that NLRP3 was a unique and multifunctional protein with diverse activators including urate crystals, RNA, and toxins, with significant roles in several inflammatory and infectious diseases including gout, contact dermatitis, and Salmonella. These mice have continued to be studied over the last 17 years to expand our knowledge of the increasing number of roles for NLRP3 in the pathophysiology of diseases involving almost every organ. In fact, descriptions of conditions where NLRP3 does not play a role are the exception, rather than the rule.¹²⁸

To better understand the function of the LRR region, we developed a unique mouse model with deletion of only the C-terminal LRR domain of NLRP3 in collaboration with Regeneron Pharmaceuticals. We showed that the LRR domain is necessary for NLRP3 function in both in vivo models (i.e., gout) and in ex vivo responses to several known activators by comparing this LRR deletion mouse to a full Nlrp3 knockout.¹²⁹ In order to understand the role of NLRP3 in specific cells and tissues in different disease models, we also developed and published the first conditional Nlrp3 knockout mouse in collaboration with Ariel Feldstein, showing that loss of Nlrp3 expression in myeloid cells is crucial for the development of acute and chronic liver disease.¹³⁰ The availability of the conditional Nlrp3 knockout combined with increased accessibility of specific Cre mice has allowed for increased understanding of the role of NLRP3 in non-hematopoietic cells and in other immune cells outside of the myeloid compartment.¹³⁰

7.3 |. CAPS associated gain-of-function mutations in Nlrp3 in mice produce a CAPS phenotype

While deletion of Nlrp3 in mice established the role of NLRP3 in numerous mouse disease models, knockout mice did not provide direct information related to CAPS, where patients have gain-of-function mutations in NLRP3. Although we had access to CAPS patient cells and already had three effective therapies for patients, questions remained concerning disease mechanisms, particularly the response to cold in FCAS patients.¹³¹ Pre-clinical models to test novel therapies were also desirable. Therefore, in collaboration with Susannah Brydges and Dan Kastner, James Mueller and I generated three different conditional mutant Nlrp3 mouse models to cover the mild, moderate, and severe aspects of the CAPS spectrum and attempt to understand differences between the phenotypes observed with different mutations.¹¹⁴

The conditional nature of the mutations in these mice was fortuitous and allowed for a variety of useful experimental manipulations. Our initial approach was to recreate heterozygous mutations in all cells similar to most CAPS patients using a universal Cre, resulting in significant systemic inflammation, poor growth, and reduced survival in all of the mutant models. Laboratory evaluation showed neutrophilia in blood and elevation of many proinflammatory cytokines in serum including IL-1β, IL-18, IL-6, IL-8, and granulocyte-colony stimulating factor (G-CSF), but not TNF. Histologic analysis showed neutrophilia in skin, joints, conjunctiva, and the meninges, but not lungs, heart, kidneys, and gut consistent with the tissues known to be affected in CAPS. Neutrophilia was also observed in bone marrow, liver, and spleen, and muscle. Similar to CAPS patients, IL-1β and IL-6 protein expression was increased in affected skin.¹¹⁴ The primary difference between human and mouse CAPS was the overall increased severity of disease in these mice, with all of the mutations showing mortality ranging between birth and 6 weeks of life. However, the most unexpected observation was the reversal of the spectrum of severity in that the mice with the FCAS mutation (correlating to human p.L353P) were the most severely affected, and the mice with the NOMID mutation were the least severely affected, highlighting unexplained differences between humans and mice.^114,132 This curious pattern was extended with the later development of a mouse using the same construct and strategy but inserting a mutation with a more severe human NOMID phenotype. This mouse developed by Chiesa and colleagues was less severely affected than the NOMID mouse we generated, living long enough to observe the chronic sequela of systemic amyloidosis observed in some CAPS patients.¹³³ A similar reversed pattern of severity was also observed in our three mice bred to a tamoxifen-inducible Cre in which injecting these adult mice with tamoxifen resulted in systemic inflammation, rash, weight loss, and death.^134,135 While the mechanism of reversal in severity between humans and mice with mutations in NLRP3 is still unknown, the spectrum of severity in murine CAPS allow us to choose different models depending on the desired study or expected effect of a genetic or pharmacologic intervention.

7.4 |. Nlrp3 mutant mice reveal myeloid cells and neutrophils as mediating CAPS

To determine the role of specific immune cells in CAPS, we adopted two strategies. First, we bred our universal mutant mice to mice lacking specific cell types (i.e., T cells, Rag1 or mast cells, Kit^Wsh). The progeny of these crosses did not have altered phenotypes, suggesting that T cells and mast cells do not play significant roles in mouse CAPS. Second, we crossed mice whose NLRP3 mutant expression was Cre dependent with mice whose Cre expression was cell-specific, including lysozyme Cre (myeloid cells), Mcpt5 Cre (mast cells), and MRP8 Cre (neutrophils). Restriction of Nlrp3 expression to myeloid cells resulted in a similar phenotype to universal expression.¹¹⁴ This result suggested that myeloid cells are the primary drivers of CAPS phenotype and was consistent with observations in patients whose complete and typical CAPS phenotypes are due somatic mosaicism and expression of NLRP3 mutations primarily in myeloid cells. Similarly, breeding our Nlrp3 mutant mice to MRP8 Cre mice resulted in a phenotype similar to the lysozyme Cre mice suggesting an important role for neutrophils in mouse CAPS.^136,137 In contrast, breeding the mutant CAPS mouse to Mcpt5 Cre had no apparent phenotype indicating a minimal role for mast cells.¹³⁶ despite the urticarial nature of the CAPS rash and previous data suggesting a role for mast cells in another mouse model.¹³⁸

7.5 |. Nlrp3 mutant mice support both IL-1 dependent and independent pathophysiologies in CAPS

The discovery of NLRP3 as a platform for caspase 1 activation and the success of IL-1 targeted therapies in patients with CAPS pointed toward a major pathologic role for IL-1β, so it was natural that we would investigate the effect of blocking the IL-1 pathway downstream using genetic and pharmacologic approaches in murine models. Therefore, we bred our Nlrp3 mutant mice to IL-1β and IL-1R knockouts.^114,139 While there was some rescue, we still observed persistent rash and decreased growth and survival in the first few weeks of life in these mice. Interestingly, we observed some differences between IL-1β and IL-1R knockouts suggesting a possible role for IL-1α in mouse CAPS, which remains to be investigated. The MWS mutant IL-1β or IL-1R deficient mice that survived past 6 weeks had long-lasting scarring due to rash, but they had near normal growth and survival, more similar to human CAPS patients treated with IL-1 targeted therapy. We also treated mutant pups with a mouse rilonacept and showed partial rescue similar to the early rescue of the IL-1 targeted genetic approaches.¹¹⁴ We and others have also treated mutant adult inducible mice with recombinant IL-1RA (anakinra) with partial rescue of phenotype, but this was not as effective as an IL-1RA gene therapy in this model.¹⁴⁰

The identification of IL-1 independent mechanisms in the pathophysiology of murine CAPS directed our attention to other pro-inflammatory cytokines, beginning with IL-18, another inflammasome-associated cytokine requiring caspase 1 for cleavage and activation. Mutant mice treated with an IL-18 binding protein demonstrated significant improvement in rash, weight gain, survival, and liver pathology.¹⁴¹ Similarly, mutant Nlrp3 mice on an IL-18 or IL-18R deficient background revealed a strong phenotypic rescue in pups with complete resolution of rash, and normal growth and survival through the first 2 months of life in the MWS model. However, we did observe a later onset phenotype after 8 weeks of age characterized by weight loss, systemic and tissue inflammation, and ultimately reduced survival suggesting that at least in MWS mice, IL-18 drives the early CAPS phenotype and IL-1 drives the later CAPS phenotype. The logical next step was to generate an Nlrp3 mutant mouse on an IL-1β/IL-18 double knockout background to investigate a synergistic role for these cytokines, hypothesizing that the phenotypic rescue would be better than each cytokine alone. While our hypothesis was correct, to our surprise, we did identify IL-1/IL-18 independent mechanisms involved and demonstrated that these were at least partially driven by NLRP3-dependent pyroptotic cell death.¹³⁹

Using similar strategies, we also evaluated other pro-inflammatory cytokines besides those specifically regulated by NLRP3 including the downstream cytokines TNF, IL-17, and IL-6. Historically, treating CAPS patients with TNF targeted therapy did not have beneficial effects, but treating our Nlrp3 mutant mice with etanercept, a soluble TNF receptor, resulted in an unexpected rescue.¹⁴² Consistently, breeding the Nlrp3 mutant mice to TNF knockout mice also improved our previously reported primary outcomes in the pups including early tissue inflammation and fibrosis,^142,143 but similar to mutant IL-18 knockout mice, this rescue was short-lived as we observed the development of a late onset inflammatory phenotype. While IL-17 plays a major role in several inflammatory diseases and can be driven by IL-1β, we observed little change in the mouse CAPS phenotype when we bred our mutant mice on an IL-17a deficient background.¹⁴³ Similarly, IL-6, another well-known IL-1 driven pro-inflammatory cytokine had no obvious effect when deleted from the Nlrp3 mutant mice,¹⁴⁴ consistent with the lack of clinical effect when a patient with CAPS was treated with an IL-6 targeted therapy.¹⁴⁵

We have used similar genetic and pharmacologic approaches to tease apart upstream inflammasome pathways. Breeding our Nlrp3 mutant mice on an ASC-deficient background resulted in the only complete and long-lasting phenotypic rescue that we have identified to date.¹¹⁴ We have observed time-limited clinical improvement in our mice bred on caspase 1 knockout backgrounds, suggesting that several inflammasome independent pathways are driven by NLRP3. Finally, we and others have treated our mice with NLRP3 inhibitors demonstrating significant efficacy and providing the pre-clinical data for ongoing and planned clinical trials in CAPS patients.^97,146

8 |. CAPS AND NLRP3 ARE A CONSUMMATE EXAMPLE OF BEDSIDE TO BENCH AND BACK

The hallmark of autoinflammatory diseases has been a nearly side-by-side understanding of patient clinical phenotypes, human genetics and molecular modeling, and translation to therapy in patients. Over the last several decades, the collective contributions from physicians, scientists, and patients around the world have resulted in the approval of therapies blocking IL-1-induced inflammation in CAPS, with ongoing industry investment pursuing upstream targets, including NLRP3.

8.1 |. Early IL-1 targeted therapy in CAPS

The observation that cold exposure led to reproducible and easily observed inflammatory symptoms and laboratory changes in patients with FCAS led to an investigator-initiated trial in a small number of patients to determine whether pre-treatment with an anti-IL-1 therapeutic could prevent the NLRP3-specific cold-induced symptoms.¹¹⁶ At the time, the only approved drug in the IL-1 pathway was anakinra, a recombinant version of the naturally occurring IL-1R antagonist. This short acting injectable medicine was developed for the treatment of sepsis, but approved only for rheumatoid arthritis at the time. Self-appointed the “anakinra chicks,” three women with FCAS underwent purposeful cold exposure—45 min in a laboratory cold room—with and without pre-treatment with two doses of anakinra. Cold exposure prior to treatment led to the typical pattern of fever, rash, arthralgias, with rapid increases in WBC and serum IL-6 within hours. Symptoms failed to develop when patients were pre-treated for 24 h with anakinra. This somewhat unconventional approach demonstrating that IL-1 blockade could successfully reduce acute inflammatory symptoms in patients with gain-of-function NLRP3 mutations was similar to results observed in a more traditional study measuring chronic symptom and laboratory inflammation control in two patients with the more severe MWS.¹⁴⁷ These two proof of concept trials were the basis of a more complex and extremely successful clinical trial in patients with NOMID leading to ultimate approval by the FDA.¹⁴⁸

8.2 |. Currently approved IL-1 targeted therapies in CAPS

To date, three drugs targeting the IL-1 pathway are approved by the FDA and EMA for the treatment of CAPS spectrum disorders, namely anakinra, rilonacept, a recombinant IL-1R that binds to and inhibits IL-1α, IL-1β, and IL-1RA, and canakinumab, a human monoclonal antibody that binds specifically to IL-1β.^149,150 While each drug has different structure and mechanisms including the ability to block IL-1α and very different pharmacokinetics, they all have similar safety profiles and are well tolerated by patients. Decisions of which medicine to choose are often based on patient preference, availability, and costs/insurance limitations. Combined with their success in treating these rare disorders, the expansion of diseases implicating IL-1 in inflammatory disease pathogenesis has made this pathway a lucrative target for the pharmaceutical industry. Beyond inhibition of IL-1 and the IL-1R, development of other biologics and small molecule inhibitors targeting the different steps from NLRP3 assembly to IL-1 release is an active area, with CAPS patients often playing an integral part in the process (Table 3).

TABLE 3.

Integral role of CAPS patients in clinical trials.

NCT Number	Study Status	Conditions	Interventions
NCT05186051	Completed	CAPS	ZYIL1 capsule
NCT00991146	Completed	CAPS	Canakinumab
NCT04868968	Completed	FCAS	DFV890
NCT00288704	Completed	FCAS, MWS	Rilonacept vs. Placebo
NCT01302860	Completed	CAPS	ACZ885
NCT00465985	Completed	MWS	ACZ885 vs. Placebo
NCT01105507	Completed	CAPS	Canakinumab (ACZ885D)
NCT00214851	Completed	FCAS	Kineret (anakinra)
NCT01576367	Completed	CAPS	BIOLOGICAL: ACZ885
NCT00685373	Completed	CAPS	Canakinumab (ACZ885)
NCT04086602	Completed	CAPS	IZD334 vs. Placebo
NCT01045772	Completed	MWS	Rilonacept
NCT03923140	Recruiting	CAPS	Tranilast
NCT05812781	Recruiting	CAPS	VTX2735
NCT05670301	Recruiting	Autoinflammatory Disease, incl CAPS	Cytokine and lipidomic profiling

Abbreviations: CAPS, cryopyrin-associated periodic syndrome; FCAS, Familial cold autoinflammatory syndrome; MWS, Muckle Wells syndrome.

8.3 |. Other potential drug targets in the NLRP3 pathway

Despite the efficacy of IL-1 drugs in CAPS and other inflammatory disease, there has been a keen interest in other targets for a variety of reasons including finding oral available treatments and theoretical advantages of targeting specific mechanisms upstream of IL-1 and the IL-1R in order to preserve some IL-1 function through other inflammasomes. For example, specific inhibition of the NLRP3 pathway may reduce the risk of infections associated with more general IL-1 blockade.¹⁵¹ There was early interest in the blocking the enzymatic function of caspase 1 as a strategy to block IL-1 release resulting in inhibitors with reasonable specificity reaching later stages of development. We demonstrated that one of these compounds VX-765 was very effective at blocking ex vivo IL-1β release from FCAS patient cells.¹¹⁵ In vivo studies were initiated, but never completed nor reported. There has also been steady interest in the signaling pathways downstream of IL-1R such as MEK, IRAK4, and MK2 with initiation of pre-clinical studies in CAPS; however, the latter is the only published report.¹⁵² Other strategies to block the NLRP3 inflammasome pathway that are being studied and developed include ASC, NEK7, and gasdermin D but the largest focus has been on inhibiting NLRP3 directly beginning with the most prevalent NLRP3 targeting molecule initially called CRID3 (now known as MCC950).⁹⁷ While clinical development in this drug was halted years ago, it is a commonly used effective tool compound¹⁵³ and has been used to guide development of dozens of related and unrelated compounds currently in different stages of development. Many of these drugs have been studied in CAPS mice and patient cells,¹⁴⁶ and a few have been administered in vivo to CAPS patients.¹⁵⁴ In addition to small molecule inhibitors of NLRP3, we have also studied anti sense therapy as a novel approach to the treatment of CAPS and other NLRP3 driven diseases.¹⁵⁵ (Table 4).

TABLE 4.

NLRP3 targeted drugs in development.

Drug	Mechanism	Clinical trial
BMS-986299	Agonist	NCT03444753
Dapansutrile	Small molecular inhibitor	NCT03595371, NCT04540120
IZD334	Small molecule inhibitor	NCT04086602
ZYIL1	Small molecule inhibitor	NCT04731324
IZD174	Small molecule inhibitor	NCT04338997
AC-201	Small molecule inhibitor	NCT02287818
Tranilast	Small molecule inhibitor	NCT05130892, NCT03923140
Oridonin	Small molecule inhibitor	NCT05130892
VTX2735	Small molecule inhibitor	NCT05812781
DFV890	Small molecule inhibitor	NCT05552469, NCT04886258

9 |. EXPANDING ROLE FOR NLRP3 IN HUMAN DISEASE

The discovery of NLRP3 began with CAPS, but the extent of the roles that NLRP3 plays in the etiology of other diseases has extended well beyond this monogenic autoinflammatory disease. The first complex disease shown to be mediated by NLRP3 was gout, as monosodium urate (MSU) was the first of many crystals shown to activate the NLRP3 inflammasome leading to IL-1β mediated inflammation.⁹⁵ Early in vitro studies were later confirmed in vivo using NLRP3 deficient mice in models of gout.¹²⁹ This was later translated to humans in studies using IL-1 inhibitors in acute and chronic gout,¹⁵⁶ that preceded clinical trials of oral small molecule NLRP3 inhibitors currently in development.

International interest in the role of IL-1 mediated inflammation quickly linked the NLRP3 inflammasome to other common disorders. Early data suggested a role for the IL-1 pathway in nonalcoholic fatty liver disease (NAFLD) based on mouse models with IL-1RA deficient mice that developed worse liver pathology.¹⁵⁷ We hypothesized that NLRP3 drives the IL-1 mediated inflammation which was supported by liver pathology in our NLRP3 gain-of-function (CAPS) mice including neutrophilic inflammation, hepatic stellate cell activation, collagen formation, and fibrosis that was similar to that observed in diet-induced NAFLD mouse models. We also showed that diet-induced NAFLD models were protected in mice that were deficient for NLRP3.^158,159 This began a now decade long investigation into the upstream and downstream NLRP3-d riven mechanisms leading to NAFLD and the use of known and novel oral NLRP3 inhibitors for murine NAFLD, with a goal of translation to human patients.^160,161

In cardiovascular disease, the role of IL-1, macrophages and inflammation in coronary artery disease, atherosclerosis and myocardial infarction was similarly recognized¹⁶² with multiple investigators aiming to reduce morbidity and mortality by targeting the IL-1β pathway. In one of the largest studies to date, the CANTOS trial showed a modest but significant reduction in nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death in patients with a history of previous myocardial infarction and elevated C-reactive protein (CRP) who received canakinumab compared to placebo.¹⁶³ A specific role of NLRP3 activation in the mechanisms of ischemic heart disease and ventricular dysfunction was shown using our inducible mutant Nlrp3 mice.¹⁶⁴ In pediatrics, the role of NLRP3-mediated IL-1 signaling was also shown to play a key role in the development of aneurysms in Kawasaki disease, the most common cause of acquired cardiac disease in children in the United States.^165,166 These studies suggest that NLRP3 may be also be an appropriate therapeutic target across the spectrum of human heart disease.

Beyond these disorders, the role of NLRP3 and its downstream effects through IL-1 have been implicated in nearly every organ in humans. While some are perhaps more obvious given clear associations with known inflammasome triggers such as in silicosis¹⁶⁷ or asbestosis,¹⁶⁸ others such as neurodegenerative disorders¹⁶⁹ and ovarian aging¹⁵³ have been linked to excess NLRP3 activation and expression (Figure 8). For these challenging disorders, inhibitors of NLRP3 have been shown to reduce inflammation in vitro and in vivo, lending new opportunities for intervention.

FIGURE 8.

Expanding role of NLRP3 in human disease. The NLRP3 inflammasome has been linked via expression or mechanistic studies to numerous, more common human diseases including neurologic, ophthalmologic, cardiopulmonary, hepatic, renal, osteal and joint, as well as disorders of metabolism and reproduction. CNS, central nervous system; COPD, chronic obstructive pulmonary disease; HEENT, head, eyes, ears, nose, and throat; NAFLD, nonalcoholic fatty liver disease; NASH, Nonalcoholic steatohepatitis. Figure created with Biorender.com.

10 |. ONGOING QUESTIONS

Translationally, CAPS has demonstrated a true bench to bedside circle, with the identification of NLRP3, our understanding of the inflammasome and IL-1 release, and subsequent use of targeted therapies leading to heightened understanding of innate immune mechanisms, as well as providing a much-needed therapy to CAPS patients and others with IL-1 mediated disease. However, the expansion of NLRP3 association with other disorders, and increased sequencing of patients with signs and symptoms of autoinflammation, but features of non-monogenic disease has led to new challenges. For example, patients with NLRP3 variants and atypical CAPS phenotypes, including patients with variants in the LRR domains, may have hearing loss, with less prominent systemic inflammatory symptoms or urticarial-like rash.^170,171 Furthermore, increased recognition of low-penetrance variants,³⁵ oligogenic or digenic presentations,¹⁷² somatic mosaicism^29–32 and “mutation-negative” patients continue to be clinically challenging in both the diagnosis and long-term therapeutic management. These cases further highlight the need for molecular clinical testing to determine whether a variant or collection of variants are pathogenic, and to determine severity of the phenotype. Understanding how these complex genetic presentations lead to disease will be important to fully appreciate the role of NLRP3 in regulating innate immunity.

Additionally, with NLRP3-mutation positive patients as proof-of-principle that inflammasome-induced inflammation could be successfully targeted with anti-IL-1 therapies, there was excitement that many more common diseases could similarly be treated with one of the three approved compounds: anakinra, canakinumab, or rilonacept. In theory, the different mechanisms of action would allow better understanding of inflammation in these disorders, as it has for CAPS. In clinical experience, however, the results and successes have not necessarily been as clear, likely due to polygenic and environmental contributions to these disorders, as well as incompletely understood intra-IL-1 family member regulatory mechanisms. Harnessing this pathway, rather than simply inhibiting it, could be equally useful in more complex, evolving diseases such as cancer¹⁷³ where precise timing of turning on and turning off inflammation may better direct immune responses, inhibit disease progression, and identify adjunct therapies.

11 |. FUTURE DIRECTIONS AND CONCLUSIONS

Although we now have effective biologic therapies for CAPS, there remains an unmet medical need for less expensive small molecule inhibitors that target the inflammasome directly or the downstream inflammatory pathways of IL-1. Such targeted therapy would likely have significant impact on the treatment of more common inflammasome-mediated disorders as well. While the last several decades have led to significant advances in our understanding of the role of the inflammasome in innate immunity and human disease, unanswered questions remain concerning CAPS pathophysiology including the unique tissue involvement of these systemic inflammatory disorders, the specific hematopoietic and somatic cells involved in autoinflammatory disease pathogenesis, and the best way to target NLRP3 involvement in related but polygenic disorders. The integration of patients, physicians, and scientists will continue to be integral to understanding how one unique protein can carefully orchestrate the innate immune response.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available at publicly available repositories.

The NLRP3 mutations in CAPS and undefined autoinflammatory diseases were derived from the Infevers database at https://infevers.umai-montpellier.fr/web/index.php. The structures of NLRP3, ASC pyrin domain, Caspase 1 CARD domain were downloaded from the RSCB Protein database at https://www.rcsb.org/ with accessions 5fna, 6npy, 7keu, 7pzc, 7pzd, 7zgu, and 8ej4. The sequences of the mammalian NLRP3 homologs were downloaded from the NCBI protein sequence database at https://www.ncbi.nlm.nih.gov/protein/ with accessions MK829787.1, NM_145827.4, XM_006162028.3, XM_010807177.3, XM_023540331.1, XM_033095764.1, XM_03544093.1, NM_004895.5, XM_004481057.3, XM_008579966.1, XM_016193428.1, XM_023618258.1, XM_036904180.2, and XM_042782806.1. The clinical trial information were downloaded from https://clinicaltrials.gov/ with accessions NCT05186051, NCT00991146, NCT04868968, NCT00288704, NCT01302860, NCT00465985, NCT01105507, NCT00214851, NCT01576367, NCT00685373, NCT04086602, NCT01045772, NCT03923140, NCT05812781, NCT05670301. NCT03444753, NCT03595371, NCT04540120, NCT04086602, NCT04731324, NCT04338997, NCT02287818, NCT05130892, NCT03923140, NCT05130892, NCT05812781, NCT05552469, and NCT04886258.