NLRP3 inflammasome and its inhibitors: a review

1The mammalian immune system defends against internal and external threats using innate immunity and adaptive immunity. The innate immune response relies on pattern-recognition receptors (PRRs) to target pathogenic microbes and other endogenous or exogenous pathogens. PRRs are expressed mainly in immune and inflammatory cells such as monocytes, macrophages, neutrophils, and dendritic cells (DCs). They present antigens to the adaptive immune system to generate long-lasting protection. Pathogen-associated molecular patterns (PAMPs), which are antigens common to a given group of pathogens, are normally recognized by at least three PRRs: Toll-like receptors (TLRs), C-type lectins (CTLs), and Galectins. The innate immune system is evolutionarily conserved across vertebrates and invertebrates, which means that both human and animal studies can provide valuable insights into innate immunity.

2A newly identified PRR, first described in detail in 2002, is the inflammasome. Numerous inflammasomes have been identified, including NLRP1, NLRP2, NLRP3, double-stranded DNA (dsDNA) sensors absent in melanoma 2 (AIM2) and NLRC4. The best characterized is the NLRP3 inflammasome, so named because the NLRP3 protein in the complex belongs to the family of nucleotide-binding and oligomerization domain-like receptors (NLRs) and is also known as “pyrin domain-containing protein 3”. In addition to the NLRP3 protein, the NLRP3 inflammasome also contains adapter protein apoptosis-associated speck-like protein (ASC) and procaspase-1. Interactions among these three proteins tightly regulate inflammasome function in order to ensure immune activity only when appropriate.

3In the absence of immune activators, an internal interaction occurs between the NACHT domain and leucine-rich repeats (LRRs), suppressing the interaction between NLRP3 and ASC, thus preventing assembly of the inflammasome. In the presence of immune activators such as PAMPs, danger-associated molecular patterns (DAMPs), other exogenous invaders or environmental stress, NLRP3 opens up and allows interaction between the pyrin domains (PYDs) in NLRP3 and ASC. Subsequently the caspase recruitment domain (CARD) of ASC binds to the CARD domain on procaspase-1, giving rise to the NLRP3 inflammasome. Formation of this complex triggers procaspase-1 self-cleavage, generating the active caspase-1 p10/p20 tetramer and inducing the conversion of proinflammatory cytokines interleukin (IL)-1β and IL-18 from their immature “pro” forms to active forms that are secreted. Formation of the inflammasome also triggers a process of inflammation-related cell death termed pyroptosis.

4The NLRP3 inflammasome is present primarily in immune and inflammatory cells following activation by inflammatory stimuli; these cells include macrophages, monocytes, DCs, and splenic neutrophils. Activation of the NLRP3 inflammasome appears to occur in two steps (;;; Figure 1). The first step involves a priming or initiating signal in which many PAMPs or DAMPs are recognized by TLRs, leading to activation of nuclear factor kappa B (NF-κB)-mediated signaling, which in turn up-regulates transcription of inflammasome-related components, including inactive NLRP3, proIL-1β, and proIL-18. This priming step is often studied in vitro using lipopolysaccharide (LPS; ). The second step of inflammasome activation is the oligomerization of NLRP3 and subsequent assembly of NLRP3, ASC, and procaspase-1 into a complex. This triggers the transformation of procaspase-1 to caspase-1, as well as the production and secretion of mature IL-1β and IL-18.

5Three models have been proposed to describe the second step of inflammasome activation, as described in detail by (shown in Figure 1). Briefly, all models assume that NLRP3 does not directly interact with exogenous activators, consistent with its ability to sense various pathogens. In the first model, extracellular adenosine triphosphate (ATP), which acts as an NLRP3 agonist, induces K+ efflux through a purogenic P2X7-dependent pore consisting of a pannexin-1 hemichannel. This process leads to NLRP3 inflammasome activation and assembly. Consistent with this model, K+ efflux is a major activator of the NLRP3 inflammasome, while extracellular ATP and pore-forming toxins are the major triggers of IL-1β secretion by the inflammasome. Fluxes of intracellular and endoplasmic reticulum (ER)-related Ca2+ may also activate the NLRP3 inflammasome.

6In the second model, all known PAMPs and DAMPs, including the activators mentioned above, trigger the generation of reactive oxygen species (ROS), which in turn induce assembly of the NLRP3 inflammasome. For example, damage to NADPH oxidase and other oxidative systems by mitochondrial ROS can activate the inflammasome.

7In the third model, assembly and activation of the NLRP3 inflammasome is thought to be triggered by environmental irritants (such as silica, asbestos, amyloid-β, and alum) which form crystalline or particulate structures when engulfed by phagocytes. These aggregates cause lysosomal rupture and release of lysosomal contents via a mechanism mediated by cathepsin B. Consistent with this model, crystalline stimuli such as silica are major triggers of IL-1β secretion by the inflammasome.

8Other factors can also activate the NLRP3 inflammasome. These include mitochondrial damage or dysfunction caused by mitochondrial Ca2+ overload, lysosomal disruption, autophagic dysfunction and the activity of thioredoxin-interacting protein (TXNIP;; ).

9While the innate immune response to insults can efficiently protect against disease and death, inappropriate activation of the NLRP3 inflammasome can contribute to the onset and progression of various diseases, particularly age-related diseases such as metabolic disorders and metabolic syndrome. Increased production of IL-1β and IL-18 by the NLRP3 inflammasome contributes to atherosclerotic plaque progression and instability in atherosclerotic patients and animal models. For example, showed that genetic ablation of the NLRP3 inflammasome suppressor known as the inhibitor of κB kinase epsilon (IKBKE) enhanced the acute phase response and down-regulated cholesterol metabolism in cultured macrophages and hypercholesterolemic mice. Atherosclerosis and other inflammatory diseases were more severe in animals with the ablation.

10Studies in macrophages and animal models have shown that oxidized low-density lipoprotein and cholesterol crystals trigger NLRP3 inflammasome activation. In macrophage and animal models of type II diabetes, hyperglycemia, and free fatty acids trigger inflammasome activation, which harms glucose metabolism and strengthens insulin resistance. In macrophage and animal models of uric acid accumulation, monosodium urate crystals activate the NLRP3 inflammasome, causing gout. Taken together, these findings suggest that during the progression of many metabolic diseases, the accumulation of abnormal metabolic products activates the NLRP3 inflammasome. Studies in animal models suggest a similar picture in Alzheimer’s disease and obesity induced by a high-fat diet.

11In macrophages and in animal models, studies have also defined a role for the NLRP3 inflammasome in the initiation and development of cerebral and myocardial ischemic diseases, including cerebral ischemia/stroke and myocardial ischemia. Inflammasome activation appears to contribute to post-ischemic inflammation after stroke. For example, showed that using ibrutinib to inhibit Bruton’s tyrosine kinase (BTK), an essential component of the NLRP3 inflammasome, reduced infarct volume, and neurological damage in a mouse model of cerebral ischemia/reperfusion injury. In addition, it is reported by that activation of nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10 inhibited ATP-mediated IL-1β release by human and rat monocytes, helping protect them from collateral damage. NLRP3 inflammasome-related proteins are up-regulated in myocardial fibroblasts following infarction, and this up-regulation may contribute to infarct size in ischemia-reperfusion injury. Consistent with this idea, inhibiting the NLRP3 inflammasome reduces myocardial injury after ischemia-reperfusion in mice.

12NLRP3 inflammasome activation has also been linked to various auto-immune and auto-inflammatory diseases. Work from our laboratory and others has demonstrated that NLRP3 inflammasome activation contributes to progression of multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in animal models. Severity of multiple sclerosis in patients correlates closely with levels of IL-1β, IL-18, and caspase-1; the serum levels of both ILs and of active caspase-1 (p20) are elevated in mice with EAE. Studies in macrophages and mouse models of colitis have linked abnormal NLRP3 inflammasome activation with inflammatory bowel disease, including ulcerative colitis and Crohn’s disease. Polymorphism in the NLRP3 gene is linked to colitis severity and progression in patients, and gain-of-function mutations in the NLRP3 gene that increase production and secretion of IL-1β and IL-18 are associated with cryopyrin-associated periodic fever syndrome (CAPS;;; ). This syndrome comprises several rare hereditary auto-inflammatory diseases in humans and animal models, including familial cold auto-inflammatory syndrome and Muckle–Wells syndrome. Inhibiting IL-1 using specific blocking agents effectively reduces systemic inflammation in CAPS patients.

13The extensive involvement of the NLRP3 inflammasome in such a range of diseases makes it a highly desirable drug target. Fortunately numerous promising inhibitors of NLRP3 inflammasome activation have been described, several of which are briefly described below together with their pharmacological mechanisms (shown in Table 1).

14Two small-molecule inhibitors of the NLRP3 inflammasome were described in groundbreaking reports in Nature Medicine this year. discovered that MCC950, a diarylsulfonylurea-containing compound known to inhibit caspase-1-dependent processing of IL-1β, also inhibits both canonical and non-canonical activation of the NLRP3 inflammasome. MCC950 inhibits secretion of IL-1β and NLRP3-induced ASC oligomerization in mouse and human macrophages. It reduces secretion of IL-1β and IL-18, alleviating the severity of EAE and CAPS in mouse models. further showed that MCC950 acts specifically on the NLRP3 inflammasome: it does not inhibit the activation of NLRP1, AIM2, or NLRC4 inflammasomes. have shown that MCC950 inhibits LPS-induced production of IL-1β via a mechanism dependent on the cytoplasmic LPS sensors caspase-4 and caspase-5. demonstrated that hypertension in mice treated with salt and deoxycorticosterone acetate can be reversed by treating them with MCC950, and this reversal depends on the inhibition of inflammasome activation and inflammasome-related IL-1β production.

15discovered that the ketone metabolite β-hydroxybutyrate (BHB), but not acetoacetate or the short-chain fatty acids butyrate and acetate, reduced IL-1β, and IL-18 production by the NLRP3 inflammasome in human monocytes. Like MCC950, BHB appears to block inflammasome activation by inhibiting NLRP3-induced ASC oligomerization. Their in vivo experiments showed that BHB or a ketogenic diet alleviate caspase-1 activation and caspase-1-mediated IL-1β production and secretion, without affecting the activation of NLRC4 or AIM2 inflammasomes. BHB inhibits NLRP3 inflammasome activation independently of AMP-activated protein kinase, ROS, autophagy, or glycolytic inhibition. These studies raise interesting questions about interactions among ketone bodies, metabolic products, and innate immunity. BHB levels increase in response to starvation, caloric restriction, high-intensity exercise, or a low-carbohydrate ketogenic diet. Vital organs such as the heart and brain can exploit BHB as an alternative energy source during exercise or caloric deficiency. Future studies should examine how innate immunity, particularly the inflammasome, is influenced by ketones and other alternative metabolic fuels during periods of energy deficiency.

16Although both MCC950 and BHB inhibit NLRP3 inflammasome activation, their mechanisms differ in key respects. BHB inhibits K+ efflux from macrophages, while MCC950 does not. MCC950 inhibits both canonical and non-canonical inflammasome activation, while BHB affects only canonical activation. Nevertheless both inhibitors represent a significant advance toward developing therapies that target IL-1β and IL-18 production by the NLRP3 inflammasome in various diseases.

17In contrast to these newly described, NLRP3-specific inflammasome inhibitors, type I interferons (IFNs), including IFN-α and IFN-β, have been used for some time to inhibit the NLRP3 and other inflammasomes in various auto-immune and auto-inflammatory diseases. These diseases include multiple sclerosis, systemic-onset juvenile idiopathic arthritis caused by gain-of-function NLRP3 mutations, rheumatic diseases and familial-type Mediterranean fever. Type I IFNs are produced by specialized immune cells such as macrophages and DCs in response to extracellular stimuli such as bacteria and virus as well as various environmental irritants. These IFNs are recognized by the type I IFN receptor (IFNAR), which is a member of the TLR family and is composed of the subunits IFNAR1 and IFNAR2. IFNAR activation involves several proteins, including Janus kinases, tyrosine kinase 2, and several kinds of signal transducers and activators of transcriptions (STATs). However, how type I IFNs affect NLRP3 inflammasome and its production of IL-1β and IL-18 remains unclear, despite numerous studies aimed to improve IFN-based treatments of NLRP3 inflammasome-related diseases. To provide an example of progress in this area, we focus below on studies of IFN therapy against multiple sclerosis in patients and EAE in mice, since type I IFN therapy has been used as a first-line or standard treatment of multiple sclerosis for 15 years.

18classified 97 patients with multiple sclerosis into those who responded to IFN-β therapy and those who did not, based on clinico-radiological criteria at 12 and 24 months of treatment. They found that expression of NLRP3 protein and levels of IL-1β were significantly lower among responsive patients who had relapsing-remitting multiple sclerosis than among other patients. found that IL-1β production by primary monocytes was lower in multiple sclerosis patients on IFN-β treatment than in healthy subjects, supporting the value of IFN-β therapy. Studies in mouse bone marrow-derived macrophages by suggest that IFN-β may inhibit IL-1β production through at least two mechanisms. In one pathway, phosphorylation of STAT1 transcription factor leads to repression of NLRP1 and NLRP3 inflammasomes, which in turn inhibits caspase-1-dependent IL-1β maturation. In the second pathway, type I IFNs induce IL-10 production via a STAT-dependent mechanism, and the IL-10 works in an autocrine fashion to reduce levels of pro-IL-1α and pro-IL-1β via a mechanism dependent on STAT3 signaling.

19Type I IFN treatment is not effective for all types of multiple sclerosis, and the NLRP3 inflammasome may be a key determinant. conducted studies on mouse primary macrophage cultures as well as EAE mice and concluded that IFN-β therapy is effective only when the NLRP3 inflammasome contributes directly to the disease process. Their studies further showed that IFNAR activation could be inhibited using the suppressor of cytokine signal 1 (SOCS1), which inhibited Rac1 activation and ROS generation, leading in turn to inhibition of NLRP3 inflammasome activity and less severe EAE.

20These studies highlight the efficacy of type I IFN therapy and the need for future studies to elucidate the mechanisms of NLRP3 inflammasome inhibition. This work may improve clinical approaches to treating multiple sclerosis and other auto-immune and auto-inflammatory diseases.

21Several additional ways for inhibiting the NLRP3 inflammasome have opened up in recent years. Autophagy, a self-protective catabolic pathway involving lysosomes, has been shown to inhibit the NLRP3 inflammasome, leading researchers to explore the usefulness of autophagy-inducing treatments. showed that the plant polyphenolic compound resveratrol, known to induce autophagy, suppresses mitochondrial damage in macrophages and thereby inhibits NLRP3 inflammasome activation and NLRP3 inflammasome-mediated IL-1β secretion and pyroptosis. showed that arglabin inhibits the production and secretion of IL-1β and IL-18 by the NLRP3 inflammasome in a concentration-dependent manner in ApoE-/- mice on a high-fat diet. The reduced IL production translates to less severe atherosclerosis. Those authors reported that arglabin exerts its effects in macrophages by inducing autophagy as well as by reducing inflammation and cholesterol levels.

22Cannabinoid receptor 2 (CB2R) is an already demonstrated therapeutic target in inflammation-related diseases. Work from our own laboratory has shown that autophagy induction may help explain why activation of the anti-inflammatory CB2R leads to inhibition of NLRP3 inflammasome priming and activation in mouse BV2 microglia stimulated with LPS and ATP as well as in a mouse model of EAE. Such CB2R activation reduces the severity of EAE in mice. Thus CB2R agonists similar to the HU-308 used in our work may become an effective therapy for treating NLRP3 inflammasome-related diseases by inducing autophagy.

23MicroRNAs may provide another route for inhibiting inflammasomes. These endogenous non-coding RNAs are 20–23 nt long and bind to the 3′ untranslated region (3′ UTR) of protein-coding mRNAs to regulate their translation. MicroRNA-223 binds to a conserved site in the 3′ UTR of the NLRP3 transcript, suppressing protein expression and thereby inhibiting NLRP3 inflammasome priming and IL-1β production. Deficiency in microRNA-223 leads to neutrophilia, spontaneous lung inflammation, and increased susceptibility to endotoxin challenge in mice. Several other microRNAs have been reported to be involved in the activation of the NLRP3 inflammasome, including microRNA-155, microRNA-377, and microRNA-133a-1. Reducing the levels of these factors may be useful for treating inflammasome-related disease.

24The past decade has witnessed tremendous progress in understanding the structure and activation of the NLRP3 inflammasome, as well as its roles in the initiation and progression of various auto-immune and auto-inflammatory diseases, including metabolic disorders, multiple sclerosis, inflammatory bowel syndrome, and CAPS. Several types of NLRP3 inflammasome inhibitors have been developed and validated in cell culture studies and animal models of NLRP3 inflammasome-related diseases, and type I IFNs have become well established in the clinic. On the other hand, several agents have proven ineffective in clinical settings, and several potential inhibitors require further development, such as autophagy-inducing and microRNA agents. This highlights the need for further research into what pathways activate the NLRP3 inflammasome and can therefore be targeted by appropriate inhibitors. There is still a long way to go toward exploiting NLRP3 inflammasome inhibitors in our fight against diseases.

25B-ZS and Z-QX were in charge of searching all the relative papers and writing this manuscript. B-ZH was in charge of drawing the picture. CL gave her valuable and professional suggestions and guide in organizing and drafting this manuscript. D-FS helped to revise the manuscript.

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.