1. Brain

Recent work in the field has identified sterile inflammation as a key contributor to neurological diseases. Of particular interest is the impact of NLRP3-mediated inflammation on the pathologic processes that contribute to neurological damage. Novel drugs that arrest or reverse the effects of chronic neuroinflammation are desperately required for many neurological diseases, and NLRP3 is an exciting target in this regard.

a. Parkinson disease

Parkinson disease (PD) patients exhibit substantial loss of dopaminergic neurons, chronic neuroinflammation, and α-synuclein inclusions in the form of Lewy bodies, all of which ultimately contribute to disease pathology. Patients with PD have elevated plasma IL-1β relative to healthy donors (Wang et al., 2020a). The central nervous system (CNS) inflammatory component in PD can be attributed mainly to activated microglial cells (Le et al., 2016), where MCC950 has shown potent inhibitory effects (IL-1β release IC₅₀ = 5 nM) on primary mouse microglia (Gordon et al., 2018). Early work in the field identified dopamine as a novel inhibitor of NLRP3 activity via dopamine D1 receptor (Yan et al., 2015). Macrophages from Parkinson patients with PARK2 mutations produce an elevated response to specific NLRP3 inflammasome activators, a response that was negated in the presence of MCC950 (Mouton‐Liger et al., 2018). More recently, the contributory role of NLRP3 activation in PD pathology was further validated in post-mortem PD brains and multiple preclinical animal models of PD; MitoPark, α-synuclein preformed fibril pathology and neurotoxic 6-OHDA administration (Gordon et al., 2018). Unwarranted activation of NLRP3 by fibrillar synuclein was shown to lead to dopaminergic degeneration in microglia, which in turn leads to exacerbation of the disease. Chronic administration of MCC950 effectively blocked fibrillar synuclein-mediated motor deficits, dopamine loss, and pathological synuclein spread in vivo. Very similar neuroprotective effects were observed in the 6-OHDA model (Fig. 2). Dopamine is known to reduce mRNA and protein expression of TNF-α, IL-6, and IL-1β in RAW 264.7 cells stimulated with LPS (Liu and Ding, 2019), thus providing a negative feedback loop. Dopamine treatment also inhibited the mRNA and protein expression levels of nitric oxide synthase and downregulated NLRP3 and caspase-1 expression. Both the administration of dopamine and MCC950 played a synergistic role in suppressing NLRP3 activity (Liu and Ding, 2019). MCC950 reduced neuroinflammation and preserved neurons in the substantia nigra when compared with untreated animals in Atg5-deficient mice (Cheng et al., 2020). Autophagy is a critical homeostatic process in which cytosolic components are degraded and recycled through lysosomes. A recent study demonstrated that loss of microglial autophagy led to cellular activation in an NLRP3-dependent manner. Deletion of Atg5, which is required for autophagy, promoted the activation of NLRP3 via phosphodiesterase 10A–cAMP signaling in mice. This activation results in increased inflammatory readouts in the brain and neurological impairment. Taken together, these data suggest that blocking IL-1β production and pyroptosis with MCC950 in the murine brain can rescue dopaminergic neuronal function, leading to increased levels of dopamine in the CNS, which in turn leads to downregulation of NLRP3 activation and an effective block of a chronic cycle of neuroinflammation.

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Fig. 2.

NLRP3 inhibition with oral MCC950 treatment is efficacious in the 6-OHDA model of PD and protects against nigrostriatal dopaminergic degeneration and behavioral deficits. Balance beam (B), Striatal dopamine (C), and its metabolite DOPAC (D) 28 days after PBS or 6-OHDA injection (n = 7 to 8 mice per group). Data are means ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test (C) or Kruskal-Wallis test with Dunn’s post hoc test (C). DOPAC, 3,4-dihydroxyphenylacetic acid. Reprinted (in part) with permission from Gordon R, Albornoz EA, Christie DC, Langley MR, Kumar V, Mantovani S, Robertson AAB, Butler MS, Rowe DB, O’Neill LA, Kanthasamy AG, Schroder K, Cooper MA and Woodruff TM (2018) Inflammasome inhibition prevents alpha-synuclein pathology and dopaminergic neurodegeneration in mice. Science Translational Medicine. Copyright 2018 The American Association for the Advancement of Science.

2. Heart

Atherosclerosis is generally accepted to be both a cholesterol storage disease and a chronic inflammatory disease, with chronic NLRP3 activation now implicated in disease progression.

Foam cells, which result from the uncontrolled uptake of LDL by macrophages, play a key role in the initiation and pathologic progression of atherosclerosis. Lipid retention is the primary step in this process, which is followed by cholesterol esterification, cholesterol efflux, and chronic inflammation at susceptible sites. In vitro in THP-1 cells, MCC950 attenuates macrophage foam cell formation and diminishes uptake of oxLDL and facilitates efflux of the cholesterol to either apolipoprotein A1 or high-density lipoprotein (Chen et al., 2017a). Vascular smooth muscle cells (VSMCs) are also a dominant cellular constituent of arteries and critical determinant of vascular disease. Foam cells can originate both from the circulating monocytes/macrophages and from the VSMCs migrating to the growing lesion. Molecular mechanisms for VSMC transformation toward foam cells remain unknown but do involve the uptake of lipids. In a recent study, inhibition of the NLRP3 inflammasome with MCC950 reduced the oxLDL-induced accumulation of lipid droplets and intracellular cholesterol levels in VSMCs. MCC950 also inhibited oxLDL-induced monocyte adhesion to VSMCs and impaired upregulation of monocyte chemoattractant protein-1 and vascular cell adhesion molecule 1 (VCAM-1) in response to oxLDL (Zang et al., 2019).

MCC950 is protective against trimethylamine-N-oxide–promoted atherosclerosis that activates NLRP3 in human umbilical vein endothelial cells (Chen et al., 2017b). A similar picture is also seen in vivo in the inhibition of atherosclerotic lesions. In Western-type diet-fed apoE^−/− mice with atherosclerotic plaques, MCC950 attenuated the atherosclerotic process as measured by stenosis, plaque size, and plaque volume (Fig. 3) (van der Heijden et al., 2017). Although the necrotic core size of the plaque remained unchanged, the number of infiltrating macrophages was significantly impaired, indicating a reduced inflammatory state. Furthermore, decreased mRNA expression of VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) was detected in the carotids of MCC950-treated mice (van der Heijden et al., 2017).

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Fig. 3.

MCC950 treatment is effective in an atherosclerotic model. “MCC950 treatment of apoE−/− mice resulted in (A) reduced plaque macrophage content, both relative to plaque size and in absolute area, but did not affect collagen content (B) or necrotic area (C) of the carotid artery plaques (magnification ×100). Gene expression analysis of these carotid artery plaques established that MCC950 treatment did not affect NLRP3 inflammasome–related genes (D), but reduced the expression of the adhesion molecules VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1; (E); *P < 0.05, ***P < 0.001 compared with control).” MOMA-2, anti-macrophages/monocytes antibody; Casp-1, caspase-1. Reprinted (in part) with permission from van der Heijden T, Kritikou E, Venema W, van Duijn J, van Santbrink PJ, Slutter B, Foks AC, Bot I and Kuiper J (2017) NLRP3 Inflammasome Inhibition by MCC950 Reduces Atherosclerotic Lesion Development in Apolipoprotein E-Deficient Mice-Brief Report. Arteriosclerosis, Thrombosis, and Vascular Biology 37:1457-1461. Copyright 2017 Wolters Kluwer Health, Inc.

In support of this, MCC950 showed athero-protective activity in a separate model. Age-associated mutations in a gene encoding the epigenetic modifier enzyme 10-11 translocation 2 (TET2) induce clonal hematopoiesis that is correlated with an increased risk of atherosclerosis. TET2-deficient animals can be used as models of atherosclerotic processes. Using this model, TET2-deficient cells displayed an increase in atherosclerotic plaque size linked to an increase in NLRP3 inflammasome–mediated IL-1β secretion, which could be negated with MCC950, leading to a reduction in plaque size. This study suggests that a greater therapeutic effect may be seen in TET2-deficient patients over patients not carrying mutations in TET2 (Fuster et al., 2017).

TET2 has not only been linked to atherosclerosis but also more broadly to heart failure. Partial inactivation of TET2 in hemopoietic cells induces cardiac dysfunction in two separate models of heart failure. Firstly, permanent ligation of the left anterior descending artery produces a severe ischemic model that simulates remodeling of the heart after scar formation. MCC950 treatment promoted protection against cardiac remodeling. Secondly, the TAC model involves pressure overload–causing systolic dysfunction in response to capillary rarefaction, leading to a cardiac hypertrophic response. MCC950 impaired the development of this cardiac hypertrophy and reduced heart size, cardiac function, and interstitial fibrosis when compared with the control animals (Sano et al., 2018). MCC950 also had a beneficial effect on cardiac fibrosis and remodeling in a mouse model of angiotensin II (Ang II) infusion–induced hypertension (Gan et al., 2018). MCC950 reduced IL-1β levels after Ang II infusion and reduced the extent of cardiac fibrosis. MCC950 also had beneficial effects in a second model in which wild‐type mice were challenged with a high‐fat, high‐cholesterol diet and Ang II infusion. Aortic dilatation, dissection, and rupture was reduced by MCC950 (Ren et al., 2020).

Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Although reperfusion can yield successes in limiting damage to the heart and improving overall prognosis, the patients' risk of heart failure increases both over the short term and long-term post-MI. MI-induced injury causes acute necrosis of cardiomyocytes and the generation of a fibrotic scar. Excessive interstitial fibrosis in the heart after MI might lead to progressively worse cardiac function and stiffening of the wall and septal. Inflammation is a key contributing factor to the post-MI pathology. Inflammatory processes can further aggravate cardiac remodeling and myocardial injury. Indeed, the damning role inflammation alone plays in the reoccurrence of myocardial infraction has been tested by the undertaking of the Canakinumab Anti-Inflammatory Thrombosis Outcome Study (CANTOS-2) trial (Ridker et al., 2017a), in which canakinumab, an IL-1β–neutralizing monoclonal antibody, showed a 150-mg dose could prevent adverse cardiac events over placebo in patients with a history of MI and elevated high-sensitivity C-reactive protein. Inflammasome activation has been implicated in the loss of functional myocardium during this period, leading to heart failure. MCC950 has efficacy in a mouse model of left coronary artery ligation-induced MI. The ejection fraction in the 10-mg/kg group (40.7 ± 4.2%; N = 6, P = 0.0029) was statistically preserved compared with that in the control group (14.0 ± 4.4%). Myocardial fibrosis was reduced in MCC950-treated animals (MCC950, 23.2 ± 3.0 vs. PBS, 36.2 ± 3.7; P < 0.05). Myocardial NLRP3, cleaved IL-1β, and IL-18 levels were reduced in MCC950-treated animals. Histopathology and biomarker examination revealed decreases in inflammatory cell infiltration and inflammatory factor expression in the heart. In vitro, MCC950 inhibited NLRP3, reduced caspase-1 activity, and further downregulated IL-1β and IL-18 (Gao et al., 2019).

In higher organisms, MCC950 conferred protection in a myocardial infarction porcine model (van Hout et al., 2017). Cardiac ischemia-reperfusion initiates sterile inflammation, and although this process is critical for wound healing, the resulting inflammatory response leads to an increased infarct size and has a deleterious effect on cardiac function (Arslan et al., 2011; Timmers et al., 2012). Infarct size and decreased cardiac function are long-term predictors of harmful cardiac remodeling and heart failure (Sutton and Sharpe, 2000). MCC950 treatment has been shown to dose-dependently reduce infarct size and improve left ventricular function 7 days post-MI. Interestingly the beneficial effect of MCC950 on cardiac function appeared only at later time points, suggesting that NLRP3-inflammasome inhibition acts primarily to attenuate the inflammatory response in the subacute phase after MI. Additional observations were reported for the MCC950-treated animals, such as a decrease in circulating markers of damage and inflammation and reduced myocardial infiltration by circulating neutrophils (van Hout et al., 2017).

¹⁸F-FDG positron emission tomography imaging has been used in a novel manner to determine the therapeutic effects of MCC950 in an acute MI murine model. Mice were treated with MCC950 or sterile saline by intraperitoneal after surgery and then daily for 7 consecutive days. ¹⁸F-FDG positron emission tomography (inflammation) imaging was used to monitor inflammatory changes on days 3 and 5. The MCC950-treated group showed lower ¹⁸F-FDG inflammatory uptakes at the infarct region when compared with the control MI group. Furthermore, macrophage and neutrophil infiltration into the region was reduced, and NLRP3-dependent inflammatory markers were reduced. The extent of adverse myocardial remodeling was impaired by MCC950, and mortality rate of the animals was reduced. Overall, the authors concluded that MCC950 demonstrated a signature of myocardial preservation when compared with the saline-treated controls demonstrating the therapeutic potential of NLRP3 inhibition in acute MI (Li et al., 2020d).

NLRP3-inflammasome activity has also been documented to be increased in atrial cardiomyocytes of atrial fibrillation patients (Yao et al., 2018). Cardiomyocyte mice develop spontaneous premature atrial contractions and inducible atrial fibrillation, which is attenuated by MCC950. In support of this, genetic knockdown of NLRP3 prevents atrial fibrillation development in cAMP Response Element Modulator transgenic mice. This study demonstrates a novel pathophysiological role for NLRP3-inflammasome signaling in cardiomyocytes with a mechanistic link to the pathogenesis of atrial fibrillation (Yao et al., 2018).

MCC950 protected against cardiac injury induced by metabolic dysfunction induced by high-caloric diets. Genetic ablation of NLRP3 protected against adverse effects from high-sugar and high-fat obesogenic diets. Not only did MCC950 impair weight gain, but it also decreased cardiomyocyte area and perivascular fibrosis, which are a feature of these diets. Other pathologic features of obesogenic diets, such as elevated levels of adiponectin and the promotion of favorable leptin and leptin/adiponectin levels, were observed in the MCC950-treated animals. Systemic markers of tissue damage and inflammation were ameliorated in MCC950-treated animals (Pavillard et al., 2017).

Heart disease can present in postmenopausal women, and some reports suggest that it may be attributed to a fault in estrogen signaling leading to NLRP3 activation (Wang et al., 2019a). The estrogen receptor, G-protein–coupled estrogen receptor (GPER), is deactivated by its ligand. Loss of this estrogen deactivation results in the increased expression of NLRP3 and IL-18. Therefore, GPER KOs can be used as a model for heart failure in this context. When compared with vehicle-treated KO mice, intraperitoneal dosing of MCC950 for 8 weeks significantly reduced pathologic readouts, including hypertrophic remodeling, which was quantified by decreased heart/body weight and improved systolic and diastolic functional indices.

Located at the interface between blood and interstitial tissues, endothelial cells play a role in maintaining homeostasis in the vascular system. Endothelial dysfunction is a key stage for the development of cardiovascular diseases. Cigarette smoking is a major preventable risk factor for cardiovascular diseases, and causative links exist between cigarette smoke and endothelial dysfunction. Proinflammatory processes and endothelial cell death by pyroptosis are two major pathologic mechanisms in cardiovascular disorders, such as hypertension, diabetes, and atherosclerosis. Cigarette smoke induces endothelial cell pyroptosis through activation of an ROS/NLRP3 axis in an in vitro human umbilical vein endothelial cell model, wherein pretreatment with MCC950 attenuated smoke-induced pyroptotic cell death. Furthermore, rats exposed to cigarette smoke had increased NLRP3 inflammasome activation in carotid arteries, which was inhibited by the attenuation of ROS/NLRP3 signaling by melatonin (Wang et al., 2019d).

3. Lung

There is a growing body of evidence supporting a role for NLRP3 in many pulmonary conditions because NLRP3 is highly expressed in the lung and can be activated by inhaled toxic particulate matter (Kim et al., 2015). In murine and human macrophages in vitro, exposure to cristobalite-bearing volcanic ash from the Soufriere Hills volcano led to the activation of NLRP3 and subsequent release of IL-1β, a response that could be downregulated with MCC950 (Damby et al., 2018). Particulate matter 2.5 (PM2.5) exposure has become a topical concern for public health in recent years. In a PM2.5-induced model of lung inflammation, MCC950 was shown to reduce lung injury (Jia et al., 2021). Similarly, another particulate, silica, induced epithelial-to-mesenchymal transition in human bronchial epithelial cells and contributed to fibrosis via the activation of NLRP3 (Li et al., 2018). Silica exposure induced phosphorylation of NF-κB, increased expression of Snail and was inhibited by MCC950. This indicated that NLRP3 could mediate silica-induced epithelial-to-mesenchymal transition via IL-1β–TGF-β activated kinase 1 (TAK1)–mitogen-activated protein kinase–Snail/NF-κB (Li et al., 2018). MCC950 also elicited robust macrophage-guided myofibroblast transformation in response to fibrogenic carbon nanotubes and silica (Hindman and Ma, 2019). In lung ischemia-reperfusion (IR) injury, a study has shown benefits of MCC950 in reducing lung IR injury, albeit they were not as strong as the effects seen in cardiac IR models. MCC950 was able to negate the effects of ROS-dependent inflammasome-activated damage by blocking pyroptosis and disrupting NLRP3–NIMA-related kinase 7 (NEK7) interactions (Xu et al., 2018b).

In human asthma, the expression of NLRP3 and IL-1 is associated with neutrophilic airway inflammation, disease severity, and steroid resistance (Kim et al., 2015). Readouts for NLRP3 activation have also been detected in Chlamydia and Haemophilus respiratory infection models of ovalbumin-induced severe, steroid-resistant allergic airway disease. In this case, NLRP3 activation promotes steroid-resistant neutrophilic inflammation and airway hyper-responsiveness. MCC950 treatment reduced steroid-resistant features of disease, whereas IL-1β administration recapitulated the detrimental effects (Kim et al., 2017). In two other studies (Primiano et al., 2016; Rossios et al., 2018), IL1R-like 1 gene expression was associated with eosinophilic severe asthma, whereas NLRP3 inflammasome expression was highest in patients with neutrophilic severe asthma. In associated murine models, house dust mite allergen (HDM) and Complete Freund's Adjuvant were used to induce severe asthma. MCC950, albeit at a heroic dose (200 mg/kg), prevented the development of the pathology and concurrently reduced the expression of disease mediators, such as IL-1β, TH2-derived cytokines, and neutrophil- and eosinophil-associated chemokines (Fig. 4) (Primiano et al., 2016). The efficacy of MCC950 has also been shown in a Toluene diisocyanate model of asthma, in which it effectively inhibited NLRP3 activation and reduced levels of downstream inflammatory mediators, such as caspase-1, IL-1β, and IL-18 (Chen et al., 2019a). MCC950 again alleviated airway hyper-reactivity, airway inflammation, and airway remodeling and significantly suppressed TH2/TH17 responses in this model. Efficacy with MCC950 has been shown in an OVA-sensitized model (Wang et al., 2018b). In two fungus-induced murine asthma models (Aspergillus fumigatus and Alternaria alternate pulmonary infection), MCC950 reduced inflammatory cell infiltration and IL-1β and TH2 cytokine levels (Jeong et al., 2018). Furthermore, in a classic OVA-induced model of eosinophilic asthma, MCC950 attenuated pathology via downregulation of the retinoid X receptor and correlated apoptosis in airway epithelial cell (Lv et al., 2018).

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Fig. 4.

MCC950 modulates HDM-induced acute airway inflammation. “MCC950 (CP-456,773) treatment reduces mouse airway inflammation following acute HDM challenge. (A–I) BAL cellularity and BAL cytokines/chemokines in mice challenged with intratracheal saline or HDM, and treated with saline, Dex (2 mg/kg), or CP-456,773 (200 mg/kg twice daily). Total cellularity (A) and differential numbers of neutrophils (B) or lymphocytes (C) in BAL samples 24 h after HDM challenge. (D–I) Concentrations of cytokine/chemokine proteins CXCL1/KC (D), CXCL5/GCP-2 (E), CXCL10/IP-10 (F), CCL7/MARC (G), CCL3/MIP-1α (H), and TNF-α (I) in BAL samples 24 h after HDM challenge. All data are representative of two to three independent experiments with 5–10 mice per group. Statistical significance was determined using a one-way ANOVA followed by a Dunnett test for multiple comparisons where all groups were compared with HDM/vehicle-treated animals. For the determination of individual P values, a Mann–Whitney U test was used to compare a specific group to HDM/vehicle-treated animals. For all groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with HDM/vehicle animals.” Reprinted (in part) with permission from Primiano MJ, Lefker BA, Bowman MR, Bree AG, Hubeau C, Bonin PD, Mangan M, Dower K, Monks BG, Cushing L, Wang S, Guzova J, Jiao A, Lin L-L, Latz E, Hepworth D and Hall JP (2016) Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation. The Journal of Immunology 197:2421. Copyright 2016 The American Association of Immunologists, Inc.

Allergic rhinitis is a common chronic respiratory disease. It is characterized principally by elevated IgE, but NLRP3 has been implicated in the downstream inflammatory responses. MCC950 was shown to protect mice in an OVA-induced model of allergic rhinitis. Intranasal administration of MCC950 significantly reduced sneezing, nasal rubbing, inflammatory cytokines, and inflammatory cells and NLRP3, caspase-1, ASC, IL-1β, and IL-18 expression levels in treated mice compared with untreated mice (Zhang et al., 2020).

Sarcoidosis is a disease that commonly manifests in the lungs and is characterized by the development of inflammatory granulomas. There is an unmet medical need, as current treatments work only to alleviate symptoms. NLRP3 has recently been shown to have a role in sarcoidosis (Huppertz et al., 2020a). In this study, an increase in cleaved caspase-1 and IL-1β was detected in sarcoidosis BAL and skin biopsy samples when compared with healthy donors. MCC950 treatment of BAL fluid from patients reduced inflammatory readouts as determined by Western blot, reverse-transcription polymerase chain reaction, and ELISA. Pharmacological inhibition of NLRP3 using MCC950 decreased lung granuloma formation in the trehalose-6,6-dimycolate–granuloma murine model.

Cystic fibrosis (CF) is characterized by pulmonary inflammation and chronic polymicrobial infection. It is a complex, autosomal-recessive chloride channelopathy arising from mutations in the cystic fibrosis transmembrane conductance regulator gene. It is the most common lethal genetic disorder affecting Caucasians and represents an archetypal model for chronic neutrophilic inflammation. When compared with healthy donors, CF neutrophils displayed increased aerobic glycolysis in the systemic circulation with increased IL-1β, suggesting NLRP3 inflammasome inhibition may have a role in CF pathology (McElvaney et al., 2019). This effect was driven by low-level endotoxemia and was resolved post-transplant. The increased pro–IL-1β produced was processed to its mature active form in the LPS-rich CF lung by NLRP3 and caspase-1 activation. MCC950 treatment inhibited IL-1β in the lungs of CF mice, resulting in significantly reduced airway inflammation and improved Pseudomonas aeruginosa clearance. Platelet-driven lung disease has also been described in CF. Activated platelet NLRP3 has been implicated in other inflammatory settings, which may warrant the further exploration of NLRP3 inhibition as a therapeutic avenue in this pathology.

4. Gut

NLRP3 has emerged as a key regulator of intestinal homeostasis. Defective NLRP3 inflammasome signaling in the gut contributes to the inflammatory bowel diseases, Crohn disease, and ulcerative colitis (UC). In a dextran sulfate sodium (DSS)-induced acute colitis model, MCC950 alleviated NLRP3 activity and attenuated disease-related weight loss and injury of the colon, as summarized by an improvement in the Disease Activity Index (DAI) score (Wu et al., 2018). The role of microRNAs and specifically miR-223 in regulating NLRP3-driven intestinal inflammation has been explored. DSS colitis mice deficient in miR-223 exhibited exacerbated disease pathology. miR-223^−/− DSS colitis mice treated with MCC950 or anakinra showed improved outcomes in pathology relative to vehicle-treated animals (Neudecker et al., 2017). NOD2 is known to be protective in DSS-induced colitis, as NOD2-deficient mice show increased NLRP3-activation and reduced DAI scores. MCC950 treatment reduced DSS-induced intestinal inflammation in NOD2^−/− mice, which was correlated with decreased disease severity. These data provide evidence that NLPR3 is a driver of intestinal inflammation in the absence of functional NOD2. The inhibition of NLRP3 signaling thus represents a strategy to impair deleterious intestinal inflammation in the absence of robust NOD2 signaling (Umiker et al., 2019). MCC950/metformin combination therapy had a protective effect in DSS-induced colitis in rats with an improvement in DAI, macroscopic dynamic index, and mitigated colon shortening (Saber and El-Kader, 2020).

MCC950 was efficacious in a spontaneous model of chronic colitis that mimics human UC in mice. Winnie mice possess a mutation in Mucin 2 that manifests in altered mucus production, with ensuing colonic inflammation and intestinal dysbiosis representative of human UC. Oral administration of MCC950 in this model significantly improved readouts for inflammation and colonic injury. A decrease in IL-1β production in isolated tissues was also detected in MCC950-treated animals (Fig. 5) (Perera et al., 2018).

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Fig. 5.

MCC950 treatment improves colitis in 10-week-old Winnie. “MCC950 treatment improves colitis in 10 week old Winnie. Winnie at 10 week were weighed on the day of termination. Lengths of the freshly removed colons from each group were measured from ileocecal junction to rectum. (A) Macroscopic appearances of colon. (B) Disease activity index of control and treatment Winnie mice. Data are represented as means ± S.E.M. (n = 10 per group) *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).” Reprinted (in part) from Perera AP, Fernando R, Shinde T, Gundamaraju R, Southam B, Sohal SS, Robertson AAB, Schroder K, Kunde D and Eri R (2018) MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice. Scientific Reports 8:8618. This content is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Copyright 2020 The Authors.

5. Liver

Inflammasome activation has been associated with the development of drug-induced and obesity-associated liver disease (Wree et al., 2014a). Viral infection of the liver and nonalcoholic fatty liver disease coupled with chronic hepatic damage and inflammation can result in NLRP3 activation (Suzuki and Diehl, 2017). The accumulation of lipids, such as saturated ceramide and palmitate fatty acids, and cholesterol crystals cause hepatocellular damage by activating NLRP3. Severe liver failure resulting from hepatocyte dysfunction can only really be resolved via liver transplant. Alternative treatments, such as mesenchymal stem cell therapy, show potential, with anti-inflammatory effects mediated by downregulation of proinflammatory cytokines and upregulation anti-inflammatory cytokines (Wang et al., 2018a). Its upregulation of IL-10 may be the mechanism needed to bring NLRP3 back in line, as NLRP3-linked pyroptosis exacerbates the inflammatory component of the disease. MCC950 increased survival rates by downregulation of pyroptosis (Wang et al., 2018a). More specifically, fulminant hepatitis, an acute liver injury caused by severe necrosis of hepatocytes in the absence of pre-existing liver disease, is also regulated via NLRP3. This condition can be triggered by different stimuli, but an overdose of acetaminophen causing hepatocellular death releases DAMPs, which activate NLRP3. In an LPS/D-GalN hepatic-injury mouse model, Rev-erbα has been identified as a novel regulator of the NLRP3 inflammasome that is expressed in a circadian pattern. Using a peritonitis animal model, they demonstrated that MCC950 could regulate the condition when Rev-erbα was dysfunctional. Circadian regulation of immunity has been proposed as a mechanism to treat NLRP3-driven diseases (Pourcet et al., 2018).

In patients with nonalcoholic steatohepatitis (NASH), elevated levels of NLRP3, gasdermin-D (GSDMD), and IL-1β have been observed (Wree et al., 2014a). Beyond inflammation, activation of the NLRP3 inflammasome also promotes liver fibrogenesis during NASH (Wree et al., 2014b). GSDMD-mediated pyroptosis appears to be an important factor in the development of NASH, as steatohepatitis is attenuated in GSDMD^−/− mice compared with wild-type (WT) mice (Xu et al., 2018a). MCC950 has been successfully used to reduce liver inflammation and fibrosis in experimental NASH in mice (Mridha et al., 2017). In vitro, pretreatment with MCC950 reversed inflammasome activation and the subsequent pyroptotic cascade (Zhang et al., 2019b). In vivo, foz/foz mice with NASH (an overnutrition model) treated with MCC950 blocked hepatic NLRP3 expression and activation. MCC950 also improved outcomes directly related to liver injury in two murine steatohepatitis models. In atherogenic diet-fed foz/foz mice, MCC950 reduced the increase in markers of liver injury, ALT, and AST; did not cause weight loss; and had no effect on hepatomegaly, fasting blood glucose, plasma insulin, adiponectin, cholesterol, or triglyceride or cholesterol crystal numbers. Although no effect was seen on metabolic readouts, MCC950 lowered plasma monocyte chemoattractant protein-1, IL-6 levels, liver inflammation, and, most importantly, the Nonalcoholic Fatty Liver Disease Activity Score. In a methionine and choline-deficient diet model, in which steatohepatitis unlinked to overnutrition insulin resistance is observed, MCC950 was able to reduce caspase-1 and IL-1β levels. Both models showed a reduction in liver fibrosis and the expression of profibrotic markers. MCC950 was also able to arrest established liver fibrosis in foz/foz mice.

In models of liver fibrosis driven by infectious stimuli, differential outcomes are observed relative to when NLRP3 inhibition is evoked. In a study of liver fibrosis caused by the parasite Schistoma japonicum, MCC950 had beneficial effects on schistosomiasis-induced liver fibrosis if administered on the day of infection; however, it had a detrimental effect in the pathology if administered on day 22 postinfection (Zhang et al., 2019c). Furthermore, the use of MCC950 in another model of liver injury, common bile duct ligation (BDL) causing cholestatic liver injury, MCC950 abrogated BDL-induced liver injury by reducing IL-1β and IL-18 production and impairing neutrophil infiltration and hepatic cell death. Transcriptome analysis of the samples implicated TLR signaling as an axis that was potentially involved in the protective effects of MCC950 in cholestatic liver injury (Qu et al., 2019).

Although inhibition of ischemia-reperfusion injury (IRI) in cardiac failure models has been well explored, MCC950 may have benefits in other areas in which ischemia-induced injury occurs, such as organ transplantation. In the case of liver transplantation, donors after circulatory death need to be considered because of the shortage of donors. Unfortunately, this type of organ donation is impacted greatly by IRI, with associated increased risk of complications and poorer prognosis in recipients. The addition of MCC950 to a hypothermia machine-perfusion system used during the transplantation process and i.v. administration of MCC950 after pig liver transplantation promoted better organ acceptance and recipient survival (Yu et al., 2018). In this model, MCC950 improved outcomes by reducing hepatocyte apoptosis, leading to superior liver function postsurgery.

6. Kidney

In addition to NLRP3-driven processes in the kidney caused by diabetic mellitus (covered in Diabetes), detrimental NLRP3 activity in the kidney has been documented in other contexts. Kidney fibrosis is a common consequence of renal injury and scarring, which can be amplified by aberrant proinflammatory signaling. Crystalline substances have long been recognized as drivers of NLRP3. Crystal deposition in the kidney, in particular the accumulation of oxalate and adenine crystals, and subsequent NLPR3 activation have been associated with the development of renal fibrosis. The fibrotic scarring process can lead to progressive loss of renal function and ultimately result in kidney failure. In a murine model of chronic crystal nephropathy, CD11c dendritic cells play a critical role in propagating oxalate crystal-induced kidney fibrosis. MCC950 attenuated the crystal-driven NLRP3 activation in renal dendritic cells both in vitro and in vivo. Furthermore, MCC950-treated mice displayed improved renal function and decreased tissue injury and fibrosis relative to untreated mice. Quantitative markers of fibrotic scarring, such as smooth muscle actin histology and renal expression levels of fibronectin, collagen I, and transforming growth factor-β, were significantly reduced by MCC950 treatment (Ludwig-Portugall et al., 2016). Attenuation of markers of renal fibrosis in response to MCC950 have also been observed in the multiple-cisplatin-induced murine model. Genetic deletion of NLRP3 has similarly been shown to halt the progression of multiple-cisplatin-induced kidney fibrosis (Li et al., 2019).

Podocytes are highly specialized cells of the kidney glomerulus that form the major component of the glomerular blood filtration barrier. Podocyte injury critically contributes to the pathogenesis of obesity-related glomerulopathy. Recently, lipid accumulation and inflammatory responses have been found to be involved in podocyte injury. Obesity-related glomerulopathy mice developed proteinuria, podocyte injury, and hypertriglyceridemia accompanied with deregulated lipid metabolism, renal ectopic lipid deposition, activation of NLRP3, and secretion of IL-1β. Inhibition of NLRP3 with MCC950 alleviated podocyte injury and IL-1β release, but there was no change in the expression of CD36, adipose differentiation-related protein, and intracellular lipid droplets (Zhao et al., 2019).

The physiological mechanisms controlling the relationship between systemic arterial pressure and systemic sodium concentration (pressure-natriuresis) are a critical feature of renal function. Increases in blood pressure induce a compensatory reduction in sodium reabsorption in the proximal tubules, and as such, the kidneys play a critical in blood-pressure homeostasis (Rodriguez-Iturbe and Johnson, 2010). However, renal inflammation impairs the pressure-natriuresis relationship and can cause hypertension. NLRP3 has been implicated in the development of kidney inflammation and blunting of the pressure-natriuresis response. MCC950 reversed established elevated blood pressure in the one kidney/deoxycorticosterone acetate (1K/DOCA)/salt-dependent model of hypertension in mice (Krishnan et al., 2016, 2018). However, in a separate study from the same group, which used a different model of age-related hypertension (one induced by a dose of angiotensin II), mice were indifferent to MCC950 treatment. This suggests that NLRP3 activation may not contribute to the pathology seen in this form of hypertension. The authors speculate that increased vascular angiotensin II type 1 receptor:angiotensin II type 2 receptor expression may be more relevant than inflammasome activation in angiotensin II and age hypertension (Dinh et al., 2017).

7. Skin

Imidazoquinolines, such as imiquimod and R848, are topical immune response modifiers that are used in the clinic as antiviral and antiallergic creams. Imiquimod and R848 induce IL-1β production by immune cells in an NLRP3-dependant manner. In vitro, MCC950 inhibits imidazoquinoline-induced IL-1β release in purified human monocytes (Primiano et al., 2016). In human PBMCs, MCC950 impaired the release of R848- and imiquimod-induced IL-1β at IC₅₀ values of 26 and 10 nM, respectively. In a mouse model of imiquimod cream–induced skin inflammation, oral twice-daily dosing of mice with 200 mg/kg of MCC950 significantly reduced ear thickness that resulted from the application of imiquimod cream. Several cytokines (IL-22, IL-17A, and IL-17F) that have been correlated with deleterious inflammation in human psoriatic skin samples were elevated in the inflamed imiquimod cream–challenged ears (Primiano et al., 2016). These data provide a potential link between NLRP3 inflammasome and its downstream inflammatory mediators in the pathogenesis of imiquimod cream–induced skin inflammation and in the induction of cytokines associated with psoriasis.

Wound healing is a complex process involving the sequential involvement of proinflammatory and anti-inflammatory mediators and growth factors, each of which is essential at specific phases of the process. Evidence exists to support the notion that inhibition of NLRP3 at specific phases may prove beneficial in wound healing. However, no significant change was observed with MCC950 treatment in obese mice, in which wound healing takes longer, or in healthy animals (Lee et al., 2018). There was also a note of possible impairment of wound healing in one treatment group (obese animals treated with a topical application of MCC950). This result is in alignment with effects previously seen with wound-healing models in NLRP3 KO mice (Ito et al., 2018).

8. Eyes

In addition to diabetic retinopathy discussed in Diabetes, there are data to support the use of NLRP3 inhibitors in several eye conditions. Age-related macular degeneration (AMD) results from the loss of retinal pigment epithelium (RPE) cells that serve to support the rods and cones of the eye. Loss of RPE cells leads to degeneration of photoreceptors in the eye and ultimately to profound visual impairment. NLRP3-dervied IL-18 has been implicated in the pathogenesis of AMD, but the evidence to support NLRP3 inhibition as a clinical strategy in age-related macular generation AMD remains controversial. Using an in vitro system, NLRP3 inhibitors, such as MCC950, have shown efficacy in human and murine RPE cells, providing promising evidence for future evaluation of pharmacological inhibitors of NLRP3 in atrophic AMD (Wang et al., 2019c). However, a more recent comprehensive investigation in vivo delivered opposing results, demonstrating that MCC950 treatment had no effect on the preservation of retinal function (Wooff et al., 2020).

NLRP3 activation is required for ocular neovascularization. In a recent murine model of oxygen-induced ischemic retinopathy, MCC950 treatment was shown to impair the formation of retinal neovascularization, reduce the number of acellular capillaries, and decrease leakage from retinal vessels (Sui et al., 2020a).

Scutellaria baicalensis Georgi is a common plant that is widely used in traditional Chinese medicine to treat a variety of indications. Baicalin, a flavonoid compound derived from the plant, possesses anti-inflammatory, antimicrobial, and antioxidant activities. Of particular note is the negative regulation of NLRP3 and NF-κB signaling by baicalin. Baicalin exerts a therapeutic effect in ocular diseases, such as glaucoma, retinopathy, and AMD, via antiangiogenesis, antiapoptosis, anti-inflammatory, and antireactive oxygen species. In an in vitro Aβ-induced ARPE-19 model of AMD, baicalin achieves an anti-inflammatory effect via the upregulation of miR-223 and resulting downregulation of NLRP3, thus suppressing pyroptosis triggered by NLRP3 inflammasome signaling. The beneficial effects of baicalin were attenuated by miR-223 silencing but restored by the inhibition of NLRP3 by MCC950, implicating NLRP3 signaling in the pathology and providing evidence to support targeting NLRP3 in AMD (Sun et al., 2019).

9. Reproductive System

Inflammation detrimentally affects reproductive physiology and female fertility. In a recent study (Navarro-Pando et al., 2021) NLRP3 was found to be increased in the ovary of aged mice. Increased expression of NLRP3 and other key downstream markers, caspase-1 and IL-1β, were also detected in granulosa cells from patients with primary ovarian insufficiency. Inhibition of NLRP3 with MCC950 improved fertility in female mice to levels comparable to those of Nlrp3^−/− mice, suggesting NLRP3 inhibition may have utility in the treatment infertility.

Inflammatory processes have been associated with spontaneous preterm labor and birth. In particular, intra-amniotic inflammation/infection is the leading cause of perinatal mortality and morbidity worldwide. A link to NLRP3 has been identified by several groups. Clinically, evidence indicates that NLRP3 is involved in pregnancy dysfunction, particularly in pregnancy-specific hypertensive syndromes, such as pre-eclampsia (Shirasuna et al., 2020). Markers of NLRP3 activation have also been detected in an animal model of LPS-induced intra-amniotic inflammation (IAI). Increased priming of NLRP3 and an increase in IL-1β were observed in both the fetal membranes and decidua basalis prior to preterm birth. Increased concentrations of IL-1β were also quantified in the amniotic fluid prior to IAI-induced preterm birth. MCC950 reduced the occurrence of both IAI-induced preterm birth and neonatal mortality, implicating NLRP3 activation and spontaneous preterm labor and birth in the context of intra-amniotic inflammation (Faro et al., 2018). In support, a further study identified a correlation between S100B-induced inflammation in the amniotic cavity that propagates NLRP3 activity in fetal membranes of pregnant C57BL/6 mice, with MCC950 treatment preventing preterm labor/birth by 35.7% and reduced neonatal mortality by 26.7% (Gomez-Lopez et al., 2018).

NLRP3 activation has recently been implicated in primary dysmenorrhea, a common gynecological disease among young women. In a mouse model of primary dysmenorrhea, intraperitoneal injection of MCC950 decreased uterine prostaglandin F2 α and prostaglandin E₂ and the expression of NLRP3, caspase-1, IL-1β, IL-18, phosphorylated–NF-κB p65, NF-κB p65, and cyclooxygenase-2 (Tang et al., 2020).

1. Innate Immunity

NLRP3 plays a significant part in innate immunity in response to a variety of activators. Activators such as tissue injury, infection, strenuous exercise, or certain drugs can induce the mobilization of stem cells from the bone marrow into peripheral blood. This mobilization is critical in the response to activation to provide innate immune effector cells. The majority of the stem cells involved in this egress are hematopoietic stem/progenitor cells, but other stem cells, such as mesenchymal stroma cells, endothelial progenitor cells, and very small embryonic-like stem cells, may also be involved. The movement specifically triggered by “sterile inflammation” in the bone-marrow microenvironment activates the complement cascade. The NLRP3 inflammasome is instrumental in this process, undergoing activation in an ATP-dependent manner that orchestrates bone-marrow egress via sequential activation of complement cascade. In support of this hypothesis, administration of the NLRP3 activator nigericin induced bone-marrow mobilization in mice. The opposite effect was observed by administration of MCC950 to mice mobilized by granulocyte colony-stimulating factor or AMD3100. These results support a role of innate immunity, bone-marrow sterile inflammation, and novel role of the ATP-NLRP3-complement cascade axis in the egress of stem cells into peripheral blood (Lenkiewicz et al., 2019).

2. Adaptive Immunity

The exact molecular mechanisms bridging sterile inflammation and induction of adaptive immunity have not been fully elucidated. There is, however, emerging evidence that although NLRP3 is formally classified as a signaling complex of the innate immune system, it may also play a key role in T-cell crosstalk and induction of an adaptive immune response to nonself or damaged-self antigens at the site of injury. Using a heat-induced sterile skin injury model, local cell death and caspase-1 activity were detected in the absence of the disruption of skin integrity. Importantly, CD8+ lineage dendritic cells were implicated in the induction of OVA-specific CD8+ T-cell responses after heat injury. MCC950 was introduced into this model to clarify the involvement of NLPR3 in the CD8+ T-cell priming process, confirming that this process was NLRP3-dependent and suggesting a therapeutic role for NLRP3 inhibition in sterile inflammation after heat injury (Chakraborty et al., 2018).

A role for NLRP3-driven T helper 1 immunity has also been described. Human CD4+ T cells possess the ability to signal through NLRP3, and indeed require complement-driven NLRP3 activity for effector functions. Specifically, human CD4+ T cells express the C5 complement receptor and generate intracellular C5a upon T-cell receptor activation. The accumulation of C5a results in an increase in ROS and the assembly of a functional NLRP3 complex within the CD4+ T cell. This allows for autocrine IL-1β secretion and activity that promotes IFN-γ production by T cells. The NLRP3-dependent nature of this axis has been demonstrated by the use of MCC950, providing intriguing evidence of a lesser-known function of NLRP3 outside of innate immunity. Activation of CD4+ T cells from CAPS patients in the presence of MCC950 led to a reduction of both IL-1β and IFN-γ secretion, supporting the hypothesis that dysregulation of this pathway occurs in human autoinflammatory disease and that NLRP3 inhibition may be beneficial beyond the well described myeloid inflammatory processes (Arbore et al., 2016).

3. Autoimmunity and Autoinflammation

Systemic lupus erythematosus is a prototypical autoimmune disease. Lupus nephritis is a major manifestation of systemic lupus erythematosus and can cause kidney injury and failure. As NLRP3 activation has been directly implicated in models of renal injury, it is possible that it also plays a role in the pathogenesis of lupus nephritis. This putative link was examined by the use of a P2X7 inhibitor upstream of NLRP3 (Zhao et al., 2013). A further study used MCC950 to impair NLRP3 activation and damaging autoinflammation in podocytes from lupus-prone mice and from patients with lupus nephritis (Fu et al., 2017).

In the original paper in which the MCC950 was shown to be a specific inhibitor of NLRP3, the drug was efficacious in a murine model of human multiple sclerosis: experimental autoimmune encephalomyelitis (EAE). EAE is induced by immunization with autoantigens that induce T cell–mediated demyelination and inflammation. Treatment of mice with MCC950 delayed the onset and reduced the severity of EAE. Numbers of IFN-γ cells and particularly of IL-17–producing cells were also reduced in both the CD4+ and γδ+ subpopulations of CD3+ T cells (Coll et al., 2015). In a separate study, it was hypothesized that autophagy and inflammation work collectively in multiple sclerosis and that targeting both pathways would be a viable therapeutic strategy (Xu et al., 2018c). Rapamycin, which targets autophagy, was combined with MCC950 to reduce disease severity scores to a greater extent than treatment with rapamycin alone in the murine EAE model. However, these authors did not test MCC950 alone, so whether the effect is due to MCC950 entirely or whether rapamycin has any additive affect is unknown (Xu et al., 2018c). In a separate study, a combination treatment of rapamycin and MCC950 in an EAE model restored the abundance and diversity of gut microbiota of EAE mice to close to that found in healthy WT mice (Xu et al., 2020). Recent work has shown that NLRP3 plays a role in the cognitive impairments observed in EAE and that MCC950 treatment can improve spatial and contextual memory deficits in late-phase EAE (Hou et al., 2020).

IL-1β is an important mediator of cartilage destruction in rheumatic diseases, particularly osteoarthritis and rheumatoid arthritis (RA). In osteoarthritis, clinical IL-1β inhibition with the mAb canakinumab in the CANTOS-2 study reduced rates of hip and knee replacements (Schieker et al., 2018). In rheumatoid arthritis, the markers highlighting NLRP3 activation have been documented in both synovia from RA patients and collagen-induced arthritis mice. In patients and nonclinical models of RA, negative regulation of NLRP3 by A20 (a regulator of ubiquitylation also known as TNFAIP3) protects against arthritis (Vande Walle et al., 2014). In a follow-up murine model study, NLRP3 activation was detected mainly in the infiltrating monocyte/macrophages in synovia. MCC950 treatment significantly reduced IL-1β production from these cells along with decreasing joint inflammation and bone destruction (Guo et al., 2018).

4. Monogenic Nod-Like Receptor Family Pyrin Domain Containing 3 Diseases

CAPS are a group of rare hereditary autoinflammatory conditions caused by an activating mutation in NLRP3. CAPS is characterized by IL-1β–mediated systemic inflammation causing recurrent episodes of fever and symptoms involving skin, joints, central nervous system, and eyes. CAPS is broadly categorized into three groups: familial cold autoinflammatory syndrome (FCAS, formerly termed familial cold–induced urticaria), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (also called chronic infantile neurological cutaneous and articular syndrome). This disease exists on a spectrum of severity, with FCAS being the most benign and neonatal-onset multisystem inflammatory disease presenting as the most severe form. Mice expressing the NLRP3 A350V mutant that mirrors a mutation seen in certain human MWS patients can be used as an experimental mouse model of CAPS. These mice die in the neonatal period from inflammation-induced multiorgan failure and produce increased concentrations of IL-1β and IL-18. MCC950 inhibited NLRP3 activation in these animals and promoted survival well beyond their PBS-treated littermates. Work from the same study also investigated the effect of MCC950 on PBMCs from patient with MWS’s ex vivo. PBMCs from CAPS patients secrete IL-1β ex vivo in response to LPS alone, and MCC950 was shown to impair this (Coll et al., 2015). A separate study confirmed that patients with low-penetrance NLRP3 variants (Q703K and V198M) displayed enhanced IL-1β levels after inflammasome activation compared with healthy controls. This work also demonstrated that IL-1β release could be decreased by MCC950 treatment (Schuh et al., 2019). One study has provided some contradictory evidence that suggests gain-of-function NLPR3 mutations found in CAPS patients cannot be deactivated by MCC950 in an in vitro and in vivo setting. Attempts to inhibit levels of NLRP3 activation markers in the FCAS-associated Nlrp3^L351P mutant (corresponding to human L353P) with MCC950 were unsuccessful in both in vivo models and ex vivo samples in this case (Vande Walle et al., 2019).

Sickle cell disease describes a group of monogenic inflammatory conditions that exhibit a clinical hallmark of chronic hemolysis and abnormal coagulation. Hemolysis promotes the release of hemoglobin from red blood cells, which is then oxidized into free heme. Several studies suggest that heme can act as a DAMP to activate inflammatory processes (Dutra et al., 2014). Platelet NLRP3 has been shown to be activated in patients with sickle cell inflammatory vaso-occlusive crisis, with increased caspase-1 activity, ASC, and NLRP3 colocalization together with elevated HMGB1 expression compared with sickle cell patients with steady-state disease. MCC950 treatment was shown to significantly reduce caspase-1 activity in platelets isolated from healthy donors, patients with steady-state disease, and patients in inflammatory crisis. In this study, increased caspase-1 activity and platelet aggregation was also observed in a mouse model of the disease, in which intravenous MCC950 effectively reduced these readouts (Vogel et al., 2018). The upregulated platelet aggregation responses during acute crisis were annotated to the heme/NLRP3/Bruton's tyrosine kinase pathway, as evaluated by the use of the heme scavenger hemopexin in both patient samples and mouse model of sickle cell disease (Vogel et al., 2017).

Although less common, another inheritable disease that features aberrant NLRP3 activation is valosin-containing protein (VCP) disease. VCP-associated diseases are characterized by inflammation and muscle atrophy. Mutations in VCP can cause an associated disease that affects the muscle, bone, and brain. Patients exhibit progressive proximal limb girdle muscular weakness and reduced life expectancy (40–50 years of age). NLRP3 has been implicated in the pathology of the disease, as NLRP3 activation has been detected in myoblasts of patients with VCP-associated disease. Promisingly, MCC950 treatment of both human VCP myoblasts ex vivo and VCP^R155H/+ heterozygote mice reversed markers of NLRP3 activation and disease pathology, leading to a significant improvement in muscle strength in vivo (Nalbandian et al., 2017).

1. Metabolic

Pancreatic β-cells are continually depleted as type 2 diabetes progresses, which has been linked to the dysregulation of NLRP3 and production of IL-1β and, ultimately, pancreatic β-cell death (Masters et al., 2010). Efforts have been made to develop compounds that have a dual mechanism of action, thereby simultaneously acting as insulin secretagogues and NLRP3 inhibitors in vitro (Hill et al., 2017). However, in vivo, db/db diabetic mice treated with MCC950 showed no effect on markers of NLRP3 signaling or the immune cell profile. There was no evidence of improved glucose metabolism or a reduction in pancreatic islet failure (Kammoun et al., 2018).

2. Cognitive

Diabetes can lead to the development of cognitive dysfunction, which is termed diabetic encephalopathy. Obese and type 2 diabetic db/db mice develop NLRP3-driven hippocampal inflammation that manifests as anxiety- and depression-like behaviors and cognitive disorders. Expression levels of NLRP3, ASC, caspase-1, and IL-1β are increased in these mice, and MCC950 has been shown to ameliorate the detrimental neuropsychiatric disability in this model (Zhai et al., 2018). In contrast to the Kammoun et al. (2018) study, MCC950 treatment also significantly improved insulin sensitivity in db/db mice.

Diabetes can also elevate the risk factor for cognitive impairment via occurrence of small vessel disease and stroke. The elevated level of hippocampal neurovascular unit remodeling after remote ischemic injury in diabetes is NLRP3-dependent. The endothelial cells at the center of the hippocampal neurovascular unit produce trophic factors, such as brain-derived neurotrophic factor, which promotes neuronal survival. MCC950 improved cognitive function and vascular integrity after MCAO-induced stroke in diabetic rats and prevented hypoxia-mediated decrease in brain-derived neurotrophic factor secretion. Further work is needed to understand the therapeutic potential of brain-penetrant NLRP3 inhibitors in prevention of neurovascular remodeling and cognitive decline in diabetic patients after stroke (Ward et al., 2019).

3. Ocular

In surgically excised proliferative retinal membranes from diabetic retinopathy patients, elevated expression of NLRP3, caspase-1, and IL-1β was evident. MCC950 impaired the expression of these NLRP3-driven inflammatory markers and prevented apoptosis in high-glucose-stimulated human retinal endothelial cells with associated downregulation of interactions between NIMA-related kinase 7 (NEK7) and NLRP3 (Zhang et al., 2017b).

4. Cardiovascular

Cardiovascular-related events are the leading cause of death for patients with type 2 diabetes mellitus. Low-grade inflammation is a feature of vascular complications in diabetic patients. MCC950 has recently been shown to improve vascular function and protect against atherosclerosis in a streptozotocin-induced diabetic apolipoprotein knockout (ApoE−/−) mouse model (Sharma et al., 2021). NLRP3 activation is increased in type 2 diabetic mice, and MCC950 treatment augmented endothelial-dependent vasorelaxation in aortic segments (Zhang et al., 2017a). Hypoadiponectinemia is associated with impaired endothelium-dependent vasodilation. Markers of NLRP3 activation and vascular endothelial injury are elevated in adiponectin-KO mice compared with WT mice in diabetes. MCC950 reduced diabetic vascular endothelial dysfunction to comparable levels in adiponectin-KO mice and WT mice. Adiponectin also directly impaired NLRP3-dependent inflammation and attenuated endothelial cell injury, which was abolished by NLRP3 inflammasome overexpression. Taken together, these data suggest that hypoadiponectinemia-induced NLRP3 inflammasome activation is a novel mechanism of diabetic vascular endothelial dysfunction and targeting of NLRP3 in the treatment of cardiovascular complications of type 2 diabetes mellitus may present a promising therapeutic strategy (Zhang et al., 2017a). Recent work in a visfatin/eNampt-induced model of endothelial dysfunction and vascular inflammation also illustrated beneficial outcomes after MCC950 treatment (Romacho et al., 2020).

5. Kidney

The kidney has significant involvement in diabetic processes. High glucose levels can promote NLRP3 inflammasome activation in mesangial cells, which comprise 30–40% of cells in the glomerulus. When treated with high glucose, these cells produce elevated IL-1β and IL-18 levels, reduced by MCC950 treatment (Chen et al., 2018b). Furthermore, MCC950 and VX-765 (a caspase-1 inhibitor) showed significant interaction with ginsenoside compound K on the decrease of IL-1β concentration in glomerular cell line HBZY-1 (Song et al., 2018).

1. Bacterial Infection

Staphylococcus aureus is a Gram positive pathogen with resistance to many commonly used antibiotics. It colonizes the nares of the nose, skin, and respiratory tract and uses an armory of virulence factors to evade host immune response. Expression of the virulent α toxin (AT) correlates with poor prognosis in patients diagnosed with respiratory infections (Bonesso et al., 2016). Staphylococcus aureus uses AT to evade detection and destruction by activating NLRP3, which allows the bacteria to uncouple from the mitochondria and avoid phagocytic-induced death in macrophages. MCC950 administration improved the survival rate and reduced (3-log colony forming unit reduction) the S. aureus burden recovered from the lungs in a murine pneumonia model (Fig. 7). NLRP3 inhibition with MCC950 was superior to blocking IL-1β and/or IL-18 using anakinra and an anti-IL-18 mAb, which did not affect bacterial burden and had significantly less benefit for survival (Cohen et al., 2018).

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Fig. 7.

Alpha toxin and NLRP3 Inhibition Promotes Bacterial Clearance. AT and NLRP3 Inhibition Promotes Bacterial Clearance. (A) S. aureus CFUs recovered from the lungs of infected (1e8 CFUs) mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hr prior to infection. (B) IL-1β levels in BALF 4 and 24 hr following infection (1e8 CFUs) in mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hr prior to infection. (C) S. aureus CFUs recovered from the lungs of infected (5e7 CFUs) mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hr prior to infection.(D) IL-1β levels in BALF 4 and 24 hr following infection (5e7 CFUs) in mice treated with MEDI4893* (15 mg/kg) or c-IgG 24 hr prior to infection. (E) Survival of mice treated with Anakinra or c-IgG and infected with 1e8 CFUs S. aureus. (F) S. aureus CFUs recovered from the lungs of infected (5e7 CFUs) mice treated with Anakinra or c-IgG 24 hr prior to infection.(G) Survival of mice treated with anti-IL-18 mAb or c-IgG and infected with 1e8 CFUs S. aureus. (H) S. aureus CFUs recovered from the lungs of infected (5e7 CFUs) mice treated with anti-IL-18 mAb or c-IgG 24 hr prior to infection. (I) Survival of mice treated with both Anakinra and anti-IL-18 mAb, or c-IgG and infected with 1e8 CFUs S. aureus. (J) S. aureus CFUs recovered from the lungs of infected (5e7 CFUs) mice treated with both Anakinra and anti-IL-18 mAb, or c-IgG 24 hr prior to infection.(K) Survival of mice treated (1 hr prior to infection) with MCC950 or vehicle control (DMSO) and infected with 1e8 CFUs S. aureus. (L) S. aureus CFUs recovered from the lungs of infected (5e7 CFUs) mice treated (1 hr prior to infection) with MCC950 or vehicle control (DMSO). Statistical significance was determined by (A–D, F, H, J, and L) Mann-Whitney test or (E, G, I, and K) log-rank test. All data are representative of greater than 2 independent experiments and n ≥ 10 mice per group per experiment. Reprinted (in part) from Cohen TS, Boland ML, Boland BB, Takahashi V, Tovchigrechko A, Lee Y, Wilde AD, Mazaitis MJ, Jones-Nelson O, Tkaczyk C, Raja R, Stover CK and Sellman BR (2018) S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking. Cell Reports 22:2431-2441. This content is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Copyright 2018 The Authors.

S. aureus can also cause bacterial pneumonia after influenza infection. Higher mortality rates are observed in animals with both influenza A and S. aureus infections (superinfection) than in mice infected with S. aureus alone (Kudva et al., 2011). MCC950 treatment after establishment of influenza and S. aureus superinfection in mice led to a decrease in bacterial load in the lungs, but this did not correlate with any benefit in survival. When MCC950 was coadministered at the same time as influenza inoculation, an increase in bacterial load was observed (Robinson et al., 2018).

Bacillus cereus is a common anaerobic Gram positive bacterium that can cause serious and potentially fatal intestinal illness. B. cereus expresses three subunits of hemolytic enterotoxin BL (HBL) that binds to cell membrane, forming a lytic pore in target cells. This pore forming activity results in the efflux of potassium, which induces NLRP3 activation, IL-1β, IL-18, and pyroptosis. MCC950 protected B. cereus-infected mice against HBL-induced lethality (Mathur et al., 2018). However, NLRP3 could also be activated independently of HBL, and MCC950 was efficacious against a strain of B. cereus deficient in HBL (Fox et al., 2020) in which the drug attenuated secretion of IL-18 in the circulation and the peritoneal cavity (Fox et al., 2020).

Salmonella typhimurium, another Gram negative pathogen, is also suspected to be an activator of the NLRP3 inflammasome. Its role in mice suggests it might be redundant in helping to clear S. typhimurium in vivo (De Jong et al., 2014). However, in human monocytes, MCC950 suppressed IL-1β produced in response to S. typhimurium challenge in an NLRP3-dependent but pyroptosis-independent manner (Diamond et al., 2017).

Group A Streptococcus (GAS) also evades NLRP3 activation by an independent mechanism. Patients with autoimmune diseases who are treated with the IL-1β inhibitor anakinra have a much higher susceptibility to GAS infections, hospitalization, and death from necrotizing fasciitis (LaRock et al., 2016). The GAS cysteine protease streptococcal pyrogenic exotoxin B can induce mature IL-1β production in the host in an inflammasome-independent manner, thus eliciting an immune response to clear the pathogen. Suppression of IL-1β in anakinra-treated patients blocks IL-1β from all sources, so even nonpathoadaptive strains could establish infection (LaRock et al., 2016). In contrast, the authors speculated that in contrast to anakinra, specific inhibition of only one inflammasome, NLRP3, could still be effective in treatment of autoimmune conditions but allow other IL-1β–producing pathways to be activated and assist in clearance of GAS infections.

The treatment of Mycobacterium tuberculosis infection requires use of an extended multidrug regimen with a high rate of noncompliance and associated prevalence of resistant mutants. A potential alternate strategy focuses on the manipulation of host responses to promote bacterial clearance in combination with direct antimicrobial treatments. NLRP3 activity is increased in M. tuberculosis pathology, with clinical isolates and lineages of M. tuberculosis promoting IL-1β production from murine macrophages in a NLRP3-dependent manner (Subbarao et al., 2020). MCC950 treatment reduced bacterial load in this model, and combined treatment with the antimycobacterial drug rifampicin further promoted bacterial clearance. Targeting NLRP3 should be further explored as an adjunct therapy to current traditional approaches for M. tuberculosis infection (Subbarao et al., 2020).

Uropathogenic Escherichia coli is the primary causative agent of common urinary tract infections, with variable host-mediated responses elicited to different strains of bacteria. For example, in murine macrophages, the UTI89 strain of uropathogenic E. coli activated cell death but only at a high multiplicity of infection, whereas the CFT073 strain promoted a NLRP3-dependent response. MCC950 was used to show that in human macrophages NLRP3 is required only for IL-1β production in response to these strains but not for pyroptosis (Schaale et al., 2016). This is analogous to results obtained by another group showing that human monocytes respond in a similar manner during S. typhimurium infection, in which α-hemolysin is the primary trigger for cell death and IL-1β release in mouse macrophages, but these cellular responses are largely toxin-independent in human macrophages (Murthy et al., 2018).

In polymicrobial sepsis, the need for an immediate response without knowledge of the etiological agent(s) driving pathology is problematic, and survival rates are low. Ideally, a host-directed strategy offers a generic approach to tackle this “right drug for the right bug” problem. A cecal ligation and puncture (CLP) model that mimics the clinical features of polymicrobial infection observed in human sepsis was used to induce experimental sepsis. WT animals had a ∼60% survival rate compared with ∼80% in the NLRP3^−/− knockout animals, potentially implicating a detrimental role for NLRP3 in polymicrobial sepsis (Kang et al., 2016). This study also reported that mutations in phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (Pink1) and parkin RBR E3 ubiquitin protein ligase (Park2), which are implicated in the development of PD, further increased susceptibility to polymicrobial sepsis. Pink1^−/− mice had a survival rate of ∼30%, and Park2^−/− mice had a survival rate of ∼35%. This susceptibility was driven by NLPR3, as double knockout of NLRP3 and Pink1 and Park2 raised the survival rate to ∼60% and 70%, respectively. MCC950 treatment conferred a similar level of protection to that seen in the knockout. Pink1^−/− animals treated with MCC950 had a survival rate of 60%, and Park2^−/−animals dosed with MCC950 had a survival rate of 70% (Kang et al., 2016). The efficacy of MCC950 in the CLP murine model has also been observed in a CLP rat model of sepsis. In this study, platelet NLRP3 activation was implicated in the pathology, and NLRP3 inhibition was suggested to be a promising treatment of exploration (Cornelius et al., 2020). Further work is required to ensure the inhibition of NLRP3 does not play a detrimental role during the secondary immunosuppressive phase of sepsis.

Streptococcal toxic shock–like syndrome (STSLS) is an acute, severe infection caused by group A hemolytic Streptococcus. The clinical hallmark is swift progressive septic shock and multiple organ failure with associated high rate of lethality. STSLS is caused by infection with Streptococcus suis, which can be modeled in mice by infection with the SC-19 strain of the bacterium. In this model, MCC950 significantly depressed IL-1β production in treated animals relative to vehicle controls. Other downstream mediators of NLRP3-driven inflammation, such as IL-6 and IFN-γ, were also significantly decreased by MCC950 treatment. MCC950 also reduced creatine kinase and aspartate aminotransaminase levels in the blood and alleviated organ injury with decreased mortality. NLRP3 could be an important target to restrict the cytokine storm and associated damage caused by S. suis infection in STSLS (Lin et al., 2019).

The basis of protracted bacterial bronchitis in young children is still unclear. Patients present with a wet cough, prominent airway IL-1β expression, and infection. Nontypeable Haemophilus influenzae (NTHi) is one of the common infections to present in these children. In vitro, NTHi produces IL-1β predominantly through caspase-1 and NLRP3 at the site of infection. No change was observed in the PBMC response between PBMCs from healthy children and those with protracted bacterial bronchitis, suggesting this is not a systemic response. MCC950 was able to reduce the IL-1β levels, confirming this pathway was involved. However, AIM2 was upregulated, and NLR family CARD domain-containing protein 4 (NLRC4) was downregulated, so NTHi may therefore be using multipathways to survive (Chen et al., 2018a).

2. Viral Infection

In certain human cells, NLRP3 has been shown to functionally replace AIM2 in viral DNA sensing. Although AIM2 has been shown to bind DNA directly in murine cells, DNA detection in human myeloid cells involves the activation of cyclic GMP–AMP (cGAS)-stimulator of interferon genes (STING) signaling, which then induces potassium efflux to trigger the NLRP3 inflammasome and pyroptotic processes. NLRP3 activation markers were recorded in response to both the classic NLRP3 stimulator nigericin and also in response to transfected double-stranded DNA. MCC950 was used to confirm NLRP3 dependence of this process (Gaidt et al., 2017).

In vivo, NLRP3 neutralization is beneficial for clearance of a hypervirulent influenza A virus strain (IAV) infection in a murine model. One study (Coates et al., 2017) showed that IAV matrix 2 proton channel activation of NLRP3 can be blocked with MCC950 in a juvenile murine model of IAV infection. MCC950 treatment starting 3 days after infection improved survival in juvenile mice with ∼70% surviving at day 14 versus ∼20% in untreated animals. Treatment with MCC950 also reduced NLRP3 expression in lung homogenates, reduced IL-18 secretion into the alveolar space, and impaired NLRP3 activation in alveolar macrophages. MCC950 treatment did not reduce viral clearance but impacted the morbidity associated with IAV infection, suggesting NLRP3 inhibition promotes recovery from IAV infection rather than prevention of IAV-induced lung injury. This result was echoed in a study establishing a link between the temporal evolution of the disease and the stages at which NLRP3 is protective and detrimental in the progression of the infection. Early use of MCC950 to impair NLRP3 activation exacerbated disease. Conversely, inhibition of NLRP3 by MCC950 late in the model reduced disease severity (Tate et al., 2016). The same group (Pinar et al., 2017) subsequently demonstrated that with a lethal strain of IAV, H7N9, PB1-F2 activated NLRP3 and may be a contributing factor to the hyperinflammation associated with human H7N9 infections. MCC950 inhibited H7N9 PB1-F2–driven lung inflammation and cellular influx, suggesting inhibition of NLRP3 once infection is established may improve mortality associated with pathogenic H7N9 IAV infection.

MCC950 has shown efficacy within another group of RNA viruses, the α viruses. The chikungunya virus activates NLRP3 in humans and mice. In an α virus–induced musculoskeletal disease animal model, MCC950 reduced chikungunya-induced inflammation markers and osteoclastogenic bone loss and myositis but did not reduce in vivo viral titers. MCC950 also impaired pathology in mice infected with another arthritogenic alphavirus, Ross River virus. However, the severity of infection induced by a flavivirus, the West Nile virus, was not affected by NLRP3 inhibition, which supports existing evidence suggesting nod-like receptor family pyrin domain containing 6 may be functionally more relevant in the response in this case (Chen et al., 2017c).

3. Fungal Infection

In contrast to bacteria, the role for NLRP3 in fungal infection remains somewhat unexplored. NLRP3 is activated by Candida albicans and required for host defense against disseminating infection; however, the activating component of the pathogen remains to be identified (Hise et al., 2009). Delineation of the role of NLRP3 in C. albicans infection is further convoluted by the observation that pathogenic C. albicans escapes macrophages in vitro by triggering NLRP3-dependent pyroptosis and host cell death. Synthetic Candidalysin peptide was sufficient to drive NLRP3 activation in THP-1 cells, and MCC950 provided excellent efficacy in the impairment of IL-1β release (Lowes et al., 2020). Furthermore, MCC950 impaired NLRP3 activation and associated pyroptosis during C. albicans infection, leading the authors to propose that NLRP3 modulation during infection may be a therapeutic avenue to exploit (Tucey et al., 2016).

4. Parasite Infection

Although Toxoplasma gondii infections are not of great concern in healthy individuals, they are in immunocompromised individuals who may develop severe or life-threatening disease. In vitro studies in human monocytes show elevated IL-1β release within 4 hours of infection (Gov et al., 2017). However, human macrophages from the same donors did not respond in the same way (NLRP3 was in fact downregulated during the differentiation of monocytes to macrophages). In monocytes, MCC950 significantly reduced IL-1β cleavage and release in response to Toxoplasma gondii infection.

MCC950 has also shown efficacy in an in vivo model of helminth (intestinal worm) infections. Mice treated daily with 20 mg/kg of MCC950 starting on the day of Trichuris infection showed nonsignificant trends in serum IL-18 and IL-1β levels. However, MCC950 did significantly increase serum titers of Trichuris Ag-specific IgG1 and lowered worm burden at day 21 postinfection (Alhallaf et al., 2018).

In Leishmania infection, NLRP3 drives CD8+ T cell–mediated cytotoxicity and disease progression (Novais et al., 2017). Mice coinfected with Leishmania major and the lymphocytic choriomeningitis virus Armstrong strain, recapitulate the immunopathology of human leishmaniasis. In this model, the effect of NLRP3 modulation was explored using MCC950 and glyburide. Treated animals were protected from the severe disease that developed in the untreated coinfected mice. NLRP3 inhibition had no effect on parasite numbers, implying that the reduced severity of disease was not due to parasite control (Novais et al., 2017).

A nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental cerebral malaria was used to show that IL-33 was a critical regulator of neuroinflammation and cerebral pathology in this condition. Administration of IL-33 alongside antimalarial drugs in an adapted version of the conventional Plasmodium berghei ANKA strain experimental cerebral malaria model significantly enhanced standard antimalarial treatment of cerebral malaria. This was reported to involve inhibition of microglial- and intracerebral monocyte-associated NLRP3 by IL-33. Cotreatment of MCC950 and antimalarial drugs reproduced the protective effect of IL-33 therapy and improved recovery from established experimental cerebral malaria. Modulation of the IL-33–NLRP3 axis may thus suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of cerebral malaria (Strangward et al., 2018).