NLRP3 inflammasome, an immune-inflammatory target in pathogenesis and treatment of cardiovascular diseases

3.1.1 Endothelial injury

Endothelial injury or intima integrity loss is the initial step of atherosclerosis. Different types of injuries, including mechanical, immune-mediated, and chemical-induced injuries can lead to endothelial injury. NLRP3 inflammasome activation in ECs is a critical event in several endothelial injuries. Cigarette smoking is one of the major risk factors of atherosclerosis. Nicotine increases the expression of both NLRP3 and ASC, and triggers NLRP3 inflammasome activation and pyroptosis in human aortic endothelial cells.²⁵ Pollution is another important reason for NLRP3 inflammasome activation-mediated endothelial injury. Compared to mice exposed to filtered air, mice exposed to PM2.5 showed exacerbated atherosclerotic plaque formation and enhanced NLRP3 inflammasome activation in the aorta, as indicated by increased serum levels of IL-1β and IL-18.²⁶ Acrolein and cadmium, two common environmental pollutants, also cause NLRP3 inflammasome-mediated EC injury.^{27, 28} In addition, NLRP3 inflammasome has been reported to be involved in endothelial injury and EC loss due to metabolic etiologies, including abnormal uptake of lipids such as oxidized low-density lipoprotein (oxLDL) and cholesterol. In fact, oxLDL treatment upregulated the protein levels of NLRP3, caspase-1, and IL-1β in vascular ECs in a dose-dependent manner and increased the number of PI-positive cells, suggestive of increased pyroptosis. Further investigation revealed that oxLDL inhibited tet methylcytosine dioxygenase 2 (TET2), which significantly downregulates the expression of caspase-1, NLRP3, and IL-1β by improving mitochondrial functions and blocking the NF-κB pathway.²⁹ Colocalization assays revealed that cholesterol crystal (ChC) or 7-ketocholesterol (7-Keto) considerably increased NLRP3 inflammasome generation and activation in mouse carotid arterial ECs.³⁰ In addition, ChCs induced severe endothelial dysfunction in coronary arteries of Nlrp3^+/+ mice, compared with the coronary arteries of transgenic Nlrp3^−/− mice.³¹ Interestingly, short-chain fatty acids, the metabolites from gut microbes, also exert anti-atherosclerotic effects by preserving endothelial function. A study involving partial-ligated carotid artery mouse model showed that butyrate significantly decreased cholesterol-induced NLRP3 inflammasome activation in the arterial walls.³² The protective role of butyrate involves blockage of the lipid raft redox signaling pathway and decrease in ChC- and 7-Keto-mediated production of free radicals.

3.1.2 Monocyte differentiation into macrophages and engulfment of lipoproteins

After endothelial dysfunction, monocytes adhere to the lesion sites, differentiate into macrophages, and engulf lipoproteins, including oxLDL and ChCs. Such macrophages with engulfed lipids transform into foam cells. NLRP3 inflammasome activation is involved in monocyte adhesion and foam cell formation. oxLDL was reported to upregulate SREBP-1, thereby promoting oxLDL-induced lipid accumulation and foam cell formation. Moreover, IL-1β, the product of NLRP3 inflammasome activation, increased the RNA and protein expression of SREBP-1 in monocytes and monocyte-derived macrophages.³³ Another study evaluated foam cell formation by assessing intracellular lipid droplets, cholesterol content, and acyl-coenzyme mRNA levels and investigated monocyte adhesion by determining the number of monocytes adhering to vascular smooth muscle cells. This study showed that NF-κB inhibition using BMS-345541 or NLRP3 inflammasome inhibition using MCC950 decreased oxLDL-induced foam cell formation and monocyte adhesion.³⁴ ChCs also trigger arterial inflammation and destabilization of atherosclerotic plaques. An equilibrium is maintained between esterified cholesterol (ESC) available in the sub-intima and the free cholesterol (FRC) generated by macrophages via the action of cholesteryl ester hydrolases on ESC. However, disequilibrium between ESC and FRC affects foam cell and ChC formation. Growth of ChCs activates NLRP3 inflammasome and induces inflammation and plaque destabilization.³⁵ Cholesterol efflux is mediated by the cholesterol transporters ATP-binding cassette A1 and G1 (ABCA1/G1), which transform cholesterol into high-density lipoprotein. Cholesterol efflux mediated by inflammasome also plays fundamental roles in atherogenesis. Bone marrow transplantation from mice with myeloid Abca1/g1 deficiency along with deficiency of the inflammasome components Nlrp3 or Caspase-1/11 into LdIr^−/− mice fed with a western-type diet showed that Nlrp3 and Caspase-1/11 deficiency alleviated atherosclerotic lesions in myeloid Abca1/g1-deficient Ldlr^−/− mice. The study also indicated that inflammasome activation promoted neutrophil recruitment and neutrophil extracellular trap formation in plaques.³⁶ Collectively, these reports indicate that accumulation of lipids such as oxLDL and ChCs induces NLRP3 inflammasome activation, which promotes monocyte adhesion, macrophage transformation into foam cells, and the subsequent development of atherosclerotic lesions. Intriguingly, microbial pathogens augment atherosclerosis via NLRP3 inflammasome. Chlamydia pneumoniae induced extracellular IL-1β release, which decreased GPR109a and ABCA1 expression and cholesterol efflux and induced cholesterol accumulation and foam cell formation in Ldlr^−/− mice. Conversely, Nlrp3 knockout alleviated the C. pneumoniae-accelerated atherosclerosis.³⁷ Similarly, Porphyromonas gingivalis, a periodontal disease pathogen, interacts with PRRs, activates NLRP3 inflammasome, and promotes atherogenesis.³⁸

3.2.1 Fibroblasts: Initial activation sites for NLRP3 inflammasome in the heart

Fibroblasts are generally considered vital in the healing process after I/R injury owing to their changing phenotypes and secretion of cytokines and growth factors.⁴¹ However, fibroblasts also show earlier and more dramatic activities during NLRP3 inflammasome activation in response to cardiac I/R circumstances. In fact, inflammasome activation in cardiac fibroblasts was reported to be essential for myocardial I/R injury. The initial site of inflammasome activation in myocardial I/R is in cardiac fibroblasts and not cardiomyocytes. Hypoxia/reoxygenation clearly induces a considerable increase in IL-1β production in cardiac fibroblasts but not in cardiomyocytes. Signals for inflammasome activation were also explored. ROS production and K⁺ efflux were found to mediate NLRP3 inflammasome activation in cardiac fibroblasts during I/R injury.⁴² The expression of NLRP3, IL-1β, and IL-18 mRNA was found to be increased in cardiac tissue post myocardial infarction, and expression in non-cardiomyocytes altered more dramatically in an NF-κB–dependent manner. Further investigation showed that ATP and K⁺ efflux promoted NLRP3 inflammasome assembly and IL-1β secretion in fibroblasts, whereas NLRP3-knockout mice showed better cardiac function and reduced infarct size.⁴³ Nevertheless, more studies are required to demonstrate the role of NLRP3 activation in fibroblasts during myocardial I/R injury.

3.2.2 Crucial molecules involved in NLRP3 inflammasome-mediated cardiac I/R injury

TXNIP is a well-known contributor of I/R injury, and increased TXNIP levels have been reported in the myocardium during I/R.⁴⁴ Furthermore, immunoprecipitation assays showed a direct interaction between TXNIP and NLRP3, and Txnip knockdown decreased NLRP3 activation and infarct size in mouse myocardial tissues. Further exploration showed that I/R stimulated NLRP3 inflammasome activation in cardiac microvascular endothelial cells (CMECs) but not cardiomyocytes, and Txnip knockdown inhibited NLRP3 inflammasome activation in CMECs. The TXNIP-mediated NLRP3 inflammasome activation occurred via ROS stimulation and was inhibited by the ROS scavenger EUK134.⁴⁵ Activated protein C (aPC) exerts a cardioprotective effect on myocardial I/R injury not only via apoptosis⁴⁶ but also via NLRP3 inflammasome.⁴⁷ aPC treatment before or after myocardial I/R injury significantly reduced infarct size and reduced NLRP3 inflammasome activation. Kinetic in vivo analyses indicated that NLRP3 inflammasome activation preceded apoptosis in a mouse I/R model. In vitro experiments involving macrophages, cardiomyocytes, and cardiac fibroblasts showed that aPC comprehensively inhibited inflammasome activation via the proteinase-activated receptor 1 and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways.⁴⁷ This study suggested that aPC exerts a fundamental and comprehensive effect on different kinds of cardiac resident cells in the context of NLRP3 inflammasome activation.

3.3.1 HF induced by pressure overload

Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) plays an important role in HF pathogenesis. CaMKIIδ expression and activation were enhanced in rabbit and human HF,⁴⁹ whereas treatment with CaMKII inhibitors (KN-93 and AIP) improved contractility and preserved cardiac functions in the human failing myocardium.⁵⁰ CaMKIIδ-dependent activation of NLRP3 inflammasome mediated fibrosis during the failing heart conditions. Such CaMKIIδ-dependent inflammasome activation occurs at an early point during angiotensin Ⅱ (AngⅡ) infusion, before the evident recruitment of macrophages. In addition, NLRP3 knockout or MCC-mediated inhibition of NLRP3 inflammasome alleviated inflammation, indicated by decreased macrophage accumulation and fibrosis during AngⅡ infusion, which resembles the effect of CaMKIIδ deletion.⁵¹ Another study also revealed that CaMKIIδ deletion in cardiomyocytes attenuated pressure overload-induced accumulation of CD68⁺ macrophages and cytokines such as IL-1β and IL-18. The cardiomyocytes isolated from mice subjected to cardiomyocyte-specific CaMKIIδ deletion and transverse aortic constriction (TAC) showed decreased NLRP3 levels, caspase-1 activity, and IL-18 activation, indicating that CaMKIIδ activation in response to pressure overload triggers NLRP3 inflammasome activation.⁵² Somatic mutations in hematopoietic stem cells also influence HF incidence and severity via NLRP3 inflammasome. Mutation in TET2, an epigenetic regulator, was reported to promote HF, and transplantation of bone marrow with TET2-deficient cells or conditional myeloid-restrictive TET2 inactivation significantly augmented cardiac remodeling in mouse models of TAC- and chronic ischemia-induced HF. In addition, TET2 deficiency enhanced IL-1β expression, whereas MCC950 treatment significantly decreased HF development.⁵³ Recently, increased S-nitrosylation of muscle LIM protein (MLP) in pressure-overloaded HF was reported to induce the formation of a complex between Toll-like receptor 3 (TLR3) and receptor-interacting protein kinase 3 (RIP3), thereby activating NLRP3 inflammasome and promoting the progression of myocardial hypertrophy.⁵⁴ However, some studies have reported contradictory findings. One study showed that NLRP3 knockout accelerated the decrease of cardiac function, manifested by enhanced level of inflammation, fibrosis, and hypertrophy in animal models with pressure overload interruption, indicating that NLRP3 negatively regulated cardiac remodeling.⁵⁵ Therefore, further investigation is warranted to explain the discordance between these studies.

3.3.2 Specific HF due to cardiomyopathy and myocarditis

Cardiomyopathy literally means disease of the heart muscle. Although the etiologies of cardiomyopathy are mainly idiopathic, they may be associated with infectious, metabolic, genetic, and immune factors. Cardiomyopathy usually shows clinical symptoms similar to those of HF, which is characterized by reduced cardiac outflow, and is one of the prognostic outcomes of myocardial infarction. Cardiomyopathy and myocarditis, along with myocardial infarction, induce HF via myocardium morbidity, and NLRP3 inflammasome contributes to such myocardium morbidity-induced HF.

Diabetic cardiomyopathy prevalence is increasing rapidly and is associated with the high morbidity rate of diabetes mellitus, which occurs due to metabolic disorders leading to inflammation, fibrosis, and cardiomyocyte death. NLRP3 inflammasome was confirmed to mediate fructose cytotoxicity in cardiomyocytes of fructose-fed rats. Furthermore, the fructose-fed rats and fructose-treated H9c2 cells showed increased CD36, TLR4, TLR6, NLRP3, IL-1β, Smad2/3 phosphorylation, and Smad4, whereas NLRP3 knockdown significantly alleviated fructose-induced cardiac inflammation and fibrosis.⁵⁶ In addition, H3 relaxin, an active peptide with protective roles in several CVDs, significantly inhibited NLRP3 inflammasome activation and collagen synthesis in cardiac fibroblasts under high glucose conditions. Moreover, ROS overproduction and ligand-gated ion channel 7 (P2X7) were crucial in the NLRP3-mediated promotion of high glucose-induced fibrosis.⁵⁷ Knockdown experiments using animal models of diabetic cardiomyopathy showed that noncoding RNAs, such as miRNA30d and lncRNA Kcnq1ot1, are important in mediating NLRP3 activation and pyroptosis, respectively.^{58, 59}

Myocardial infarction, which is extremely fatal in the acute phase, can also gradually progress to chronic HF. Therefore, treatment after myocardial infarction is critical to delay HF onset. Colchicine significantly decelerated HF development after myocardial infarction, as evaluated by heart weight/tibial length, lung wet weight/dry weight ratio, and BNP expression. In particular, colchicine alleviated left ventricular remodeling and preserved contractile function. Furthermore, colchicine decreased NLRP3, ASC, and caspase-1 levels in the MI area, thus attenuating NLRP3 inflammasome expression and activation.⁶⁰ Intriguingly, diabetes was demonstrated to further deteriorate cardiac functions and augment HF after myocardial infarction by enhancing the activation of AIM2 and NLRC4 inflammasome instead of NLRP3 inflammasome.⁶¹

Several pathogens, including bacteria, viruses, and parasites cause heart diseases, and coxsackievirus B3-induced myocarditis is the most common and important infectious heart disease. NLRP3 inflammasome is strongly associated with CVB3-induced myocarditis. A continuous increase in the production of IL-1β, cleaved caspase-1, and ASC was reported in the first 7 days after CVB3 infection. Conversely, inhibiting caspase-1 using specific inhibitor or blocking the IL-1β pathway using a neutralizing antibody decreased the activities of myocardial enzymes, improved cardiac functions, and resulted in higher survival rate in animal models, indicating amelioration of the symptoms of CVB3-induced myocarditis.⁶² Cathepsin B, an important regulator in the lysosome-associated pathway of NLRP3 inflammasome activation, promotes CVB3-induced myocarditis via activation of NLRP3 inflammasome and pyroptosis.⁶³ Furthermore, in addition to cardiomyocytes, macrophages also show NLRP3 inflammasome activation in CVB3-induced myocarditis, and instead of viral RNAs, two CVB3 capsid proteins, VP1 and VP2, trigger NLRP3 inflammasome activation in macrophages.⁶⁴ In contrast, another study reported severe lesions, worse cardiac functions, and higher cardiac virus titers in CVB3-infected NLRP3 knockout mice, compared with the controls.⁶⁵

Septic cardiomyopathy is defined as acute cardiac dysfunction that occurs in patients with sepsis, and is caused by etiologies other than the generally observed cardiac etiologies. NLRP3 inflammasome activation in cardiac fibroblasts also significantly contributes to the development of septic cardiomyopathy. For example, lipopolysaccharide (LPS) treatment was reported to increase NLRP3 and IL-1β activation in cardiac fibroblasts, and this activation was inhibited by both NLRP3 knockdown and treatment with a pharmacological inhibitor (glyburide). Interestingly, LPS-induced activation of NLRP3 inflammasome in cardiac fibroblasts downregulated intracellular cAMP response to dobutamine in cardiomyocytes, highlighting the potential of NLRP3 inflammasome as a therapeutic target.⁶⁶ Chagas cardiomyopathy, a parasite-induced cardiomyopathy, is also associated with NLRP3 activation. IL-1β and NLRP3 upregulation has been reported in patients’ indeterminate clinical form of Chagas disease.⁶⁷ Moreover, single nucleotide polymorphism association analysis revealed that Chagas disease presenting Chagas cardiomyopathy is associated with inflammasome genes, including NLRP1 and CARD; however, NLRP3 was not investigated in this study.⁶⁸

Several other cardiomyopathies such as idiopathic dilated cardiomyopathy,⁶⁹ genetic structural cardiomyopathy,⁷⁰ and autoimmune myocarditis⁷¹ are associated with NLRP3 inflammasome activation as well. The mechanisms of all previously mentioned CVDs involving NLRP3 inflammasome and pyroptosis are summarized in Figure 3.