Review
Inflammasome Activation and Neutrophil Extracellular Traps in Atherosclerosis
2025 Volume 32 Issue 5 Pages 535-549
Details
2025 Volume 32 Issue 5 Pages 535-549
The deposition of cholesterol containing cholesterol crystals and the infiltration of immune cells are features of atherosclerosis. Although the role of cholesterol crystals in the progression of atherosclerosis have long remained unclear, recent studies have clarified the involvement of cholesterol crystals in inflammatory responses. Cholesterol crystals activate the NLRP3 inflammasome, a molecular complex involved in the innate immune system. Activation of NLRP3 inflammasomes in macrophages cause pyroptosis, which is accompanied by the release of inflammatory cytokines such as IL-1β and IL-1α. Furthermore, NLRP3 inflammasome activation drives neutrophil infiltration into atherosclerotic plaques. Cholesterol crystals trigger NETosis against infiltrated neutrophils, a form of cell death characterized by the formation of neutrophil extracellular traps (NETs), which, in turn, prime macrophages to enhance inflammasome-mediated inflammatory responses. Colchicine, an anti-inflammatory drug effective in cardiovascular disease, is expected to inhibit cholesterol crystal-induced NLRP3 inflammasome activation and neutrophil infiltration. In this review, we illustrate the reinforcing cycle of inflammation that is amplified by inflammasome activation and NETosis.
Abbreviations: AIM2, absent in melanoma; ADAM, a disintegrin and metalloprotease; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CANTOS; canakinumab antiinflammatory thrombosis outcome study; CARD, caspase recruitment domain; COLOT, colchicine cardiovascular outcomes trial; CPPD, calcium pyrophosphate dihydrate; CPPs, calciprotein particles; CRP, C-reactive protein; DAMP, damage/danger-associated molecular pattern; ESCRT, endosomal sorting complexes required for transport; GSDMD, gasdermin D; HAMPs, homeostasis-altering molecular processes; IL, interleukin; IL-1R, interleukin-1 receptor; LoDoCo, low-dose colchicine; LPS, lipopolysaccharide; LRR, leucine-rich repeat; MSU, monosodium urate; NETs, neutrophil extracellular traps; NINJ1, ninjurin-1; NLRP3, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3; oxLDL, oxidized LDL; PAD4, peptidyl arginine deaminase 4; PAMPs, pathogen-associated molecular patterns; PYD, pyrin domain; RCD, regulated cell death; ROS, reactive oxygen species; SFA, saturated fatty acid; TLR, toll-like receptor; VSMC, vascular smooth muscle cell
Atherosclerosis is an inflammatory disease characterized by the infiltration of various immune cells1, 2). Among immune cells, macrophages have been regarded as central players in the development of atherosclerosis3). Moreover, recent advances in transcriptome analysis at single-cell resolution have provided a detailed landscape of immune cells in atherosclerotic lesions1, 2). Various types of immune cells, including macrophages, neutrophils, dendritic cells, T cells, and B cells, are involved in complex immune responses in the development of atherosclerosis. Besides the infiltration of immune cells, the accumulation of cholesterol is an essential feature of atherosclerotic lesions4). Initially, cholesterol accumulates in foam cells derived from macrophages. Infiltrated macrophages incorporate lipoproteins and store esterified cholesterol in lipid droplets. However, excessive incorporation of cholesterol results in the deposition of cholesterol crystals in atherosclerotic lesions. Although the role of deposited cholesterol crystals had not been uncovered, Latz and his colleagues identified that cholesterol crystals are potent activators of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome, a molecular complex that regulates the processing of interleukin (IL)-1β and IL-18 5). Whereas cholesterol crystals activate NLRP3 inflammasome in macrophages, cholesterol crystals induce NETosis, a form of cell death accompanied by the release of neutrophil extracellular traps (NETs), in neutrophils6). NETs formed by cholesterol crystals, in turn, provide priming signals for NLRP3 inflammasomes. Moreover, recent studies have implied a critical role of inflammasomes in neutrophil recruitment into atherosclerotic lesions7, 8). Thus, inflammasome activation in macrophages and NETosis in neutrophils amplifies the inflammatory feedback loop during the development of atherosclerosis.
Inflammasomes are large molecular complexes that function as scaffolds for caspase-1 activation9, 10). Active caspase-1 initiates an inflammatory response via processing of the potent inflammatory cytokines IL-1β and IL-18. In addition, caspase-1 processes the pore-forming protein gasdermin D (GSDMD), which induces pyroptosis, an inflammatory cell death characterized by the release of cytosolic content11). Multiple sensor molecules form inflammasome complexes upon stimulation with pathogen-associated molecular patterns (PAMPs) and damage/danger-associated molecular patterns (DAMPs)12). Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, NLR, and caspase recruitment domain containing 4 (NLRC4), and absent in melanoma (AIM2) function as sensor molecules in inflammasome complex assembly. Among these, NLRP3 is regarded as the most critical sensor for DAMPs in atherosclerosis13). Furthermore, recent investigations have suggested that AIM2, which recognizes double-stranded DNA, is involved in the development of atherosclerosis14, 15).
The NLRP3 inflammasome consists of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (Fig.1). NEK7 participates in K＋ efflux-dependent NLRP3 inflammasome complex assembly9, 16). The components of the NLRP3 inflammasome complex possess domains that are essential for interaction and oligomerization. NLRP3 employs a pyrin domain (PYD) for oligomerization, whereas caspase-1 employs CARD for oligomerization. A notable feature of ASC is that it carries both PYD and CARD, which enables it to form a polymeric assembly called “Speck” with NLRP3 and caspase-1 17). Recent studies have provided a structural basis for the NLRP3 inflammasome complex and revealed that NLRP3 forms an inactive “CAGE” structure in steady states by forming homo-oligomers via leucine-rich repeat (LRR)-mediated interactions18). In the CAGE structure, PYD is assumed to be packed inside the complex. Upon activation stimuli, NLRP3 forms “DISC” like complex which allows the polymeric assembly of ASC to activate caspase-1 19). Active caspase-1 cleaves GSDMD to initiate pyroptosis.
The canonical NLRP3 inflammasome complex is composed of NLRP3, ASC, and caspase-1. Upstream HAMPs, such as K+ efflux, mitochondrial ROS production, and lysosomal destabilization, promote the assembly of the NLRP3 inflammasome complex. Active caspase-1 processes the inflammatory cytokines IL-1β and IL-18, and the pore-forming protein GSDMD. GSDMD-pore mediate the release of IL-1β, IL-18, and IL-1α. The Ca2＋ influx induced by GSDMD pores activates calpain to promote IL-1α processing. The GSDMD pore formation induces NINJ1 oligomerization, which causes plasma membrane rupture. The NLRP3 inflammasome also activates caspase-8 and promotes the GSDME processing of downstream apoptotic caspases.
Pyroptosis was originally reported as caspase-1-dependent necrosis-like regulated cell death20). However, after the identification of GSDMD as an executor of pyroptosis21, 22), it has been redefined as gasdremin-dependent necrotic cell death11). Multiple gasdermin family members share structural features with GSDMD. GSDMA, GSDMB, GSDMC, GSDME, and PJVK are gasdermin family members11). Gasdermin family members harbor a pore-forming amino (N)-terminal domain that can induce pyroptosis. The pore-forming activity of the N-terminal domain is prevented by the auto-inhibitory carboxyl (C)-terminal domain connected by linker sequences23). Inflammatory caspases, including caspase-1, caspase-4, caspase-5, and caspase-11 (orthologs of caspase-4 in mouse and rat) cleave the linker sequence of GSDMD and induce pyroptosis. Proteinases other than caspases, such as cathepsin G and elastase, are also involved in the processing of GSDMD24, 25). Moreover, apoptotic caspases are involved in gasdermin processing. Caspase-8 is activated and processes GSDMD to induce pyroptosis during Yersinia infection26, 27). While caspase-3 negatively regulates GSDMD-dependent pyroptosis by cleavage of the alternative target site, caspase-3 processes GSDME to induce pyroptosis28). The caspase-3-mediated processing of GSDME is considered a cause of secondary necrosis during apoptosis.
The processed N-terminal domain of gasdermins binds to inositol phospholipids, such as PIP1 and PIP2 on the plasma membrane and oligomerizes to form a large β-barrel pore23, 29). Moreover, the N-terminal domain of gasdermin interacts with cardiolipin in the mitochondrial membrane23). Both GSDMD and GSDME damage the mitochondria to induce apoptosis30). A recent report suggested that GSDMD interacts with cardiolipin exposed on the outer mitochondrial membrane and foam pores on the mitochondrial membrane to accelerate pyroptosis31). GSDMD pores cause mitochondrial dysfunction and the production of the reactive oxygen species (ROS). ROS production from mitochondria is essential for the palmitoylation of GSDMD, which enables its binding to the plasma membrane32, 33). Palmitoylation of GSDMD promotes binding of not only the N-terminal domain of GSDMD but also full-length GSDMD to the plasma membrane. The repair mechanism of the GSDMD pores has also been reported. Endosomal sorting complexes required for transport (ESCRT) III-machinery remove plasma membranes damaged by GSDMD pores in response to Ca2＋ influx34). Annexin-V＋ microvesicles are produced during the ESCRT-dependent membrane repair process. Microvesicles mediated by GSDMD pores are involved in the activation of tissue factors and the coagulation process35, 36). Another important role of GSDMD pores is the release of inflammasome-dependent cytokines29, 37). Since IL-1 family cytokines lack signal peptides, their release of IL-1 family cytokines requires unconventional mechanisms11). Mechanistically, the polybasic region of mature IL-1β mediates its translocation to the plasma membrane through interaction with PIP2. Processed cytokines located near the plasma membranes are released from the GSDMD pores. Although the release of IL-1β requires caspase-1-dependent processing, the release of IL-1α does not require processing38). IL-1α is released via GSDME pores during inflammasome activation even in the absence of caspase-1 39). Although unprocessed IL-1α has activity against receptors, GSDMD pore-mediated Ca2＋ influx promotes calpain-mediated IL-1α processing40). Moreover, a mediator of plasma membrane rupture (PMR), which is thought to be driven by osmotic pressure, has been identified. The cell surface protein ninjurin-1 (NINJ1) mediates PMR during pyroptosis41). While NINJ1 is monomeric in the membrane of living cells, NINJ1 forms a filamentous cluster during pyroptosis42). Because the size of the GSDMD-pore is insufficient for the release of large cellular contents, such as HMGB1 and LDH, NINJ1-mediated PMR is critical for the release of cytosolic contents as DAMPs. Taken together, the downstream effectors of inflammasomes work in coordination to initiate inflammatory responses.
NLRP3 inflammasome activation is regulated by multiple steps. In addition to the direct regulation of inflammasome assembly, a regulatory mechanism that modulates NLRP3 inflammasome-dependent inflammatory responses has been suggested. In particular, the priming step is critical for the full activation of the NLRP3 inflammasome. Among inflammasome components and downstream effectors, NLRP3 and IL-1β are markedly regulated during the priming step. Signaling from TLRs and IL-1Rs transcriptionally upregulates NLRP3 and IL-1β to enhance inflammasome response. Moreover, post-translational regulation of NLRP3, such as phosphorylation, ubiquitination, and deubiquitination, is essential for full activation of the NLRP3 inflammasome. The primed NLRP3 inflammasome is assembled in response to activators including PAMPs and DAMPs12). In most cases, the activators do not directly interact with NLRP3 but activate NLRP3 inflammasome-specific cellular processes. Recently, these processes have been suggested as homeostasis-altering molecular processes (HAMPs)12, 43). K＋ efflux, Ca2＋ influx, lysosomal destabilization, and mitochondrial ROS production are well-known HAMPs that activate NLRP3 inflammasome. Cholesterol crystals, calcium phosphate crystals, calciprotein particles (CPPs), and saturated fatty acids are putative DAMPs associated with atherosclerosis10, 13, 44, 45). Crystals and particulates are common patterns that cause lysosomal destabilization. Although phagocytes, such as macrophages, can incorporate these crystals and particles by phagocytosis, an excess load of them in the lysosome results in indigestion called “frustrated phagocytosis” which causes lysosomal destabilization4). Leakage of the lysosomal enzyme cathepsin B into the cytosol initiates NLRP3 inflammasome activation. Cathepsin B leakage likely activates the NLRP3 inflammasome via the induction of K＋ efflux, which is regarded as a common HAMPs that activates NLRP3 inflammasome activation46). Specific descriptions of each DAMP are provided below.
Cholesterol CrystalsCholesterol crystals are thought to be the central DAMPs that activate the NLRP3 inflammasome, which is associated with atherosclerosis. Although cholesterol crystals are thought to accumulate in advanced atherosclerosis, Duewell et al. revealed that the crystals are deposited in the early stage of atherosclerosis development and activate the NLRP3 inflammasome5). Although the detailed mechanisms remain unclear, several processes regarding the formation of cholesterol crystals in atherosclerosis have been reported. Oxidized LDL (oxLDL) is incorporated into macrophages via the scavenger receptor CD36 and promotes the intracellular crystallization of cholesterol to trigger NLRP3 inflammasome activation47). Another study suggested that vascular endothelial cells are the origin of cholesterol crystals in atherosclerosis in hyperlipidemic mice48). In contrast, ACAT1 deficiency results in accumulated free cholesterol, which promotes cholesterol crystal deposition and aggravates the development of atherosclerosis in an NLRP3 inflammasome-dependent manner49).
Free Fatty AcidsSaturated fatty acids (SFAs) are risk factors for cardiovascular diseases. The replacement of dietary saturated fatty acids with monounsaturated fatty acids and polyunsaturated fatty acids lowers cardiovascular and all-cause mortality50). The pro-inflammatory effects of saturated fatty acids have been investigated as mechanisms of low-grade inflammation associated with metabolic diseases51). Activation of TLR2/4 is a possible mechanism underlying SFA-mediated pro-inflammatory effects. Although direct interaction of TLR4 and SFA had been expected based on the similarity between SFAs and lipid A contained in lipopolysaccharide, a ligand for TLR4, indirect mechanisms of activation have been suggested52). In addition to upregulating IL-1β priming, SFAs activate the NLRP3 inflammasome via multiple mechanisms. Wen et al. suggested that SFAs impair mitophagy, resulting in mitochondrial ROS production and NLRP3 inflammasome activation53). Other studies have suggested that SFAs trigger NLRP3 inflammasome activation via lysosomal destabilization44, 54). In contrast, supplementation with unsaturated fatty acids prevents SFA-induced NLRP3 inflammasome activation44, 55). With regard to SFA-induced lysosomal destabilization, we previously reported that excess SFAs cause lysosomal destabilization via intracellular crystallization of SFAs44). Although previous studies have suggested that fatty acid oxidation via NOX4- or UCP2-mediated fatty acid synthesis activates the NLRP3 inflammasome, these studies have been retracted56, 57).
CPPs and Calcium Phosphate CrystalsCPPs are defined as aggregates of serum fetuin-A laden with tiny calcium phosphate precipitants58). CPPs are dispersed in the blood as colloids and circulating CPPs are elevated in patients with chronic kidney diseases. Increased CPP levels are associated with increased aortic calcification in CKD patients59). Furthermore, CPPs have been correlated with the volume of lipid-rich plaques in coronary atherosclerosis60). CPPs also have the potential to activate both TLR4 signaling and the NLRP3 inflammasome. CPPs directly bind to TLR4 and activate downstream signaling61). Moreover, we found that CPPs activate the NLRP3 inflammasome via lysosomal destabilization45). Jäger et al. suggested that CPPs activate CaSR to induce NLRP3 inflammasome activation62). Notably, CPPs induce IL-1α release in an NLRP3 inflammasome-independent manner45). Calcium crystals, including calcium phosphate, calcium carbonate, and calcium oxalate, are also present in atherosclerotic lesion63). Calcium phosphate crystals also activate the NLRP3 inflammasome via lysosomal destabilization64, 65).
Extracellular ATPExtracellular ATP is presumed to be a DAMP associated with tissue damage that is released from dying cells. Extracellular ATP interacts with P2X7 receptors and causes K＋ efflux, resulting in NLRP3 inflammasome activation66). The contribution of the P2X7 receptor to inflammasome activation and atherosclerosis development has been confirmed in animal models67).
Non-Canonical Inflammasome ActivationCaspase-11, an inflammatory caspase, functions as a sensor molecule and is activated in a ligand-dependent manner12). Active caspase-11 processes GSDMD to induce pyroptosis and triggers NLRP3 inflammasome activation21). This process is known as non-canonical inflammasome activation (Fig.2). The pannexin 1 channel- or GSDMD-dependent K＋ efflux mediates the caspase 11-triggered non-canonical inflammasome68). Although the involvement of caspase-11 in the development of atherosclerosis has been demonstrated, the upstream regulators of caspase-11 in atherosclerosis have not been determined. Caspase-11 was initially reported as an intracellular sensor for lipopolysaccharide (LPS) in E. coli 69). Caspase-11 recognizes hexa-acetylated lipid A in LPS. The following studies have reported that oxidized phospholipids (oxPAPCs) are endogenous ligands of caspase-11 70). However, a recent report suggested that oxPAPC failed to activate caspase-11 and functioned as an inhibitor of LPS-mediated activation of caspase-11 71). Thus, the endogenous activators of caspase-11 remain controversial. Ligand-independent spontaneous activation of caspase-11 is a possible mechanism. A previous study suggested that the absence of SERPINB1 induces spontaneous activation of caspase-11 72). Because the expression of caspase-11 is elevated in advanced atherosclerotic and unstable plaques, spontaneous activation of caspase-11 should be considered7).
The non-canonical NLRP3 inflammasome is initiated by inflammatory caspases including human caspase-4 and murine caspase-11. Stimulation with LPS activates caspase-4/11 and promotes the processing of GSDMD. Ion mobilization by GSDMD-pore mediates various cellular processes; Ca2＋ influx causes the processing of IL-1α, ESCRT-mediated membrane repair, and microvesicle formation. K＋ efflux promotes assembly of the NLRP3 inflammasome and processing of IL-1β and IL-18. Pannexin 1 has also been suggested to be a mediator of the K＋ efflux induced by caspase-4/11 activation.
Multiple studies have shown that NLRP3 inflammasome deficiency prevents the development of atherosclerosis in mice5, 8, 64, 73). Duewell et al. reported that the deficiency of NLRP3 inflammasome components or the combined deficiency of IL-1β/α in bone marrow cells prevents the development of atherosclerosis in LDLR-deficient mice. Moreover, a subsequent study confirmed that deficiency of NLRP3 inflammasome components in LDLR-deficient mice or Apoe-deficient mice prevented atherosclerosis. In contrast, Menu et al. reported that the development of atherosclerosis is unchanged by inflammasome deficiency in cholesterol-rich diet (1.25%)-fed Apoe-deficient mice74). IL-1β and IL-1α are likely to be central effectors of the NLRP3 inflammasome because IL-1β/α-deficiency in bone marrow cells prevents atherosclerosis development to a similar extent as NLRP3- and ASC-deficiency5). Inhibition of IL-1β prevents the development of atherosclerosis75). This study clearly demonstrated the distinct roles of IL-1β and IL-1α76). While blocking IL-1α with an antibody prevents early atherogenesis, blocking IL-1β reduces the area of established atheroma. Notably, one study has proposed that IL-1 receptor signaling in vascular smooth muscle cells (VSMC) plays a protective role in advanced atherosclerotic lesions77). Lack of IL-1 receptor signaling causes a reduction in the number of VSMCs and the collagen content required for the fibrous cap. The role of IL-18, another inflammasome-dependent cytokine, in atherosclerosis is debatable, probably because of its systemic effects. Multiple studies suggest that the administration of IL-18 promotes atherosclerosis78, 79). Accordingly, a report suggests that IL-18 deficiency prevents the development of atherosclerosis despite increased cholesterol levels80). However, another study suggested that IL-18 deficiency promotes atherosclerosis with increased cholesterol levels81).
Although few studies have investigated the detailed mechanisms of the inflammatory response in inflammasome-deficient mice, Westerterp et al. revealed that the exacerbation of atherosclerosis by the combined deficiency of myeloid Abca1 and Abcg1 in bone marrow cells was inhibited by deficiency of NLRP3 and caspase-1 8). Furthermore, the infiltration of neutrophils and the formation of NETs by myeloperoxidase＋ citrullinated histone H3＋ was dramatically prevented by the deficiency of inflammasome components. Furthermore, our recent study clarified that a deficiency of caspase-11 prevented necrotic core formation and neutrophil infiltration into atherosclerotic plaque7). In acute inflammatory responses, crystals and particulates induce neutrophil infiltration in an NLRP3 inflammasome-dependent manner5). The aforementioned studies in atherosclerotic mice revealed that neutrophil infiltration in atherosclerosis is mediated by inflammasome activation7, 8).
The involvement of neutrophils in the development of atherosclerosis has been underestimated because of their low lifespan and frequency in atherosclerotic plaque82, 83). However, clear visualization of neutrophils using anti-Ly6g antibody or myeloperoxidase staining indicates the presence of neutrophils in mouse atherosclerotic plaque82). Staining with anti-CD177 or CD66b clarified the presence of neutrophils in human rupture-prone plaques. Recent studies have shown the involvement of neutrophils in various steps of atherosclerosis development, including the initiation of atherosclerosis, destabilization of atherosclerotic plaques, and desquamation of endothelial cells83). Neutrophils play a promotive role in the initial step of atherosclerosis formation. While hypercholesterolemia induces neutrophilia, the depletion of neutrophils at the initial stage of atherosclerosis development prevents the development of atherosclerosis and macrophage infiltration84). Neutrophil-derived factors cathepsin G, cathelicidin, and α-defensin promote the adhesion of monocytes to the vascular wall85-88). In contrast, neutrophils are involved in unstable plaque formation, which is characterized by a lipid-rich necrotic core covered by a fibrous cap composed of VSMCs and an extracellular matrix83). In human carotid atherosclerotic plaque samples, the number of neutrophils is strongly associated with the histopathologic features of rupture-prone atherosclerotic lesions89). Another study showed that neutropenia increases the VSMC content and fibrous cap thickness, which decreases plaque vulnerability, whereas neutrophilia has the opposite effect90). Mechanistically, infiltrated neutrophils degrade the extracellular matrix through proteinases such as metallopeptidase (MMP)8, MMP9, and disintegrin and metalloprotease (ADAM)8 7, 82, 91). Furthermore, a recent study showed that histone H4 released by NETs induced lytic cell death in VSMCs90). Moreover, NETs function as drivers of the inflammatory responses in atherosclerosis.
Although the original function of NETs is to eliminate pathogens, recent studies have shown that NETs play a critical role in the development of atherosclerosis92). NETs are web-like structures composed of decondensed chromatin and neutrophil-derived nuclear, cytosolic, and granular proteins. Cell death accompanied by NET formation was defined as NETosis93). NETosis is mediated by multiple enzymes, including NADPH oxidase, neutrophil elastase, myeloperoxidase, and peptidyl arginine deaminase 4 (PAD4)92). PAD4-mediated citrullinated histones are a marker of NETosis. Previous studies have implicated NETosis in the development of atherosclerosis. NETs induce type I interferon responses initiated by plasmacytoid dendritic cells to promote atherosclerosis94). Furthermore, the inhibition of PAD4 by Cl-amidine prevents phorbol-12-myristate-13-acetate (PMA)-induced NETs formation and the development of atherosclerosis95). A later study revealed that the inducer of NETosis in atherosclerosis is cholesterol csytals6). To date, the induction of NETosis by multiple crystals and particulates has been reported. MSU crystals induce NETosis and IL-1β release in neutrophils96). Other crystals and particulates, such as calcium oxalate, calcium phosphate, silica particles, and asbestos also induce NETosis97). The report shows that cholesterol crystals induce NETosis in NADPH oxidase-, proteinase 3-, and neutrophils elastase-dependent manner6). In addition, the involvement of NETosis in the development of atherosclerosis has been demonstrated in mice deficient in proteinase 3 and neutrophil elastase on an Apoe-deficient background. NETs provide macrophages with priming signals that enable them to produce IL-1β in response to cholesterol crystals. NETs also induce IL-17-producing Th17 cells, which upregulate the chemokines CXCL1 and CXCL2 to promote neutrophil recruitment. Moreover, NETs can provide dsDNA, which triggers AIM2 inflammasome activation, resulting in plaque destabilization in advanced atherosclerosis15). Notably, the inhibition of PAD4 failed to inhibit cholesterol crystal-induced NETosis6). The effect of PAD4 inhibition on the development of atherosclerosis is still controversial because PAD4 deficiency in bone marrow cells does not influence atherosclerosis, but contributes to acute thrombotic complications of the intima98).
Multiple studies have investigated the involvement of GSDMD in the induction of NETosis99-101). Earlier studies have suggested that non-canonical inflammasome-mediated or neutrophil protease-mediated processed GSDMD promotes NETosis100, 101). However, a follow-up study suggested that GSDMD promotes inflammasome-induced pyroptosis in neutrophils but is dispensable for NETosis99). Further investigations are required to clarify the role of GSDMD in the induction of NETosis in atherosclerosis. Taken together, cholesterol crystal-triggered inflammatory responses in innate immune cells and crosstalk between these cells could be a potential target to regulate inflammation in atherosclerosis (Fig.3).
Crystals and particles such as cholesterol crystals and CPPs activate the NLRP3 inflammasome. Canonical and non-canonical NLRP3 inflammasome activation induces neutrophil infiltration into atherosclerotic plaques. Cholesterol crystals induce NETosis in infiltrated neutrophils. The released NETs upregulate the expression of proIL-1β to augment inflammasome-dependent inflammatory responses. NETs can be a source of dsDNA that activates the AIM2 inflammasome. Colchicine, which inhibits neutrophil infiltration and crystal-induced NLRP3 inflammasome activation, is a potential therapeutic option to prevent inflammatory responses triggered by cholesterol crystals in atherosclerotic plaques.
Multiple drugs targeting the NLRP3 inflammasome, including OLT-1177, Inzomelid, Somalix, and DFV890, are in clinical and preclinical development102). However, the efficacy of NLRP3 inflammasome-targeting drugs in human atherosclerotic diseases has not yet been evaluated. On the other hand, the drugs targeting downstream of inflammasome have been investigated. Drugs targeting IL-1, such as canakinumab (a monoclonal antibody targeting IL-1β), anakinra (IL-1 receptor antagonist), and rilonacept (a soluble decoy receptor for IL-1), are available for treatment103). The Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) is the first trial to demonstrate that inflammation could be a target for atherosclerosis104). The study showed that canakinumab (150 mg) lowered the rate of recurrent cardiovascular events in patients with previous myocardial infarction and high-sensitivity C-reactive protein (CRP) ≥ 2 mg/L (n = 10,061). Although CANTOS provides significant results that inflammation could be a therapeutic target for atherosclerotic disease, several issues remain, including cost-effectiveness and safety concerns. In particular, the incidence of fatal infections and sepsis was higher in the canakinumab-treated group than in the placebo group.
Recent studies have demonstrated the efficacy of colchicine in treating atherosclerotic diseases. Colchicine, an anti-inflammatory alkaloid, has been used for medicinal purposes since ancient Egyptian times, approximately 3500 years ago105). Currently, colchicine is used to treat gout and familial Mediterranean fever (FMF) caused by a PYRIN mutation. The anti-inflammatory effects of colchicine are mediated through multiple mechanisms. Colchicine is concentrated in leukocytes and directly binds to tubulin to inhibit its polymerization, resulting in disruption of the cellular cytoskeleton and inhibition of mitosis, intracellular trafficking, cytokine and chemokine secretion, and cell migration105-107). In particular, colchicine inhibits the migration of neutrophils to inflamed tissue105). In addition to its effect on chemotaxis, colchicine inhibits neutrophil adhesion by downregulating L-selectin105). Colchicine also alters the distribution of E-selectin on the endothelial cell surface to prevent neutrophil adhesion105). Furthermore, recent studies have suggested that colchicine inhibits NET formation108, 109). Meanwhile, the inhibitory effects of colchicine on the NLRP3 inflammasome have been suggested. Martinon et al. reported that colchicine inhibits MSU crystals and calcium pyrophosphate dihydrate (CPPD) crystal-induced NLRP3 inflammasome activation, whereas it failed to inhibit ATP-induced NLRP3 inflammasome activation110). A later study suggested that a microtubule-dependent mechanism is involved in this process111). However, it remains controversial whether colchicine can broadly inhibit NLRP3 inflammasome activation. Multiple studies have shown that colchicine successfully prevents PYRIN inflammasome activation, whereas it fails to inhibit K＋ efflux-driven NLRP3 inflammasome activation induced by Nigericin, a K＋ ionophore112-114). Unknown inhibitory mechanisms of crystal- or particulate matter-induced inflammasome activation may be involved in this process.
Based on the anti-inflammatory effect of colchicine, the Low-Dose Colchicine (LODOCO) study, an open-label pilot trial (n = 532), was conducted to investigate whether low-dose colchicine (0.5 mg/day) can reduce the risk of cardiovascular events in patients with clinically stable coronary disease115). Colchicine lowers the risk of acute coronary syndrome, out-of-hospital cardiac arrest, or non-cardioembolic ischemic stroke. A subsequent larger study, the LoDoCo2 trial was conducted to validate the findings of the LODOCO study. LoDoCo2 was designed as a multicenter, double-blind, placebo-controlled, randomized trial involving 5,522 patients with chronic coronary disease. Colchicine treatment (0.5 mg/day) significantly lowered the incidence of cardiovascular events (cardiovascular death, spontaneous myocardial infarction, ischemic stroke, or ischemia-driven coronary revascularization)116). Moreover, the Colchicine Cardiovascular Outcomes Trial (COLCOT), a randomized, double-blind trial involving 4,745 patients after myocardial infarction was performed117). Treatment with colchicine (0.5 mg/day) significantly lowers the risk of ischemic cardiovascular events. Recently, low-dose colchicine for atherosclerotic cardiovascular disease was approved by FDA in United States. Because treatment with colchicine is effective in the prevention of cardiovascular events and is cost-effective118), it is anticipated to be a therapeutic option.
Although inflammation is a key characteristic of atherosclerosis, the development of therapies that target inflammation is ongoing. The inflammasome plays a critical role in the recognition of DAMPs, including cholesterol crystals, and the initiation of the inflammatory response in the development of atherosclerosis. Inflammasome activation promotes neutrophil recruitment to atherosclerotic plaques, in which cholesterol crystals trigger NETosis in recruited neutrophils. The vicious inflammatory cycle triggered by cholesterol crystals may be a potential therapeutic target.
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