Gout is a type of arthritis associated with inflammation and is primarily caused by the deposition of monosodium urate (MSU) crystals in joints because of increased serum uric acid levels. Elevated serum uric acid levels gradually develop into hyperuricemia and lead to MSU formation, which is stimulated by various factors and leads to joint inflammation. The risk factors for gout include genetic, metabolic, and multiple comorbidities, including metabolic syndrome, cardiovascular disease, and renal disease (1). The global healthcare burden of gout has increased. Worldwide prevalence and incidence vary by study region, population, and assessment methods, ranging from <1% to 6.8%, with an incidence of 0.58–2.89 per 1,000 person-years (1). Patients with gout are primarily treated with uric acid-lowering and anti-inflammatory therapies (1, 2). Uric acid-lowering drugs such as allopurinol, febuxostat, probenecid sulfinpyrazone, and benzbromarone—typically used in combination—increase the metabolic burden on the kidney, the risk of kidney stone disease, and uric acid excretion and primarily reduce purine catabolism (3). Additionally, some patients do not accept long-term uric acid-lowering therapies due to lack compliance and, therefore, exhibit a poor treatment response. Anti-inflammatory treatments include nonsteroidal anti-inflammatory agents, colchicine, and corticosteroids. However, these drugs may induce multiple side effects; the development of new drugs has been slow (3). Additionally, currently used clinical drugs do not cover all clinical stages of gout.

Cell death commonly occurs in various physiological and pathological processes in the human body. In 2018, the Nomenclature Committee on Cell Death classified cell death as either accidental or regulated based on functional differences, which includes 11 primary forms: apoptosis, necroptosis, pyroptosis, ferroptosis, parthanatos, entotic cell death, NETotic cell death, autophagy-dependent cell death, alkaliptosis, oxeiptosis, and lysosome-dependent cell death (4). Numerous studies have characterized these types of cell death and their underlying molecular mechanisms. However, the role of cell death in certain diseases remains unclear. Different cell death patterns may be associated with the different clinical stages of gout. MSUs in gout can function as pattern recognition receptors and thus act as danger signals to activate multiple immune cells and multiple cell death pathways, possibly through mutual crosstalk (5). For example, tumor necrosis factor (TNF)-α and interleukin (IL)-1β are important molecules or products of apoptosis, necroptosis, and pyroptosis. These molecules can be secreted by monocytes and promote inflammatory cell infiltration in patients with gout (6–9). In this review, we provide insights that may be useful for developing new drugs for gout and for determining the associated pathological mechanisms by describing the multiple forms of cell death, including pyroptosis, NETosis, necroptosis, and apoptosis, in gout and their roles.

Pyroptosis is a form of pro-inflammatory cell death (10). The various key molecules involved in pyroptosis include the inflammasome [apoptosis-associated speck-like protein containing a C-terminal caspase activation and recruitment domain (ASC), NLRP3, and pro-caspase1], members of the gasdermin family, caspase1, and caspases4/5/11, which mediate the non-classical pathway (11). The activation of the NLRP3 inflammasome hydrolyzes IL-1β precursor (pro-IL-1β), IL-18 precursor (pro-IL-18) and gasdermin D via caspase-1, resulting in the maturation and release of IL-18 and IL-1β and N-terminal fragments of gasdermin D. It results in gasdermin-mediated pore formation and membrane rupture, the release of cellular contents to mediate inflammation (11). The activation of the NLRP3 inflammasome and release of IL-1β are thought to be important in the progression of hyperuricemia to gout. Both soluble uric acid and MSU in gout act as damage-associated molecular patterns (12). Soluble uric acid level and MSU accumulation depend on mitochondrial reactive oxygen species (ROS) production and the Toll-like receptor (TLR)/myeloid differentiation primary response (MyD)88-nuclear factor kappa B (NF-κB) signaling pathway to activate the NLRP3 inflammasome and pro-IL-1β transcription, respectively. When inflammasomes are activated, pro-IL-1β is cleaved by caspase1, which promotes maturation and pyroptosis to cause the release of biologically active IL-1β (12). In the following section, we focus on the potential link between pyroptosis, which is a potential therapeutic target, and gout.

Neutrophils can participate in gout inflammation and gout stones by inducing the formation of neutrophil extracellular traps (NETs), which release a range of mediators including histones, ROS, and a variety of proteases (117, 118). NETs are extracellular meshworks containing chromatin and granule proteins and were initially considered a means of targeting microorganisms. These features include disassembly of nuclear and granule membranes and hypercitrullination of histones by peptidylarginine deiminase 4, which mediates chromatin decondensation, membrane rupture, and concomitant ROS production by NADPH oxidase (119). ROS trigger the release of neutrophil elastase into the cytoplasm and activate neutrophil elastase enzymatic activity in a myeloperoxidase-dependent manner, contributing to its degradation of F-actin and translocation to the nucleus for continued degradation of core histones, such as H1 and H4, subsequently promoting chromatin depolymerization and NETosis (118, 120–122) ( Figure 3 ).

NETs and NETosis may play both anti-inflammatory and pro-inflammatory roles in gout. Histones released during NETosis may be important in the progression of gout and its related clinical symptoms; however, histones exert cytotoxic and immunostimulatory effects on glomerular cells (123), have cytotoxic effects, and increase lethality in endothelial cells and mouse models of sepsis in vitro (124). By contrast, MSU can trigger NET formation and NETosis. In standard synovial fluid from MSU-induced arthritis, non-refractive fibrous material, cells, and crystals were observed that primarily consisted of fibrin and collagen fibers that formed NETs (125). MSU and low-grade inflammation are often detected in the synovial fluid of joints of patients with asymptomatic gout, possibly in a state of clinical sub-inflammation (126, 127). Although MSU is thought to be an essential driver of inflammation, in practice it may not lead to a robust inflammatory response, which is consistent with the disease profile of clinically asymptomatic patients with gout. The robust inflammatory response also requires stimulation by other factors that lead to intense inflammation, such as the NLRP3 inflammasome (128–131). In this process, NETs may act as a physical barrier by phagocytosing MSU, thus forming a large reticulation of NETs to prevent the spread of harmful substances and influence disease progression (125). Studies have shown that neutrophils that phagocytose MSU form NETs by extruding DNA, packing crystals, and degrading cytokines to suppress excessive inflammatory responses (132). Additionally, NETs limit the inflammatory response by degrading cytokines and chemokines and inhibiting neutrophil recruitment and activation; however, the onset of NET formation leads to inflammation (133, 134).

NETs and NETosis may be linked to necroptosis (135). The critical molecules involved in necroptosis include RIPK1, RIPK3, and MLKL. RIPK1 and RIPK3 form a complex and activate MLKL, which leads to membrane pore formation, membrane leakage, and cell lysis, resulting in the release of cellular components. The detailed molecular mechanisms underlying these effects have been previously reviewed (11, 136, 137). Mulay et al. found that crystals of calcium oxalate, MSU, calcium pyrophosphate dihydrate, and cystine induce cysteinase-independent cell death in renal tubular epithelial cells, which is blocked by the RIPK1 inhibitor necrostatin-1, suggesting that crystal-induced cell death is associated with necroptosis (138). Garcia-Gonzalez et al. evaluated NETs in the synovial fluid of patients during a gout attack. Neutrophils in the synovial fluid release NETs in response to MSU, depending on the number of crystals, but not on cell density, which is accompanied by the activation of MLKL, a key molecule of necroptosis (125). Similarly, MSU-induced NETs were accompanied by RIPK3 expression and MLKL phosphorylation, and the inhibition of RIPK1, RIPK3, and MLKL inhibited NET formation in human and mouse neutrophils (139). The bridge linking the two forms of death may be ROS. Preferential activation of the three critical molecules of necroptosis leads to membrane rupture in neutrophils and ROS production, stimulating NET formation and NETosis (139). Thus, in addition to the association between necroptosis and NETs, pyroptosis of macrophages and NETs in neutrophils are interlinked. The latter may be a compensatory measure to suppress inflammation via NET formation (140). Sil et al. found that macrophage-derived IL-1β enhances the formation of MSU-stimulated NETs. Anakinra reduced NETosis in macrophages. In addition, caspase11-deficient experimental mice with gout and their source macrophages responded to MSU stimulation by significantly reducing the production of cytokines, such as IL-1β, TNFα, and IL-6. Caspase11-deficient neutrophils also failed to produce NETs, the mechanism of which may involve the effect of caspase11 on the regulation of macrophage pyroptosis in gout and NET formation (141). The P2Y6 receptor in endothelial cells in vitro activates LPS-induced transendothelial migration of neutrophils via Rho kinase (142). The P2Y6 receptor antagonist MRS2578 inhibits MSU-induced NETosis via a mechanism that may involve the P2Y6/store-operated calcium entry/IL-8 axis (92). In addition to the aforementioned link between NETosis, necroptosis, and pyroptosis, mutual crosstalk with autophagy may occur. Nuclear factor, interleukin 3 regulated (NFIL3), which is highly expressed in neutrophils of patients with gout, promotes its transcription by binding to the REDD1 promoter, which enhances autophagy and NET formation by inhibiting the mTOR pathway, to promote inflammatory responses (143). Inhibition of autophagy to prevent intracellular chromatin depletion leads to apoptosis, because intracellular chromatin depletion is necessary for NET formation and NETosis (144). Neutrophils from patients with gout form NETs in an autophagy-dependent manner, and inhibition of phagolysosome and PI3K signaling pathways prevents MSU-induced NETs and NETosis (145).

Apoptosis is a controlled form of cell death that primarily involves the exogenous death receptor and endogenous mitochondrial pathways. Its key molecules include multiple death receptors, caspase enzymes, and mitochondrial proteins that mediate apoptosis (11). The morphological features of apoptosis include cytoplasmic concentration, breakage of DNA, and formation of apoptotic vesicles. This process generally does not result in the release of cellular contents. However, many apoptotic cells lead to inefficient clearance, causing an inflammatory response (11). However, few studies have directly examined the relationship between gout and apoptosis ( Figure 4 ). Studies have shown that new macrophages are constantly recruited within the gout tophi and that these macrophages produce TNF-α and matrix metalloproteinase-2/9 to induce degradation of the stroma. However, apoptosis occurs in CD68⁺ macrophages, which limits inflammation (146). MSU stimulation can affect apoptotic processes in various cells and gout through different mechanisms, with most studies focusing on apoptosis in neutrophils and endothelial cells. MSU stimulates the release of ROS and reactive nitrogen species from fibroblast-like synoviocytes, leading to endoplasmic reticulum oxidative stress and possibly mitochondrial dysfunction, thereby promoting fibroblast-like synoviocyte apoptosis (147). The adhesion between neutrophils and endothelial cells is key to the development of acute gout (148). MSU is also gradually deposited on the vessel wall to induce a vascular inflammatory response and damage the patient’s vascular endothelial cells, thereby causing gout complications. During inflammation regression, neutrophils migrating to the inflammatory site first undergo apoptosis and are phagocytosed by phagocytic cells (149). Some apoptosis-related mechanisms in the body improve inflammation in order to cope with the inflammatory response in gout. Promoting the apoptosis of neutrophils and inhibiting the apoptosis of endothelial cells are beneficial for suppressing the inflammatory response to improve gout. CD300a is an inhibitory receptor whose expression increased in neutrophils from the knees of mice after MSU stimulation, which promotes apoptosis of neutrophils by increasing the cleavage of caspase8 of the exogenous death receptor pathway (150). Similarly, annexin A1, a glucocorticoid regulatory protein, reduced neutrophil infiltration and IL-1β release by inducing apoptosis in neutrophils in a MSU-induced gouty arthritis mouse model (151). Although multiple anti-inflammatory mechanisms exist, their actions may be insufficient to inhibit inflammation. Therefore, promoting endogenous anti-inflammatory substances or inhibiting the production of pro-inflammatory molecules—and thus promoting inflammatory cell apoptosis or inhibiting beneficial cell apoptosis through artificial agonists or inhibitors—is a potential future therapeutic direction. For example, inhibition of Rho-associated kinase, a serine/threonine kinase, attenuates neutrophil accumulation, IL-1β levels, and hypernociception in experimental models of gout through mechanisms involving reduced MYPT phosphorylation, decreased Iκβα activity, and increased caspase-dependent apoptosis of neutrophils (152). Similarly, PI3K inhibitors AS605240, GSK045, and CL27c significantly reduced MSU-induced neutrophil infiltration by increasing apoptosis and reducing myeloperoxidase activity, NF-κB activation, and IL-1β levels, thus improving inflammation (93). Simiaowan reduced neutrophil infiltration, inhibited endothelial cell apoptosis in rats with MSU-induced gout, and improved the inflammatory response by attenuating the expression of ICAM-1 (148). AT-01-KG, an artificial agonist of FRP2/ALX, inhibits inflammation by inducing neutrophil apoptosis and cytarabine in a mouse model of gouty arthritis (94). Natural traditional medicines have also been shown to inhibit apoptosis of chondrocytes in gouty arthritis, as summarized in a previous review (153). Anemonoides nemorosa and Tecomella undulata are used in traditional medicines to treat gout. Studies showed that Aqueous extracts of A. nemorosa can induce cell cycle arrest and apoptosis by regulating mitochondrial function (154). Tecomella undulata induces apoptosis in tumor cells by promoting Fas, Fas-associated death domain, caspase3/7/8, and DNA fragmentation (155). In addition, some clinical medications for gout are effective in modulating apoptosis. Uromodulin gene mutations cause premature hyperuricemia, gout, and kidney damage (156). Uromodulin gene mutations affect the processing of uromodulin/tammhorsfall glycoprotein (156). Colchicine promotes the secretion of tammhorsfall glycoprotein from the endoplasmic reticulum, which significantly reduces apoptosis, whereas intracellular endoplasmic reticulum accumulation of tammhorsfall glycoprotein promotes apoptosis (156). Allopurinol reduces apoptosis and increases osteoblast viability in an animal model of hyperuricemia and reduces the risk of vascular calcification by decreasing Wnt3a, Runx2, Sp7, Bglap, Col1a1, SM22a, and Acta2 expression in vascular smooth muscle cells (157).

Cell survival and death are fundamental processes. The molecular mechanisms and morphological characteristics of various types of cell death have been revealed to be closely related to multiple diseases. First, we explored the role of pyroptosis in gout, in which the assembly of NLRP3, a key molecule in pyroptosis, is considered a critical step in the progression from hyperuricemia to gout. In response to MSU stimulation, NLRP3 inflammasomes undergo assembly and release IL-1β, which promotes inflammation. Second, we discussed the crosstalk between NETosis and other forms of cell death and its relationship with gout. NETosis appears to require the assistance of crucial molecules involved in necroptosis for activation. The occurrence of NETs and NETosis in neutrophils appears to be a compensatory mechanism that suppresses inflammation. Finally, we described the roles of these two forms of cell death in gout, autophagy, and apoptosis, which have not been widely evaluated and require further investigation. Studies should focus on the impact of crosstalk between different forms of cell death on gout pathogenesis. In addition, using artificial agonists and inhibitors to target cell death or biological mediators that regulate cell death is a promising therapeutic approach. Whether the combined targeting of multiple pathways and molecules is beneficial for intercellular crosstalk should be examined. Forms of cell death other than those covered in this study may exist and be relevant to gout pathogenesis; however, current research seems to be limited. Thus, unexplored forms of cell death will remain a potential direction of future research. Although translating preclinical results into clinical practice remains challenging, there is great potential to target the precise mechanisms of cell death in gout.

JZ is responsible for the collection, collation and writing of the original manuscript. KW, PJ, CC, LXX, LSX and YS is responsible for the collection of the original manuscript. SG, YX, and DH are responsible for the revision and review of the manuscript. All authors reviewed and accepted with the final version.

This work was funded by the National Natural Science Funds of China (82074234 and 82071756), Shanghai Chinese Medicine Development Office, National Administration of Traditional Chinese Medicine, Regional Chinese Medicine (Specialist) Diagnosis and Treatment Center Construction Project-Rheumatology, State Administration of Traditional Chinese Medicine, National TCM Evidence-Based Medicine Research and Construction Project, Basic TCM Evidence-Based Capacity Development Program, Shanghai Municipal Health Commission, and East China Region-based Chinese and Western Medicine Joint Disease Specialist Alliance.

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

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