Gout and asymptomatic hyperuricemia (AHU) are impacting individuals worldwide, with a growing prevalence among younger people (1, 2). Acute gout arthritis (AGA) is an inflammatory disorder characterized by hyperuricemia and the deposition of monosodium urate (MSU) crystals in joints and tissues (3). Patients with AGA have episodes of severe inflammation, swelling, and pain. Without appropriate treatment, chronic gout can lead to cardiovascular disease and renal impairment (1, 3). In hyperuricemia, serum uric acid levels exceed the saturation threshold. The condition is known as asymptomatic hyperuricemia when it occurs in the absence of inflammation caused by monosodium urate crystals (4, 5). Asymptomatic hyperuricemia, traditionally considered a metabolic abnormality linked to gout and kidney stones, is now recognized as a significant risk factor for hypertension, adiposity, diabetes mellitus, hepatic steatosis, chronic nephropathy, and cardiometabolic diseases (5, 6). As safe and effective medications for AGA treatment are lacking, and the necessity of urate-lowering therapy for individuals with asymptomatic hyperuricemia is debatable, there is an urgent need to investigate the etiology of AGA and silent hyperuricemia.

Pyroptosis is a type of inflammatory programmed cell death, first proposed by Brennan and Cookson in 2001 (7). Unlike necrosis and apoptosis, it induces cellular membrane disruption and the secretion of pro-inflammatory cytokines, which amplify the inflammatory response (8). In individuals suffering from gout, the body activates innate immune cells by recognizing MSU through endogenous damage associated molecular patterns (DAMPs), which participate in pyroptosis (9). Pyroptosis is known to occur through three main pathways: the canonical pathway, the non-canonical pathway, and the Caspase-3 mediated pathway (10). The canonical pathway is mediated by inflammasomes and Caspase-1. Upon stimulation by PAMPs or DAMPs, the inflammasomes activate Caspase-1, promoting the maturation and release of pro-inflammatory cytokines like interleukin-1β (IL-1β) and IL-18, as well as the activation of gasdermin D (GSDMD). The activated effector molecule GSDMD forms non-selective pores on the cell membrane, leading to the loss of its integrity (11). Various inflammatory factors are released through these pores, while extracellular substances enter the cell, accelerating cell swelling and organelle damage, ultimately resulting in cell rupture.

Inflammasomes, composed of pattern recognition receptors (PRRs), apoptosis-associated speck-like protein containing a CARD (ASC), and inactive pro-caspase-1, are multiprotein complexes found in the cytoplasm (12). They can only function after they are activated. PRRs serve as the initial signal to induce transcription and synthesis of inactive pro-IL-1β and pro-IL-18. Inflammasomes then act as the second signal to convert pro-caspase-1 to active Caspase-1 (13). The PRRs of inflammasomes mainly include members of the nucleotide-binding oligomerization domain-like receptor (NLR) family and the absent in melanoma 2 (AIM2)-like receptor (ALR) family (14).

Absent in melanoma 2 (AIM2) constitutes a component of the AIM-like receptor (ALR) family, which contains an N-terminal pyrin domain (PYD) and a C-terminal domain that belongs to a group known as hematopoietic, interferon-inducible, nuclear proteins with a 200-amino-acid repeat (HIN-200). This receptor helps recognize cytosolic DNA via its HIN-200 region, which stimulates the adaptor protein ASC to facilitate the oligomerization and subsequent proteolytic maturation of pro-caspase-1 (15, 16). AIM2 is an intracellular DNA sensor that identifies genomic material originating from several sources, such as pathogenic bacteria, viral entities, genotoxic stress from ionizing radiation, and endogenously derived DNA disseminated via exosomal mechanisms (17–21). Researchers have found through experiments that the oligomeric assembly kinetics of the AIM2 inflammasome depends on the chain length of double-stranded DNA (dsDNA). The oligomerization is optimally induced by dsDNA that is 80–200 base pairs in length (22, 23). In the context of autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren’s syndrome (SS), dysregulated hyperactivation of the AIM2 inflammasome pathway may occur due to the continuous accumulation of self-derived DNA substrates (24–26). However, only a few studies have investigated the role of the AIM2 inflammasome in AGA. Therefore, in this study, we evaluated the expression of AIM2, Caspase 1, and GSDMD in PBMCs of patients diagnosed with AGA and AHU, estimated the levels of the inflammatory cytokines IL-1β and IL-18 in serum, hope to provide new insights into the involvement of the AIM2 mediated pyroptosis pathway in the development of AGA and strategies for treating gout.

Double-stranded DNA is the sole ligand of the AIM2 inflammasome. To assess the feasibility of this study, we first measured the plasma levels of double-stranded DNA in the healthy control (HC), AHU, and AGA groups. Plasma DNA is non-cellular DNA circulating in the blood, which can exist as DNA-protein complexes or as free DNA fragments. Due to the release of DNA from a small number of necrotic or apoptotic cells during metabolism, the generation and clearance of DNA are in dynamic equilibrium, resulting in a relatively stable low concentration of free DNA in the blood of healthy individuals. Our findings underscore the impact of obesity on chronic low-grade inflammation (see Table 1 , Figure 4 ), which may precipitate DNA damage and impair DNA repair mechanisms, as suggested by previous studies (28, 29). The accumulation of DNA damage, particularly in response to high-fat diets (30, 31), is further supported by the presence of γH2AX foci, an early indicator of DNA double-strand breaks (32). Additionally, the correlation between overweight status in adolescents and lymphocytic DNA damage (33) and the association of obesity with various DNA lesions, including double-strand breaks and oxidative base damage, which are reflective of BMI and DNA damage indices (29), align with our observations. In the AHU group, significantly elevated dsDNA levels were observed compared to the HC group (see Figure 1 ), which is consistent with the metabolic syndrome phenotype characterized by obesity, dyslipidemia, and mild inflammation (see Table 1 , Figure 4 ). Moreover, the AGA group exhibited even higher dsDNA levels (see Figure 1 ), suggesting a more pronounced inflammatory state. Despite no significant differences in metabolic factors between the AGA and AHU groups, the AGA group displayed significantly higher levels of inflammatory markers, such as platelets, ESR, and CRP (see Table 1 ). The moderate correlation between dsDNA levels and these inflammatory markers indicates a potential role for acute inflammation in the elevated dsDNA levels observed in AGA patients (refer to Table 2 ). AGA is recognized as a disease involving a complex interplay within the “metabolic-immune-inflammatory” network, where various inflammatory factors contribute to disease onset and progression (34). The aggregation of neutrophils at crystal deposition sites in AGA and their subsequent release of inflammatory mediators, such as IL-1β and IL-18, initiate a local acute inflammatory response. The activation of IL-1β further stimulates the secretion of neutrophil extracellular traps (NETs), composed of DNA, histones, and granules, which, when in excess or not promptly cleared, can induce the release of damage-associated molecular patterns (DAMPs) and trigger inflammatory responses (35). The detection of dsDNA in the synovial fluid of gout patients (36) and the association of acute gout attacks with increased oxidative stress and mitochondrial dysfunction, leading to the release of mitochondrial DNA into the circulation, further support the role of dsDNA in inflammatory processes. Additionally, the recent discovery that pyroptosis can lead to the loss of membrane integrity and leakage of intracellular contents, potentially contributing to increased dsDNA levels, highlights the complexity of dsDNA dynamics in disease states. In conclusion, the clearance of plasma dsDNA fragments emerges as a promising therapeutic target for AGA.

AIM2 is the sole inflammasome capable of detecting double-stranded DNA. Recent studies using gene-silenced cells and gene-knockout animal models have shown that AIM2 recruits ASC to form an inflammatory complex upon sensing dsDNA fragments of 80-200bp, which in turn activates Caspase-1 and facilitates the maturation and release of inflammatory cytokines such as IL-1β and IL-18 (9, 37). Our understanding of the AIM2 inflammasome’s role in pathogenesis has been furthered by observations of its upregulated expression, alongside that of ASC and Caspase-1, in PBMCs and neutrophils of rheumatoid arthritis patients. This increase correlates with the severity of the disease (25). Additionally, impaired DNase1 activity in the ductal tissues of Sjögren’s syndrome patients and non-tumor salivary gland epithelial cell lines has been implicated in the insufficient degradation of cytoplasmic DNA, leading to the persistent activation of AIM2 (26). Notably, diminished AIM2 expression in a murine lupus model corresponded to improvement in lupus symptoms (38). Discrepancies may exist, however, between the immune mechanisms of AIM2 inflammasome observed under artificial conditions and those in human physiology. Prior studies have reported elevated dsDNA levels in the peripheral blood of systemic lupus erythematosus (SLE) patients, alongside heightened AIM2 mRNA expression in PBMCs. These findings were associated with kidney involvement and disease activity (24). Su X and colleagues extended this research by examining AIM2 inflammasome expression in Chinese patients with acute and chronic brucellosis (39). Their findings indicate a disease-stage-specific modulation of AIM2 inflammasome components. Acute brucellosis patients exhibit higher AIM2 and lower ASC expression, while chronic brucellosis patients show the opposite pattern. Furthermore, acute brucellosis was associated with significantly higher serum IL-18 levels (39). In our study, we document a pronounced upregulation of AIM2 mRNA and protein in PBMCs from individuals with AGA and AHU, in contrast to healthy controls (as shown in Figures 2 , 3 ). The AHU group displayed a statistically significant higher concentration of AIM2 mRNA compared to the AGA group (indicated in Figure 2 ). Conversely, the AGA group presented with markedly elevated AIM2 protein levels compared to the AHU group, as shown in Figure 3 . These findings suggest a nuanced role of AIM2 in the pathogenesis of gout and asymptomic hyperuricemia. NLRP3 is a widely recognized inflammasome and identified as playing a crucial role in the innate immune response associated with gout. Both NLRP3 and AIM2 mediated the canonical pyroptotic pathway. The regulation of NLRP3 and AIM2, as well as the release of pro-IL-1β are associated with NF-κB activation through various receptors, culminating in the transcription of pro-inflammatory cytokine genes and the attraction of inflammatory cells to the joint. Mild-to-moderate pyroptosis can be beneficial, but severe pyroptosis can harm the organism by increasing cell death (40). Our findings suggest that the AIM2 inflammasome responds differently in different states, and the influence of AIM2 on patients with AGA and AHU may be biphasic regulation to protect the host. It deserves more attention to investigate further to clarify the impact of AIM2 inflammasome on AHU and AGA. The interplay of NLRP3 and AIM2 in cellular inflammatory dialogues is an area that requires further investigation.

Caspase-1 as one of the members of the caspase family, functions as the principal effector in the canonical pyroptotic pathway. The canonical pyroptotic pathway, also known as the caspase 1-dependent pathway, involves the activation of inflammasomes such as NLRP3, NLRC4, AIM2, Pyrin, etc. In the molecular pathways of MSU-triggered inflammation, the NLRP3 inflammasome plays a crucial role in the innate immune response associated with gout. The NLRP3 inflammasome depends on a two-signal initiation system, which avoids unregulated activation that would damage the host. The first signal stimulates NF-κB via TLR4 and TLR2, leading to the production of pro-IL-1β, pro-caspase-1, and inflammasome components (41). In the cytoplasm, pro-caspase-1 exists as an inactive zymogen. Monosodium urate crystals act as the second activation signal, causing the assembly of inflammasome and activation of Caspase-1 (42). After activation, pro-caspase-1 is cleaved and separated into its subunits, p20 and p10. These subunits combine to form a functional p20/p10 heterodimeric enzyme, which functions as the activated form of Caspase-1, which proteolyzes pro-IL-1β to mature IL-1β (43). IL-1β then interacts with the IL-1β receptor to trigger a downstream signaling cascade involving proinflammatory cytokines and chemokines, resulting in the recruitment of neutrophils and other cells to the site of crystal deposition. Several regulatory mechanisms are involved in NLRP3 inflammasome activation by MSU crystals, such as NETosis, mitochondrial ROS generation, ATP, K+ efflux, etc (41). Many researches besides our previous investigation revealed a significant upregulation of NLRP3 and Caspase-1 expression in the PBMCs of individuals with AGA and the synovial tissues of rodent AGA analogs (44). AIM2 inflammasome is the first non-NLR family member and belongs to the Caspase-1-dependent canonical pyroptotic pathway. In previous studies, elevated expression of AIM2 and Caspase-1 has been observed in rheumatoid arthritis (25), systemic lupus erythematosus (45), Sjogren’s syndrome (26), and psoriasis (46). AIM2 mRNA or protein expression is associated with Caspase-1 mRNA or protein expression and is related to disease severity. In this study, we observed an increase in the levels of Caspase-1 mRNA and protein expression not only in the PBMCs of individuals with AGA but also in those with AHU (see Figures 2 , 3 ). Moreover, the expression levels of the Caspase-1 protein positively correlated with the AIM2 protein (as shown in Figure 5 ). These findings suggest a synergistic effect between Caspase-1 expression and AIM2 inflammasome activation in the pathogenesis of AGA. Both NLRP3 and AIM2, as components of the canonical pyroptosis pathways, can activate Caspase-1. What relationship do they have in acute gout attacks and asymptomatic hyperuricemia? Further investigation is warranted.

GSDMD and GSDME are members of the Gasdermin superfamily and are closely associated with the process of pyroptosis (47, 48). GSDMD acts as a substrate of Caspase-1 and participates in inflammatory responses (49). Cleaved by Caspase-1, GSDMD converted into C-terminal and N-terminal domains during pyroptosis. The N-terminal domain can initiate pyroptosis, whereas the C-terminal domain inhibits pyroptosis by binding to the N-terminal domain. Caspase-1 can release GSDMD-N from the inhibitory state of GSDMD-C, which leads to the formation of pores on the cell membrane that are 10–20 nm in diameter. Intracellular substances flow out, and water flows in through these pores, leading to an increase in the osmotic pressure, which causes cell membrane rupture and cell pyroptosis (37, 50). Studies have shown that GSDMD-deficient cells cannot undergo pyroptosis upon stimulation, resulting in a significantly reduced mortality rate post-stimulation, indicating that GSDMD plays a crucial role in pyroptosis (51). Previous studies have also documented similar findings, albeit with the involvement of the NLRP3 inflammasome (52, 53). Accompanied by the inhibition of pyroptosis and reduced IL-1β release, the expression of NLRP3 inflammasome was suppressed both in vivo and in vitro through knockdown of GSDMD (54). In mice with AGA induced by MSU, GSDMD inhibition via siRNA markedly reduced ankle swelling and inflammation, verified by histopathological analysis (54). In this study, the mRNA and protein levels of the GSDMD increased significantly in the AGA group compared with the HC group (see Figures 2 , 3 ). GSDMD mRNA and protein levels increased in the AHU group compared to the HC group, but only the mRNA level exhibited significance (see Figures 2 , 3 ). GSDMD protein slightly increased without statistically significant, aligning with the AHU group’s characteristics. There were significantly positive correlations between the protein levels of GSDMD and AIM2 in both AGA and AHU groups. These findings suggested that GSDMD may be involved in AIM2 inflammasome-mediated pyroptosis in patients with AGA and AHU.

The cytokines IL-1β and IL-18 are common pro-inflammatory factors in the IL-1 family. IL-1β is primarily secreted by activated macrophages, which trigger downstream pro-inflammatory cytokines and chemokines by binding to IL-1β receptors. This interaction stimulates the accumulation of neutrophils and other cells at the site of crystal deposition, thus initiating and exacerbating local inflammatory reactions. IL-18 is found in monocytes in the blood of healthy individuals and epithelial cells throughout the gastrointestinal tract. Its secretion is mainly related to Caspase-1, thereby playing a critical role in inflammation and immune regulation (55). Some studies have found that the concentrations of uric acid in the serum of AGA patients and the synovium of model mice positively correlated with the levels of IL-1β and IL-18 (56, 57). Moreover, IL-1β blockers can significantly improve the symptoms of acute gouty arthritis (56, 57). In this study, we found that the levels of IL-1β and IL-18 increased significantly in the sera and PBMCs of patients with AGA, and slightly increased in the patients with AHU compared with healthy volunteers (see Figures 2 – 4 ). The expression of AIM2 protein correlated with IL-1β protein in the AGA group, on the contrary, unrelated to IL-1β protein in the AHU group (as depicted in Figure 5 ). AIM2 protein exhibited a positive correlation with IL-18 in the AHU group and no correlation was observed in the AGA groups (as illustrated in Figure 5 ). This distinction is congruent with the distinct immunopathological characteristics of asymptomatic hyperuricemia and a tendency for acute gouty arthritis spontaneous remission.

To summarize, AGA is a complex disorder characterized by metabolic, immunologic, and inflammatory dysregulation. The AIM2-mediated canonical pathway plays a significant role in the onset and development of AGA and AHU. The modulation of AIM2 inflammasome expression might be a promising target for AGA therapy. Our research has certain limitations, such as a limited sample size, a single-site design, potential selection bias, and neglecting the impact of NLRP3. Therefore, further investigations involving cultivated gene-silenced cells and gene knockout animal models are warranted to elucidate the precise contribution of the AIM2 inflammasome-mediated pyroptosis pathway in AGA and asymptomatic hyperuricemia.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Medical Ethics Committee of the General Hospital of Northern Theater Command. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

JC: Formal analysis, Investigation, Methodology, Software, Writing – original draft. JT: Funding acquisition, Supervision, Writing – review & editing. PL: Conceptualization, Formal analysis, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. DF: Data curation, Investigation, Software, Visualization, Writing – original draft. LG: Data curation, Resources, Validation, Visualization, Writing – review & editing. RS: Data curation, Investigation, Validation, Writing – review & editing.