The P2X receptors are ATP-gated ion channel receptors, and of these P2X receptors, the P2X₇ receptor, initially defined as the P2Z receptor, possesses a unique sequence and rich function despite being the latest identified (28). Activation of the P2X₇ receptor by ATP opens ion channels causing Ca²⁺ influx and K⁺ outflux to activate the NLRP3 inflammasome in the innate immune system, where Ca²⁺ may represent the second messenger of inflammasome activation (29). Also, the P2X₇ receptor has a unique structure and function, which does not only form ion channels but also forms the pore channels, allowing the passage of up to 900 Da molecules upon continuous activation by high concentrations of ATP. The formation of such pores appears to be required to activate the NLRP3 inflammasome (30, 31). Upon activation, the NLRP3 proteins interact with apoptosis-associated speck-like protein containing a CARD (ASC), which then recruits the pro-caspase-1 forming the NLRP3 inflammasome, which subsequently undergoes cleavage to form active caspase-1. This is further processed, leading to the secretion of mature IL-1β and IL-18 that exert inflammatory effects (32). Following pharmacological inhibition of the P2X₇ receptor, the level of ATP-induced IL-1β release is reduced in inflammatory cells (33). A similar reduction was observed in the levels of the P2X₇ receptor, ASC, or NLRP3-deficient mouse macrophages. These findings suggested that the activation of the ATP-mediated P2X₇ receptor plays a crucial role in promoting IL-1β secretion in inflammatory cells (34). Currently, it is recognized that the P2X₇ receptor, among the family of P2XRs, is the most relevant in the inflammatory response.

Gout is an acute inflammatory disease based on hyperuricemia. Predisposing factors, including alcohol abuse, overeating, cold, and late nights, are required to initiate a gout flare. We evaluated the pathogenic signals embedded in these factors associated with gout flares and found that under these conditions, ATP fluctuations were observed in vivo. This, for example, included mechanical stimulation during strenuous exercise promotes ATP release (35), and the adaptive febrile response of the body during cold increases ATP (36). Dramatic changes in ATP levels can activate the P2X₇R-NLRP3 signaling pathway and promote the IL-1β secretion associated with gout flares. Thus, ATP is considered to be involved in gout flares. A study by Tao et al. observed no difference in the IL-1β concentrations in peripheral blood leukocyte cultures from gout and hyperuricemia patients when stimulated with MSU alone, but upon stimulation with MSU and ATP together, IL-1β concentrations were found to be significantly higher in gout patients than that of the hyperuricemia patients (6). Thus, it is suggested that ATP is the second pathogenic signal for gout besides MSU. Importantly, the ability of the P2X₇ receptor to promote IL-1β secretion upon activation may be a major determinant of gout flares, which is influenced by the gene polymorphisms of the P2X₇ receptor. Studies have confirmed that gout patients possess single nucleotide polymorphism in functionally enhanced P2X₇ receptor compared to hyperuricemia patients (37), thus establishing an essential role of P2X₇ receptor in gout flares.

In hyperuricemic patients, synergistic action between MSU and ATP is required to fully activate the NLRP3 inflammasome, which causes sufficient secretion of IL-1β, leading to the onset of gout flares. Thus, it can be argued that as a receptor for ATP, the P2X₇ receptor is an important regulator in determining gout flares, explaining why most hyperuricemic patients do not develop gouty arthritis throughout their lives.

Besides the P2X₇ receptor, the P2X₄ receptor can also be activated by ATP, causing an inflammatory response, which is functionally similar to the P2X₇ receptor. Upon activation, it forms large-conductance pores in the cell membrane facilitating the ion flow, which subsequently activates the NLRP3 inflammasome. This further enhances the body’s inflammatory response through its synergy with the P2X₇ receptor (38).

P2Y receptors belong to the G protein-coupled receptor family and are further divided into P2Y₁-like receptors (coupled to G_q) and P2Y₁₂-like receptors (coupled to G_i), which exert regulatory effects on inflammation by affecting the activity of PLC and AC, respectively (39). Upon recognizing the corresponding nucleotide, P2Y₁-like receptors coupled to G_q activate the PLC-IP3/DAG-PKC pathway, promoting Ca²⁺ mobilization and inflammatory response. Also, the P2Y₁-like receptors can activate AC; for example, the P2Y₁₁ receptor promotes the activation of the AC-cAMP-PKA pathway to initiate the inflammatory response (40). The P2Y₁₂-like receptors coupled to G_i can exert an inhibitory effect on AC, further reducing the conversion of dephosphorylation of ATP to cAMP, thus, inhibiting the cAMP-PKA pathway-mediated inflammatory response.

The P2Y₁-like receptors, including the P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors, can promote inflammation either through the PLC-IP3/DAG-PKC pathway or through the activation of AC. Among these P2Y₁-like receptors, the P2Y₂ receptor may be closely associated with gout flares. Besides its inflammatory function, the P2Y₂ receptor can activate the NLRP3 inflammasome by stimulating the action of ATP release to further activate the P2X₇ receptor (41).

The P2Y₂ receptors are expressed on various cells, including macrophages, lymphocytes, neutrophils, and eosinophils, and are activated by multiple nucleotides such as ATP and UTP to participate in immune regulation and inflammatory responses. The activated P2Y₂ receptors exhibit upregulated expression of inflammatory cytokines and chemokines, while the release of inflammatory factors is blocked by the treatment used to inhibit the P2Y₂ receptors (42, 43). Furthermore, the activated P2Y₂ receptors open pannexin-1 channels by inducing PLC activation and Ca²⁺ mobilization, which leads to further release of ATP promoting IL-1β maturation and secretion through the P2X₇R-NLRP3 pathway (41). Thus, the binding of the P2Y₂ receptors to ligands promoted the release of inflammatory factors and initiated the inflammatory response in multiple ways. The absence of the P2Y₂ receptor is observed to reverse the increase of the nucleotide-induced IL-1β release (44).

The current study suggested that the P2Y₆ receptor activated by UDP and UTP is involved in the pathogenesis of gout. Uratsuji et al. found that the P2Y₆ receptors were involved in the MSU-induced inflammatory responses. The activation of the P2Y₆ receptors in a variety of cells was observed to contribute to the production of inflammatory cytokines; particularly, the inhibition of the P2Y₆ receptor in THP-1 cells could reduce MSU-induced IL-1β production (45). Additionally, the P2Y₆ receptor affected the MSU-induced neutrophil function associated closely with gout. Sil et al. found that the inhibition of the P2Y₆ receptors reduced neutrophil migration and IL-8 production, which is responsible for recruiting neutrophils to the joint cavity during a gout flare, thereby amplifying the effects of gout (46). In addition, treatment with the P2Y₆ receptor antagonist MRS2578 inhibited the formation of MSU-induced neutrophil extracellular traps (NETs) in gout (46). Therefore, activation of the P2Y₆ receptor may contribute to the persistence of gout flares.

The P2Y₁₂-like receptors coupled to G_i, including P2Y₁₂, P2Y_13, and P2Y₁₄ receptors, inhibit the cAMP-PKA pathway-mediated inflammatory response and theoretically inhibit gout pathogenesis. However, due to the complexity of the mechanisms of action in different internal environments, the regulation of inflammation by P2Y₁₂-like receptors is also diverse. For example, cAMP has long been considered an inducer of inflammation. Activation of the cAMP-PKA-ERK signaling pathway promotes IL-1β secretion (47). Additionally, the AC/cAMP/PKA upregulates IL-1β-induced IL-6 production by enhancing the JAK2/STAT3 pathway (48). However, cAMP has also been recently pointed out as an important factor in regulating inflammation relief (49). The cAMP inhibits NF-κB transcription by activating downstream PKA to reduce the production of pro-inflammatory factors. Also, it promotes the phosphorylation of CENP to increase the production of anti-inflammatory factors and stimulate macrophage polarization. Moreover, cAMP has been found to inhibit the assembly of the NLRP3 inflammasome by directly binding to NLRP3 proteins, and promotes their ubiquitination and degradation (50, 51), thus acting as a negative regulator of the NLRP3 inflammasome reducing the secretion of IL-1β. Therefore, the P2Y₁₂-like receptors can not only exert inhibitory inflammatory effects but can also often exhibit pro-inflammatory effects through the regulation of cAMP. In microglia, extracellular ADP was observed to act on P2Y₁₂ receptors that activated NF-κB and NLRP3 inflammasomes while the inhibition of the P2Y₁₂ receptors reduced the IL-1β levels (52). Similarly, the P2Y₁₄ receptors promoted the secretion of inflammatory factors IL-1α, IL-8, and IL-6 in response to MSU stimulation (53).

Complex regulatory effects of inflammation are manifested in the P2Y₁₄ receptor. In a gout study, Li et al. found that the P2Y₁₄ receptors on macrophages negatively regulated cAMP, which enhanced the MSU-induced activation of the NLRP3 signaling pathway. The knockdown of the P2Y₁₄ receptor then limited the activation of the NLRP3 inflammasome, which in turn attenuated the MSU-induced inflammatory infiltration of synovial tissue (54). Thus, in acute gouty arthritis, the activation of the P2Y₁₄ receptor can exert a pro-inflammatory effect through the cAMP/NLRP3 signaling pathway.

The P2Y receptors in gout often play conflicting roles both in promoting remission of inflammation and maintaining the persistence of inflammation. As mentioned earlier, the P2Y₁₂-like receptor signaling pathway that inhibits inflammation can have pro-inflammatory effects through activation of NLRP3 inflammasome. Similarly, the P2Y₁-like receptor signaling pathway that promotes inflammation may exhibit an inhibitory effect on inflammation. For example, in a gout mouse model, the activation of the P2Y₆ receptors reduces 1-palmitoyl-2-linoleyl-3-acetyl-rac-glycerol-induced neutrophil infiltration, alleviating joint symptoms (55). Thus, the P2Y receptors play a complex role in inflammatory diseases, either as a friend or foe. Besides modulating the pro-inflammatory factors in the regulation of inflammatory responses, P2Y receptors can also influence the expression of anti-inflammatory cytokines. TGF-β and IL-10 are the major cytokines responsible for gout resolution (56). The P2Y₁ receptors were found to enhance the effects of TGF-β1 and IL-10 (57, 58), while the P2Y₁₁ receptor, although reported to inhibit TGF-β1 production through activation of cAMP, was found to upregulate the IL-10 expression (57, 58). Overall, the data suggested that the P2Y receptor signaling pathway was involved in the pathogenesis of gout in different ways.

The P1 receptors belong to the family of G protein-coupled receptors and include A₁, A_2A, A_2B, and A₃ receptors. These are widely expressed on immune cells and exhibit anti-inflammatory effects. The ligand of the P1 receptor, adenosine, is the main product of sequential hydrolysis of extracellular ATP, which is catalyzed by CD39 and CD73. Under physiological conditions, the adenosine concentration is maintained at low levels, but under certain conditions such as inflammation, hypoxia, and cellular injury, the concentration of adenosine is increased by degrading extracellular ATP and ADP. Adenosine is then rapidly metabolized to AMP and inosine by the action of adenosine kinase or adenosine deaminase. The contribution of adenosine in alleviating inflammation and maintaining homeostasis in the organism has been recognized widely (10).

The P1 receptor signaling exerts anti-inflammatory effects through a variety of pathways. The adenosine-activated P1 receptors (A₁, A₃) increase the inhibition of neutrophil adhesion, reducing the production of superoxide and secretion of inflammatory cytokines (59–61). Furthermore, the interaction of adenosine with A₁, A_2A, and A₃ receptors inhibits the release of IL-1β from activated human peripheral blood mononuclear lymphocytes (62). Also, the interaction of adenosine with A₃ receptors was found to reduce TNF-α production in the macrophage cell lines (63), while the interaction of adenosine with A₁ receptors was found to reduce parasite-stimulated production of reactive oxygen species (ROS) and IL-8 from neutrophils (64). Besides its inhibitory effect on the secretion of inflammatory factors, the activation of the P1 receptor signaling pathway also promotes the secretion of anti-inflammatory cytokines, including TGF-β1 and IL-10, by the immune cells (57, 60).

The targeting of adenosine receptors has been efficient in the treatment of several inflammatory diseases. The activation of adenosine-mediated A_2A receptor contributed to block the activity of NLRP3 inflammasome, thereby reducing the production of pro-inflammatory cytokines while increasing the production of anti-inflammatory cytokines (65, 66). In post-traumatic brain injury events, agonizing the A₃ adenosine receptor could improve the neurocognitive function by inhibiting the activation of the NLRP3 inflammasome (67). Similarly, in arthritic mice, activating the A₃ adenosine receptors could reduce the production of TNF-α and alleviate arthritic symptoms (68). Thus, increased adenosine levels following an inflammatory episode could activate the P1 receptors, exerting anti-inflammatory effects through modulation of the NLRP3 inflammasome, thus, contributing to the resolution of gout flares.

Currently, only limited evidence is available to directly demonstrate the involvement of P1 receptors in suppressing gout inflammation or promoting gout resolution. However, based on the anti-inflammatory effect of the P1 receptor signaling pathway, we can see an inevitable impact on the pathogenesis of gout through changes in adenosine concentrations during the different stages of gout flares.

MSU alone is not sufficient for initiating a gout flare; the synergistic effect of high levels of extracellular ATP is also required. After the initiation of gout, an increased expression of nucleoside triphosphate dihydrolase 1 (CD39) and 5’-nucleotidase (CD73) can be seen in an inflammatory environment. CD39 dephosphorylates extracellular ATP to ADP and adenosine monophosphate (AMP), which is further converted to adenosine by CD73 (69, 70). Thus, the changes in the expression of CD39 and CD73 during a gout flare can determine the level of nucleotides and the state of inflammation that affects the course of gout disease.

The CD39 and CD73 are extracellular nucleotidases expressed on monocytes, macrophages, B cells, T cells, natural killer cells, dendritic cells, and neutrophils. They mainly maintain a balance between the extracellular ATP and the concentration of adenosine. This homeostasis is important for the persistence and extent of the inflammatory state (71). Changes in the levels of nucleotide enzymes can occur during gout flares. Inflammatory cytokines, oxidative stress, and a hypoxic environment may increase the activity of CD39 and CD73 (72), thereby reducing ATP levels and increasing adenosine concentration. Further, tissue hypoxia can also reduce the expression of nucleoside transporters, increasing the adenosine levels. CD39 is the main enzyme that metabolizes ATP. In vitro studies in macrophages have found that activated P2X₇ receptor could upregulate the expression of cytosolic CD39 by triggering a lipid raft-dependent mechanism (73). In contrast, the elevated CD39 could limit the P2X₇R-mediated pro-inflammatory response. The knockdown of CD39 results in excessive IL-1β release (74, 75). This implies that in addition to catabolizing ATP and promoting adenosine production, the interaction between CD39 and the P2X₇ receptor can modulate the upregulated inflammatory state of the organism in time to restore cellular homeostasis.

Macrophages are the primary sites of signaling events that regulate the onset and remission of gout. During the disease progression, macrophages show a shift from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Zanin et al. found (76) that M1 macrophages showed a decrease in the expression of CD39 and CD73 along with a reduction in ATP and AMP hydrolysis, whereas the M2 macrophages displayed higher CD39 and CD73 expression, as well as increased ATP and AMP hydrolysis. Also, the addition of adenosine led to an increase in the expression of the M2 macrophage gene, which in turn promoted the conversion of M1 phenotype to M2 phenotype (77). Moreover, after the initiation of inflammation, adenosine was found to decrease the production of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines by macrophages (78). These data suggested that purinergic signaling may be involved in gout resolution by regulating the conversion of macrophages from the M1 to M2 phenotype.

Besides changes in the purine metabolites, the expression of purinergic receptors seems to be altered in response to the internal environment during the inflammatory process of gout flares. Among them, the changes in the expression of the P2X₇ receptor have the most important impact. In addition to directly activating the NLRP3 inflammasome, MSU was found to stimulate the expression of P2X₇ receptors and P2X₄ receptors, secreting IL-1β and causing gout flares (11). Besides, an interesting experiment found that ethanol can upregulate the expression of P2X₇ receptors to induce NLRP3 inflammasome activation (79), which partly explains why alcohol consumption predisposes to gout flares.

The expression of the P2Y receptor is also altered during a gout flare. The MSU stimulation of human keratinocytes resulted in an increased expression of P2Y₆ receptors and P2Y₁₄ receptors (45, 53). The P2Y₆ receptor belongs to the P2Y₁-like receptor family, which promotes the inflammatory responses and the upregulation by MSU facilitates the initiation and persistence of gout. In contrast, the P2Y₁₄ receptor belonging to the P2Y₁₂-like receptors inhibits cAMP and exhibits an inflammatory suppressive effect. The upregulation of P2Y₁₄ by MSU appears to be detrimental to the development of gout. However, its upregulation can also promote gout pathogenesis through the negative effect of cAMP on the NLRP3 inflammasome (54).

The transition of macrophages from the phenotypes M1 to M2 promotes remission of gout inflammation. Upon macrophage activation, an increase was observed in the expression of P1 receptors (A₁ and A₃), which was accompanied by a decrease in the production of pro-inflammatory cytokines IL-6 and TNF-α and an increase in the production of anti-inflammatory factor IL-10. This promoted macrophage polarization and contributed to the suppression of inflammation (80, 81).

The previous section showed in vivo conversions of purine metabolites at different stages of a gout flare. Early in the flare, large amounts of ATP were released extracellularly by the necrotic, apoptotic, and inflammatory cells through pannexins or connexin channels (82). First, ATP was bound to the P2X₇ receptors in the cytosol. Then, in concert with MSU, it stimulated the activation of the NLRP3 inflammasome, thus, prompting IL-1β secretion. Next, the expressions of CD39 and CD73 were increased in the inflammatory hypoxic environment following gout initiation, which promoted the dephosphorylation of ATP to adenosine. This inhibited the inflammatory response and promotes the self-resolution of gout flares by activating the P1 receptor signaling pathway. During the process, changes in the expression of purinergic receptor induced by the internal environment contributed to the ordered changes in purine signaling that regulated both gout flare and resolution ( Figure 1 ). The main reasons for this seemingly contradictory mechanism are the ordered changes in the expression of purine metabolites and their receptors in the inflammatory environment, as well as the different purine signaling pathways they mediate in response to inflammation. Also, the accumulation of acidic metabolites in the inflammatory environment inhibits the cAMP/PKA signaling pathway reducing the IL-1β production (83), which indirectly counteracts the purinergic signaling-mediated cAMP/PKA pathway activity, favoring gout resolution.

Gout is a self-limiting disease with numerous causes contributing to its self-resolution. It is widely recognized that after initiating the inflammatory response in gout, neutrophils phagocytose MSU to form NETs. The aggregated NETs act by reducing the expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-8), which promote the production of anti-inflammatory cytokines (TGF-β1, IL-1Ra, IL-10, IL-37), attenuating the inflammatory response (1, 84). Additionally, CD14 plays a role in the self-relief of gout. The knockdown of CD14 can reduce the activation of MSU-induced NLRP3 and release IL-1β (85). Studies have observed that a reduction of CD14 expression in gout patients may contribute to gout resolution (86). Besides neutrophils and CD14, purinergic signaling pathways play a synergistic role in gout resolution through the regulation of inflammatory responses.