Gout is defined by arthritic inflammation in the joints, and the cause of the disease is due to an excess of circulating uric acid, a byproduct of purine degradation. Previous study has found that the key factor for the generation of acute gouty arthritis are the dramatic changes of extracellular ATP, which was followed by activation of P2X7R signaling pathway as well as MSU crystals-induced activation of NLRP3 inflammasome signaling pathway. These mechanisms provided a new avenue for understanding acute gouty arthritis and new methods for treatment. However, it is not everyone with hyperuicemia to develop gout. Some patients have hyperuricemia, but never developed gout. The reason could be related to genetic factors, especially variants of P2RX7 gene.
P2X7R is expressed highly in almost all tissues and organs, especially in the immune cells of monocyte–macrophage origin. The expression and function of the P2X7R are affected by the concentration of extracellular ATP. A study has shown that dilatation of the P2X7 channel to form a “pore” stimulated by extracellular ATP, that is recognized as a unique feature of the P2X7R. However, opening of the ionic channel and formation of the “pore” are two distinct processes. At this moment, short stimulation of P2X7R with extracellular ATP activates K+ efflux. Prolonged or repeated exposure to the P2X7 ligand opens a non-selective cation channel, then the formation of a cytolytic pore permeable to allows NLRP3 agonists to enter the cytoplasm to activate NLRP3 inflammasome [29, 30]. It has found that Ala348to Thr affected bipolar disorder included the gain-of-function haplotypes. The search for all SNPs in the P2RX7 gene was performed within the Chinese population in the HapMap project database and reported in the dbSNP database [National Center for Biotechnology Information (NCBI), Bethesda, MD, USA]. One major identified non-synonymous coding SNPs in P2RX7 were screened out: rs1718119 (Ala348to Thr). Several studies[31, 32] that investigate the functional characterization of P2X7R polymorphism in autoimmunity diseases are limited, and there are only two studies showed that the rs1718119 polymorphism of P2X7R were associated with Systemic lupus erythematosus(SLE) and Rheumatoid arthritis (RA). Thus, the rs1718119 polymorphism of P2X7R were associated with gout is still unknown.
Therefore, the current study first time analyzed the genotyping study in genomic DNA samples from gout and hypeluricemia patients, then analyzed the transfected Ala348to Thr mutant in HEK-293T cells using fow cytometric and in THP-1 cells using ELISA and qRT-PCR. We excluded age and gender and selected hyperuricemia patients over 5-years course of disease with serum uric acid levels > 480 µmol/L (8 mg/dl) having no history of gout flar.The results of the present genotyping study demonstrated that mutation in the P2X7R gene at the rs1718119 loci were related to gout onset. A allele and GG/(AA + AG) genotypes in rs1718119 may become dangerous genes if mutated and activate gout. A hyperuricemia patient who carries these dangerous genes at these loci may be more susceptible to a gout attack. What’s more, rs1718119 was dominant gene. Homozygous and heterozygous hyperuricemia patients with these alleles exhibit a higher risk of gout than patients without these alleles. Patients with hyperuricemia may have a relatively low risk of gout if they carry a single SNP-susceptible genotype, but the risk of gout may be increased if they both carry one or more susceptible locus.
Furthermore, the function of ATP-stimulated P2X7R detected by the amount of ethidium+ bromide. Stokes  has shown a significant association that massive K+ loss from the cell occurs on P2X7 pore dilatation, due largely to the presence of the Ala348to Thr mutation. As shown in our results, our fndings were consisted with previous studies.In our research,the transfected Ala348to Thr mutant in HEK-293T cells increased ATP-induced P2X7-dependent ethidium+ bromide uptake to values of 145% of wild type. Under the agitation of ATP, it suggested that P2X7R was activated in Ala348to Thr mutant and wild-type, and we observed the strength of P2X7R function by detecting the amount of ethidium+ bromide uptake by HEK-293 T cells. We further founded that THP-1 monocytic cells with the P2X7R carrying Ala348to Thr increased ATP-induced secretion of proinflammatory cytokines IL-1β gene. It suggested that the Ala348to Thr polymorphism has the major effect in conferring increased P2X7 receptor function and could up-regulate inflammation via ATP-stimulated P2X7R with high uric acid.Besides,the NLRP3 inflammasomes play an important role in exogenous (pathogen-associated molecular patterns) PAMPs or endogenous (damage associated molecular patterns) DAMPs in this process. Among DAMPs, extracellular ATP and other nucleotides play an undisputed role. P2X7R is activated by extracellular ATP to activates K+ efflux. NLRP3 inflammasome assembly and caspase-1 recruitment occurs spontaneously at K+ concentrations below 90 mM, but is prevented at higher concentrations. Thus, low intracellular K+ may be the least common trigger of NLRP3 inflammasome activation. Activated P2X7R promotes formation of a cytolytic pore permeable to allows NLRP3 agonists to enter the cytoplasm to activate NLRP3 inflammasome. In our research, The mRNA expression level of NLRP3 gene can directly reflect the involvement of NLRP3 inflammatory bodies in the ATP-P2X7R-IL-1β signaling pathway. The expression level of NLRP3 gene in THP-1 cells expressing the Ala348to Thr mutant via ATP-stimulated was increased and was significantly stronger than the wild type and empty virus. It shows that the Ala348to Thr mutant has increased the function of P2X7R by changing the structure of P2X7R,and the mRNA expression of NLRP3 involved the down-stream signaling of inflammasomes with this variant could regulate the development of inflammation via ATP-stimulated P2X7R with high uric acid.
Another point to note was that in our study ASC plays an important role in gout as a adaptor protein. The NLRP3 inflammasome was a molecular platform activated upon extracellular stimulator to trigger inflammation through the maturation of pro-inflammatory cytokines such as IL-1β. This study has showed that the expression of ASC has not changed significantly in wild type compared to the Ala348to Thr mutation.These findings indicate that ASC as an inflammasome adapter protein also formulates the ASC speck, a supramolecular aggregate of ASC dimers, which serves as another platform for the activation of caspase-1 after inflammasome activation[35, 36]. It also implies that since the activation of inflammasomes stimulated by the specific agonists,such as MSU and ATP, results in pyroptosis and thereby the release of most of the ASC specks in the extracellular space. It is conceivable that extracellular ASC specks were ingested by macrophages, which resulted in partial ASC loss on THP-1 cells from wild type and the variant. More worth mentioning is that inflammasome complexes are required for activation of caspase-1 have been identified. Here we have shown that the expression of caspase-1 has unchanged significantly between wild type and the Ala348to Thr mutation, suggesting that caspase-1 may be a protein that usually undergo proteolytic processing or caspase-1 signal pathway may not have important role in development of gout by ATP-stimulated P2X7R mediated biological process.
Gout attacks are the result of the interaction of MSU and ATP, and the SNPs present in the P2RX7 gene may lead to the occurrence of gout. These findings provide a new SNP to improve the pathogenesis of acute gouty arthritis and may explain why some hyperuricemia patients never develop acute gouty arthritis. In our study, we first found this SNP, rs1718119 altering Ala348to Thr, changed the functions of P2X7R in P2RX7 gene with high uric acid. In addition, the genetic variability in P2RX7 gene with this variant was involved in the process of NLRP3 inflammasome activation. It might influence susceptibility for the development of gout. These findings may provide a new therapeutic strategy for the prevention and treatment of gouty arthritis.