P2X7R Mediates the Synergistic Effect of ATP and MSU Crystals to Induce Acute Gouty Arthritis

Abstract

Activation of the nod-like receptor protein 3 (NLRP3) inflammasome by monosodium urate (MSU) crystals has been identified as the molecular basis for the acute inflammatory response in gouty arthritis. However, MSU crystals alone are not sufficient to induce acute gouty arthritis (AGA). Adenosine triphosphate (ATP) is an endogenous signaling molecule involved in the NLRP3 inflammasome activation. We aimed to explore the role of ATP in MSU crystal-induced AGA development. In peripheral blood mononuclear cell-derived macrophages obtained from gout patients, we observed a synergistic effect of ATP on MSU crystal-induced IL-1β release. Furthermore, in a rat model of spontaneous gout, we demonstrated that a synergistic effect of ATP and MSU crystals, but not MSU crystals alone, is essential for triggering AGA. Mechanistically, this synergistic effect is achieved through the purinergic receptor P2X7 (P2X7R). Blockade of P2X7R prevented AGA induction in rats after local injection of MSU crystals, and carrying the mutant hP2X7R gene contributed to the inhibition of NLRP3 inflammasome activation induced by costimulation of MSU crystals and ATP in vitro. Taken together, these results support the synergistic effect of ATP on MSU crystal-induced NLRP3 inflammasome activation facilitating inflammatory episodes in AGA. In this process, P2X7R plays a key regulatory role, suggesting targeting P2X7R to be an attractive therapeutic strategy for the treatment of AGA.

Figure 1

Synergistic effect of ATP and MSU crystals on the release of IL-1β. ELISA of IL-1β in the supernatants of the culture of PBMC-derived macrophages obtained from gout patients. (a) LPS (50 ng/mL)-primed cells stimulated with MSU crystal suspension (100 μg/mL), ATP (1 mM), and nigericin (10 μM). (b) LPS-primed cells were stimulated with MSU, ATP, MSU+ATP, nigericin, or MSU+nigericin. (c) Comparison of IL-1β levels in different groups in (b). Statistics were analyzed using the SNK-q test (a), Dunnett's t-test (b), and independent sample t-test (c). Data are presented as mean ± SEM. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 2

Manifestation of spontaneous arthritis in rats. Representative images of rat joints after the injection of sterile saline, MSU, ATP, and MSU+ATP in the tail vein of hyperuricemic SD rats (12 h). Spontaneous arthritis manifested as redness and swelling in the toe joints of the rats.

Figure 3

Characteristics of spontaneous arthritis in rats. (a) Manifestation of spontaneous arthritis in rats over time. Rats showed redness and swelling of the fourth metatarsophalangeal joint 4 h after tail vein injection of MSU+ATP; joint swelling increased 8 h after the injection; joint symptoms further increased 12 h after the injection, along with an increase in the skin temperature. (b) Representative confocal microscopy images of joint sections of rats with spontaneous arthritis, showing infiltration of neutrophils (HIS48, green) and T cells (CD3, red).

Figure 4

ATP and MSU crystals induced NLRP3 inflammasome activation in rats. (a) ELISA of serum IL-1β levels in rats. (b–d) qPCR of IL-1β, caspase-1, and NLRP3 mRNA levels in peripheral whole blood cells of rats. (e) WB of IL-1β, caspase-1, and NLRP3 protein levels in the joints and surrounding tissues of rats. Dunnett's t-test was used for statistical analysis. Data are expressed as mean ± SEM. n = 3-8. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 5

Inhibition of P2X7R alleviates MSU crystal-induced AGA in rats. Coderre gout models were divided into the control group, MSU group, and MSU+BBG group. (a) Representative images of the right ankle joint of each group of rats after treatment (12 h). (b, c) Right ankle joint circumference and joint swelling index of rats (12 h). (d) ELISA of serum IL-1β levels in rats. (e–g) qPCR of IL-1β, caspase-1, and NLRP3 mRNA levels in the peripheral whole blood cells of rats. Dunnett's t-test was used for statistical analysis. Data are expressed as mean ± SEM. n = 4. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 6

The E496A mutant inhibits ATP-induced P2X7R pore formation. HEK-293T cells were transfected with lentiviruses overexpressing WT and E496A mutant hP2X7R genes as well as empty lentiviruses. (a) Flow cytometry results of P2X7R-dependent EB uptake induced by 1 mM ATP (indicated by arrow) in HEK-293T cells overexpressing different lentiviruses. (b) Mean values of EB uptake in the three groups. Dunnett's t-test was used for statistical analyses. Data are expressed as mean ± SEM. ^∗∗∗P < 0.001.

Figure 7

The effect of NLRP3 inflammasome activation in THP-1 cells overexpressing the hP2X7R gene of the E496A mutant. Lentiviruses overexpressing the WT and E496A hP2X7R genes were transfected into THP-1 cells receiving stimuli of LPS (control group), MSU (MSU group), or MSU+ATP (MSU+ATP group). (a) ELISA of IL-1β levels in THP-1 cells after different stimulations. (b, c) qPCR of IL-1β and NLRP3 mRNA levels in THP-1 cells after different stimulations. Independent sample t-test was used for statistical analysis. Data are expressed as mean ± SEM. ^∗P < 0.05; ^∗∗∗P < 0.001.