P2X Deficiency Blocks Lesional Inflammasome Activity and Ameliorates Atherosclerosis in Mice.

Background: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X and promotes inflammasome assembly and interleukin-1β expression. We hypothesized a functional role of the signal axis ATP-P2X in inflammasome activation and the chronic inflammation driving atherosclerosis.

Methods: P2X-competent and P2X-deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X may have a role in atherosclerosis, P2X expression was analyzed in aortic arches from low density lipoprotein receptor mice consuming a high-cholesterol or chow diet. P2X and P2X low density lipoprotein receptor mice were fed a high-cholesterol diet to investigate the functional role of P2X knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X.

Results: Lipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1β in P2X-competent macrophages. In contrast, P2X-deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1β. P2X receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X-deficient mice showed smaller atherosclerotic lesions than P2X-competent mice (0.162 cm±0.023 [n=9], P2X low density lipoprotein receptor : 0.084 cm±0.01 [n=11], =0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X-deficient mice. Last, we observe increased P2X expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease.

Conclusions: P2X deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X represents an interesting potential new target to combat atherosclerosis.