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Extracellular adenosine-5'-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X

Background: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X 7 and promotes inflammasome assembly and interleukin-1β expression. We hypothesized a functional role of the signal axis ATP–P2X 7 in inflammasome activation and the chronic inflammation driving atherosclerosis. Methods: P2X 7 -competent and P2X 7 -deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X 7 may have a role in atherosclerosis, P2X 7 expression was analyzed in aortic arches from low density lipoprotein receptor -/- mice consuming a high-cholesterol or chow diet. P2X 7 +/+ and P2X 7 −/− low density lipoprotein receptor −/− mice were fed a high-cholesterol diet to investigate the functional role of P2X 7 knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X 7 . 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 7 -competent macrophages. In contrast, P2X 7 -deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1β. P2X 7 receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X 7 -deficient mice showed smaller atherosclerotic lesions than P2X 7 -competent mice (0.162 cm 2 ±0.023 [n=9], P2X 7 −/− low density lipoprotein receptor −/− : 0.084 cm 2 ±0.01 [n=11], P =0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X 7 −/− mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X 7 -deficient mice. Last, we observe increased P2X 7 expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease. Conclusions: P2X 7 deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X 7 represents an interesting potential new target to combat atherosclerosis.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2 −/− mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2 −/− mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2 +/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2 −/− animals. Insulin tolerance testing (ITT) performed in obese P2Y 2 −/− mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y

Introduction: Dying cells release damage associated molecular patterns (DAMPs) like nucleotides into the extracellular fluid, orchestrating a profound immune response. Purinergic receptors decrypt the composition of extracellular nucleotides and translate it to distinct cellular reactions. M1 and M2 macrophages are immune cells involved in many diseases which react in a completely different way. Nevertheless, how the microenvironment exactly triggers their behavior is not fully understood. Hypothesis: We hypothesize a distinct expression pattern of purinergic receptors on M1 and M2 macrophages which leads to an unique microenvironment recognition and behavior. Methods: Macrophages were generated by bone marrow isolation and cultivation with macrophage colony stimulating factor (M-CSF). Bone marrow derived macrophages (BMDMs) were differentiated to M1 via addition of IFNγ and LPS. M2 differentiation was initiated by the addition of IL-4. Gene transcription quantification was assessed by real-time PCR. Intracellular Ca 2+ signaling was quantified by Fluo-4 AM. Results: Differentiation of BMDMs with either IFNγ/LPS or IL-4 leads to the upregulation of M1 or M2 specific gene transcription. Subsequent real-time PCR analysis of purinergic receptors revealed a strong relationship between the expression pattern and the differentiation to M1 or M2 macrophages. Whereas M1 macrophages upregulate predominantly G αi coupled metabotropic P2Y receptors, M2 macrophages preferentially upregulate G αq coupled P2Y receptors. Comparison of the two ADP-dependent P2Y receptors (P2Y 1 and P2Y 13 ) showed a M1 and M2 specific expression pattern with M1 macrophages solely expressing the G αi coupled P2Y 13 and M2 macrophages exclusively expressing the G αq coupled P2Y 1 receptor. In agreement with the specific expression pattern, ADP stimulation evokes a significant Ca 2+ flux in M2 macrophages but failed to induce it in M1 macrophages. Conclusion: Our experiments revealed for the first time that M1 and M2 macrophages express a unique purinergic receptor repertoire and sense the nucleotide based microenvironment differently. With this knowledge we could interfere with one macorphage subtype without disturbing the others.

Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2XP2XLipopolysaccharide 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 P2XP2X