Macrophages are tissue-resident immune cells crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is in turn modulated by ectonucleotidases such as CD39/E-NTPDase1, expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in LPS-primed macrophages. First, we verified that ATP boosted CD39 activity and caveolin-1 expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-β-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in LPS-primed macrophages treated with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated in LPS and LPS+BzATP-treated caveolin-1 deficient macrophages. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

中文翻译：

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P2X7受体激活可增加小窝蛋白1的表达和巨噬细胞脂质筏的形成，从而增强CD39活性。

巨噬细胞是驻留在组织中的免疫细胞，对免疫反应的启动和维持至关重要。嘌呤能信号调节巨噬细胞活性并影响细胞可塑性。ATP激活的嘌呤能受体P2X7具有促炎特性，有助于巨噬细胞的激活。P2X7受体信号转导又由胞外核苷酸酶（例如CD39 / E-NTPDase1）调节，在胞膜小孔和脂质筏中表达。在这里，我们检查了P2X7受体的活性，并确定了对LPS启动的巨噬细胞对胞外核苷酸酶定位和功能的影响。首先，我们验证了ATP增强了LPS启动的巨噬细胞中CD39的活性和小窝蛋白1的表达。制霉菌素和甲基-β-环糊精等破坏胆固醇丰富结构域的药物在所有情况下都会降低CD39的酶促活性。我们注意到，在经ATP处理的LPS引发的巨噬细胞中，CD39与脂质筏标记物（flotillin-2和Caveolin-1）共定位。P2X7受体抑制作用阻止了这些ATP介导的小窝蛋白1表达的增加，并抑制了与CD39的共定位。此外，我们发现在LPS和LPS + BzATP处理的Caveolin-1缺陷型巨噬细胞中，STAT3激活显着减弱。两者合计，我们的数据表明P2X7受体触发脂质筏依赖性机制的启动，该机制上调CD39活性，并可能有助于限制巨噬细胞应答以恢复体内平衡。我们发现，在LPS和LPS + BzATP处理的Caveolin-1缺陷型巨噬细胞中，STAT3激活显着减弱。两者合计，我们的数据表明P2X7受体触发脂质筏依赖性机制的启动，该机制上调CD39活性，并可能有助于限制巨噬细胞应答以恢复体内平衡。我们发现，在LPS和LPS + BzATP处理的Caveolin-1缺陷型巨噬细胞中，STAT3激活显着减弱。两者合计，我们的数据表明P2X7受体触发脂质筏依赖性机制的启动，该机制上调CD39活性，并可能有助于限制巨噬细胞应答以恢复体内平衡。