Smith Stephanie M C, Mitchell Gordon S, Friedle Scott A, Sibigtroth Christine M, Vinit Stéphane, Watters Jyoti J
Department of Comparative Biosciences, University of Wisconsin, Madison, WI, USA 53706 ; Comparative Biomedical Sciences Training Program, University of Wisconsin, Madison, WI, USA 53706.
Program in Cellular and Molecular Biology, University of Wisconsin, Madison, WI, USA 53706.
Hypoxia (Auckl). 2013 Aug 6;2013(1):1-11. doi: 10.2147/HP.S45529.
Hypoxia and increased extracellular nucleotides are frequently coincident in the brainstem. Extracellular nucleotides are potent modulators of microglial inflammatory gene expression via P2X purinergic receptor activation. Although hypoxia is also known to modulate inflammatory gene expression, little is known about how hypoxia or P2X receptor activation alone affect inflammatory molecule production in brainstem microglia, nor how hypoxia and P2X receptor signaling interact when they occur together. In this study, we investigated the ability of a brief episode of hypoxia (2hrs) in the presence and absence of the non-selective P2X receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) to promote inflammatory gene expression in brainstem microglia in adult rats. We evaluated inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) mRNA levels in immunomagnetically-isolated brainstem microglia. Whereas iNOS and IL-6 gene expression increased with hypoxia and BzATP alone, TNFα expression was unaffected. Surprisingly, BzATP-induced inflammatory effects are lost after hypoxia, suggesting that hypoxia impairs pro-inflammatory P2X receptor signaling. We also evaluated the expression of key P2X receptors activated by BzATP, namely P2X1, P2X4 and P2X7 receptors. Whereas hypoxia did not alter their expression, BzATP upregulated P2X4 and P2X7 mRNAs; these effects were ablated in hypoxia. Although both P2X4 and P2X7 receptor expression correlated with increased microglial iNOS and IL-6 levels in microglia from normoxic rats, in hypoxia, P2X7 only correlated with IL-6, and P2X4 correlated only with iNOS. In addition, correlations between P2X7 and P2X4 were lost following hypoxia, suggesting that P2X4 and P2X7 receptor signaling differs in normoxia and hypoxia. Together, these data suggest that hypoxia suppresses P2X receptor-induced inflammatory gene expression, indicating a potentially immunosuppressive role of extracellular nucleotides in brainstem microglia following exposure to hypoxia.
缺氧和细胞外核苷酸增加在脑干中常常同时出现。细胞外核苷酸通过P2X嘌呤能受体激活，是小胶质细胞炎症基因表达的强效调节剂。虽然已知缺氧也能调节炎症基因表达，但对于单独的缺氧或P2X受体激活如何影响脑干小胶质细胞中炎症分子的产生，以及当它们同时发生时缺氧和P2X受体信号如何相互作用，人们了解甚少。在本研究中，我们研究了在存在和不存在非选择性P2X受体激动剂2'(3')-O-(4-苯甲酰苯甲酰)腺苷-5'-三磷酸（BzATP）的情况下，短暂缺氧（2小时）促进成年大鼠脑干小胶质细胞炎症基因表达的能力。我们评估了免疫磁珠分离的脑干小胶质细胞中诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子α（TNFα）和白细胞介素-6（IL-6）的mRNA水平。单独缺氧和BzATP时，iNOS和IL-6基因表达增加，而TNFα表达未受影响。令人惊讶的是，缺氧后BzATP诱导的炎症效应消失，这表明缺氧损害了促炎P2X受体信号。我们还评估了由BzATP激活的关键P2X受体，即P2X1、P2X4和P2X7受体的表达。缺氧未改变它们的表达，但BzATP上调了P2X4和P2X7的mRNA；这些效应在缺氧时被消除。虽然在常氧大鼠的小胶质细胞中，P2X4和P2X7受体表达均与小胶质细胞中iNOS和IL-6水平升高相关，但在缺氧时，P2X7仅与IL-6相关，P2X4仅与iNOS相关。此外，缺氧后P2X7和P2X4之间的相关性消失，这表明P2X4和P2X7受体信号在常氧和缺氧状态下有所不同。总之，这些数据表明缺氧抑制P2X受体诱导的炎症基因表达，表明缺氧后细胞外核苷酸在脑干小胶质细胞中可能具有免疫抑制作用。
