Bao Rui, Shui Xianqi, Hou Jiong, Li Jinbao, Deng Xiaoming, Zhu Xiaoyan, Yang Tao
Department of Anesthesiology and Intensive Care Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, P.R. China.
Institution of Surgical Teaching and Research, Changhai Hospital, Second Military Medical University, Shanghai 200433, P.R. China.
Int J Mol Med. 2016 Sep;38(3):969-75. doi: 10.3892/ijmm.2016.2679. Epub 2016 Jul 14.
The number of regulatory T cells (Treg cells) and the expression of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; also known as CD39) and 5'-ectonucleotidase (NT5E; also known as CD73) on the Treg cell surface are increased during sepsis. In this study, to determine the factors leading to the high expression of CD39 and CD73, and the regulation of the CD39/CD73/adenosine pathway in Treg cells under septic conditions, we constructed a mouse model of sepsis and separated the Treg cells using a flow cytometer. The Treg cells isolated from the peritoneal lavage and splenocytes of the mice were treated with adenosine or the specific adenosine A2A receptor agonist, CGS21680, and were transfected with specific siRNA targeting E2F transcription factor 1 (E2F-1) or cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), which are predicted transcription regulatory factors of CD39 or CD73. The regulatory relationships among these factors were then determined by western blot analysis and dual-luciferase reporter assay. In addition, changes in adenosine metabolism were measured in the treated cells. The results revealed that adenosine and CGS21680 significantly upregulated CD39 and CD73 expression (P<0.01). E2F-1 and CREB induced CD39 and CD73 expression, and were upregulated by adenosine and CGS21680. Adenosine triphosphate (ATP) hydrolysis and adenosine generation were inhibited by the knockdown of E2F-1 or CREB, and were accelerated in the presence of CGS21680. Based on these results, it can be inferred that adenosine, the adenosine A2A receptor agonist, E2F-1 and CREB are the possible factors contributing to the high expression of CD39 and CD73 on the Treg cell surface during sepsis. Adenosine and its A2A receptor agonist served as the signal transducer factors of the CD39/CD73/adenosine pathway, accelerating adenosine generation. Our study may benefit further research on adenosine metabolism for the treatment of sepsis.
脓毒症期间，调节性T细胞（Treg细胞）的数量以及Treg细胞表面外核苷酸三磷酸二磷酸水解酶1（ENTPD1；也称为CD39）和5'-核苷酸酶（NT5E；也称为CD73）的表达会增加。在本研究中，为了确定导致CD39和CD73高表达的因素，以及脓毒症条件下Treg细胞中CD39/CD73/腺苷途径的调控，我们构建了脓毒症小鼠模型，并使用流式细胞仪分离Treg细胞。从小鼠腹腔灌洗物和脾细胞中分离出的Treg细胞用腺苷或特异性腺苷A2A受体激动剂CGS21680处理，并用靶向E2F转录因子1（E2F-1）或环磷酸腺苷（cAMP）反应元件结合蛋白（CREB）的特异性小干扰RNA（siRNA）转染，E2F-1和CREB是预测的CD39或CD73转录调节因子。然后通过蛋白质免疫印迹分析和双荧光素酶报告基因检测确定这些因素之间的调控关系。此外，还检测了处理后细胞中腺苷代谢的变化。结果显示，腺苷和CGS21680显著上调CD39和CD73的表达（P<0.01）。E2F-1和CREB诱导CD39和CD73的表达，并被腺苷和CGS21680上调。敲低E2F-1或CREB可抑制三磷酸腺苷（ATP）水解和腺苷生成，而在CGS21680存在的情况下则加速生成。基于这些结果，可以推断腺苷、腺苷A2A受体激动剂、E2F-1和CREB是脓毒症期间Treg细胞表面CD39和CD73高表达的可能因素。腺苷及其A2A受体激动剂作为CD39/CD73/腺苷途径的信号转导因子，加速腺苷生成。我们的研究可能有助于进一步研究腺苷代谢用于脓毒症的治疗。
