Treg细胞中CD39-CD73-腺苷代谢通路异常在脓毒症免疫抑制发生中的作用及其机制研究

The systemic inflammatory response represents the core pathogenic event of sepsis, underlying clinical manifestations and laboratory findings in patients. Numerous studies have shown that CD4+CD25+Foxp3+ regulatory T lymphocytes (Treg cells) participate in the development of sepsis due to their ability to suppress the immune response. Our preliminary study indicated that both CD39 and CD73, two key enzymes responsible for adenosine metabolism, were significantly increased in Treg cells obtained from peripheral blood of a murine cecal ligation and puncture (CLP) model of sepsis. At the meantime, adenosine production of Treg cells and Treg cell numbers were also increased in CLP mice compared with Sham-operated mice. Furthermore, either blocking adenosine production by using CD39 knockout or CD73 knockout mice to, or using specific inhibitors for adenosine receptors, can reverse the increased Treg cell numbers during sepsis. On the basis of these results, the present proposal will verify the following work hypothesis: during sepsis, upregulation of CD39/CD73 in Treg cells will cause over-production of adenosine, which acts on Treg cells in an autocrine manner to promote Treg proliferation and enhance the immunosuppressive function of Treg. In addition, since Foxp3 as a key marker of Treg is relevant in Treg proliferation and biological function, the present proposal will investigate the signaling transduction pathways involved in adenosine modulating Foxp3 expression in Treg cells. Finally, using specific inhibitors of associated signaling pathways, we will study whether these signaling pathways play important roles in Treg proliferation and the pathogenesis of immunosuppression during sepsis. The present proposal not only clarifies the mechanisms involved in the Treg-mediated immunosuppression during sepsis, but also provides a new approach to the clinical prevention and treatment of immunosuppression from the perspective of improving metabolic Treg cells.

调节性T淋巴细胞（Treg细胞）兼具免疫低反应性和免疫抑制功能，在脓毒症免疫抑制中发挥重要作用。我们前期发现脓毒症小鼠免疫抑制期外周血Treg细胞上腺苷代谢关键酶CD39和CD73表达较假手术组增多、腺苷生成增加，同时Treg细胞数增多；而采用CD39/CD73基因敲除、或腺苷受体阻断剂均能逆转脓毒症小鼠Treg细胞增多的现象。提示在本项目中可针对如下假说进行验证：脓毒症时Treg细胞中CD39/CD73上调、腺苷生成增多，后者通过自分泌作用于Treg上的腺苷受体而促进其自身增殖和免疫抑制功能。鉴于Treg的标志分子Foxp3在其增殖和功能维持中发挥关键作用，本项目还将研究腺苷调控Treg细胞Foxp3表达的关键信号转导机制，并用相应阻断剂验证其在脓毒症小鼠Treg细胞增殖和免疫抑制发生中的作用。本项目研究有助于从改善Treg细胞代谢角度为脓毒症免疫抑制的防治提供新思路。

课题组围绕Treg细胞CD39-CD73-腺苷代谢通路在小鼠脓毒症中的作用进行了研究。研究结果显示，CLP诱导的脓毒症小鼠，外周血 Treg细胞数量及Treg细胞膜上CD39和CD73表达显著增多。脓毒症小鼠肺泡灌洗液和肺组织中观察到类似的现象。CD39-/-和CD73-/-脓毒症小鼠，外周血Treg细胞数量与野生型脓毒症小鼠相比显著下降，但较假手术组CD39-/-和CD73-/-小鼠，Treg细胞数量无显著变化。ATP或AMP处理脓毒症小鼠外周血Treg细胞1 h后，培养液上清中ADP、AMP和腺苷含量显著增加。此外，正常小鼠3种T细胞亚群只表达A2A亚型腺苷受体；而脓毒症小鼠T辅助细胞和细胞毒性T细胞表达A3亚型受体，但Treg细胞仍只表达A2A亚型。给予腺苷受体A2A亚型特异性阻断剂（ZM241385），可显著降低脓毒症小鼠外周血Treg细胞数量，增加外周血Treg细胞Foxp3表达。进一步探讨脓毒症时外周血Treg细胞数量及其CD39和CD73表达变化的机制，结果显示，CXCL4上调能增加CD4+T细胞中CD4+CD25+FOXP3+Treg细胞比例，并增加炎症相关蛋白表达，伴随STAT5磷酸化上调；而给予STAT5抑制剂能降低CXCL4对CD4+T细胞的作用，类似于CXCL4抗体对CD4+T细胞的作用。说明CXCL4可能通过靶向STAT5介导Treg细胞发育。此外，Treg细胞经腺苷和腺苷A2A受体激动剂CGS21680处理后CD39和CD73表达显著上调；E2F-1和CREB能诱导CD39和CD73表达，且腺苷和CGS21680可上调表达，而下调E2F-1或CREB能抑制ATP水解和腺苷产生，CGS21680则能加速两者的产生。说明E2F-1和CREB参与脓毒症时Treg细胞CD39和CD73表达的上调。进一步研究发现，药物抑制腺苷产生和JNK，能降低Foxp3表达。活化JNK/AP-1能增加CD4+CD25+Treg细胞的Foxp3表达。同时，AP-1能调节Treg细胞中Foxp3基因的启动子活性，诱导c-Fos或c-Jun活化，可上调Foxp3基因转录，而敲除c-Fos、Fra-2、c-Jun或JunD，则可降低Foxp3表达。说明腺苷在Foxp3的高表达中发挥了一定的作用，脓毒症时，腺苷可通过JNK/AP-1途径促进Treg细胞中Foxp3的表达。