有氧糖酵解关键酶PKM2在树突细胞活化过程中的作用及机制研究

Dendritic cell (DC) plays a central role in specific immune responses. The control of DC activation has been a fundamentally important issue in immunology. To data, this process is known to be regulated by distinct cell signaling pathways, which are mediated by pattern-recognition receptors on cell surfaces, through manipulating gene expression. Emerging studies also suggest that the change of metabolic type in cells is also critical for DC activation. Remarkably, DC activation relies on metabolic intermediates from the aerobic glycolysis, which support cytokine secretion and co-stimulatory factor expression. However, the regulation of metabolic reprogramming associated with DC activation still remains elusive. It is well-known that pyruvate kinase M2 (PKM2) is the final rate-limiting enzyme in glycolysis. Our recent data demonstrated that PKM2 plays an important role in DC activation. To comprehensively understand PKM2 function in DC, we will 1) characterize mechanism by which PKM2 facilitates glycolysis in DC; 2) investigate the role of PKM2 in cytokine secretion and co-stimulatory factor expression during DC activation; 3) reveal signaling pathway(s) regulating PKM2 functions in the process of DC activation; 4) explore the hypothesis that alterations in extracellular microenvironment modulates PKM2 function and thereby influences DC activation. By integrating metabolic regulation into the control of DC activation, our work will bring new insights into the regulation of DC functions. Meanwhile, the functional characterization of PKM2 in this work will provide a new target for DC-based immune therapy.

树突细胞（DC）处于机体特异性免疫应答的中心地位，其活化机制的研究一直是免疫学最基本的科学问题之一。目前已知，DC活化主要通过其表面模式识别受体介导的信号通路调控基因表达来实现，而新近的研究表明，这一过程也涉及到DC代谢类型的改变。一个显著的事件是：DC的活化依赖于有氧糖酵解代谢为其细胞因子分泌和表面共刺激分子表达提供必需的物质原料，但确切的调控机制仍然未知。丙酮酸激酶M2（PKM2）是公认的促进有氧糖酵解代谢的关键酶，我们的前期实验表明，PKM2在DC活化过程中发挥重要作用。在本项目中，我们拟深入探究：（1）PKM2促进DC有氧糖酵解代谢的机制；（2）PKM2促进活化DC细胞因子分泌及共刺激分子表达的机制；（3）相关细胞信号通路；（4）肿瘤微环境通过影响PKM2功能抑制DC活化的机制。本项目从代谢参与DC活化调控的新视角，力求揭示新机制；同时为设计基于DC的免疫治疗方法提供新的重要靶点。

树突细胞（Dendritic cell, DC）作为免疫系统中尤为重要的抗原递呈细胞，是连接天然免疫与获得性免疫的桥梁。当外界病原体入侵时，DC发生活化，活化的DC高表达细胞因子以及共刺激分子，介导机体免疫应答。近年来研究发现，DC在活化的过程中其自身代谢方式发生重塑，即由氧化磷酸化转变为有氧糖酵解，进而有利于生物大分子合成，为DC活化提供物质基础。丙酮酸激酶M2（PKM2）是公认的促进有氧糖酵解代谢的关键酶，我们的前期实验表明，PKM2在DC活化过程中发挥重要作用。在本项目中，我们首先以小鼠骨髓细胞来源的DC为研究模型，在LPS诱导DC活化的经典实验体系下发现，PKM2由四聚体向单体解聚，并伴随着活性下降，进而促进DC活化。进一步研究表明，解聚后的PKM2进入细胞核内通过与转录因子c-Rel结合促进了IL-12p35基因表达。随后对调控PKM2活性变化的信号分子进行筛选发现，JNK-p300通过上调PKM2 K433乙酰化水平促进PKM2的解聚和入核。此外，通过检测胞外酸化率、葡萄糖消耗量以及乳酸生成量探究PKM2的四聚体解聚对DC有氧糖酵解代谢的影响，我们发现抑制PKM2四聚体解聚阻碍了DC活化过程中有氧糖酵解代谢进程。以上结果提示，DC活化过程中的有氧糖酵解代谢的发生依赖于PKM2四聚体解聚。综上所述，本研究不但发现了在DC活化过程中PKM2可通过四聚体解聚促进有氧糖酵解代谢，还能够入核与转录因子结合介导基因表达，促进DC活化。本项目的研究不仅有助于揭示细胞代谢参与调控DC活化的作用机制，而且还将为设计新的基于DC调节的免疫治疗方法和途径提供重要的分子靶点。