Rhbdd3通过调控糖代谢介导脓毒症时DC功能障碍的研究

Immunosupression is a major feature of the late phase of sepsis. It is known that the development of dendritic cells (DC) dysfunction contributes to sepsis-induced immunosuppression. However, the underlying mechanism remains unclear. Recently, the Rhomboid domain containing protein 3(Rhbdd3) has emerged as a negative regulator of immunologic response triggered by TLR receptor signal. Our preliminary experiments found that the expression of Rhbdd3 increased in peripheral blood DC of septic patients, correlating with the severity of disease and DC function. In vitro study showed that Rhbdd3 depletion promoted the function of BMDC activated by LPS, which implyed the modulation from Rhbdd3. Meanwhile, Rhbdd3 depletion elicited an increase of glycolysis, but a decrease of oxidative phosphorylation during DC activation. However, glycolytic inhibitor can reverse the effects of Rhbdd3 depletion on DC. In regard of this, we propose the research hypothesis that Rhbdd3 mediated DC dysfunction during sepsis via regulating glucose metabolism. The program was designed to investigate our hypothesis on septic patients, Rhbdd3-deficient mice, as well as in vitro clutured cell. We will explore the expression of Rhbdd3 in DC during sepsis, and the impact of Rhbdd3 on DC function, whether the regulation via glucose metabolism or not, most of all, the probable molecular mechanism invloved in Rhbdd3-mediated glucose metabolism of DC. Collectively, with in-depth research into the role of Rhbdd3 during sepsis, our current program will contribute to further understanding of the metabolic regulation and its influence in sepsis-induced immunosuppression. It also will be of great help to enrich experimental evidence and new therapeutic target for sepsis in future.

DC功能障碍是脓毒症免疫抑制的重要原因之一，但其机制尚未阐明。Rhbdd3是新发现的TLR信号途径负向调控分子。我们的预实验发现，脓毒症患者外周血DC中Rhbdd3表达上调，且与病情严重程度和DC功能相关。进一步用Rhbdd3-/-小鼠研究发现，Rhbdd3-/-BMDC活化后的功能增强，提示Rhbdd3可调控DC功能。同时，Rhbdd3-/-BMDC活化后氧化磷酸化水平降低，糖酵解升高；抑制糖酵解可减弱Rhbdd3-/-BMDC的功能。我们推测：Rhbdd3通过调控糖代谢介导脓毒症时DC功能障碍。本项目将从临床患者、离体细胞及基因敲除小鼠等层面，系统的探索脓毒症时Rhbdd3的表达及其对DC功能的调控作用；尤其是该调控作用是否通过糖代谢途径介导；进一步探讨Rhbdd3调控DC糖代谢的关键信号通路。研究结果将有助于解读能量代谢调控网络在脓毒症免疫抑制发展中的意义，为脓毒症的防治提供新靶点。

能量代谢平衡是维持细胞正常生理功能的关键因素。脓毒症时机体能量代谢紊乱是导致免疫细胞发生功能障碍的主要原因之一。我们的研究发现：脓毒症患者外周血PBMC中Rhbdd3的表达水平显著增加，且与患者的感染状态以及最终预后密切相关。体外研究发现：Rhbdd3-/-BMDCs分泌促炎细胞因子水平显著增加，且诱导效应性T细胞增殖及分泌IFN-γ的水平显著增强。此外，我们研究发现：LPS刺激Rhbdd3-/-BMDCs显著上调糖酵解关键酶(PKM2,HK2,PFK1,PFKFB3,PDK1)的表达，且诱导Rhbdd3-/-BMDCs ECAR的水平上调，伴随着乳酸及糖消耗水平提高。同时，我们也发现，Rhbdd3也显著抑制BMDCs氧化磷酸化水平，包括抑制OCR及线粒体膜电位的水平，抑制细胞上清中ATP水平。此外，体外/体内研究证实：给予2-DG或α-cyanocimnamate调节糖代谢水平，显著影响BMDCs分泌促炎细胞因子的能力。深入研究发现，Rhbdd3可能是通过PI3K/Akt激酶信号和HIF-1α转录因子的活性，从而影响细胞糖代谢状态进而影响BMDCs的功能。在体研究发现，Rhbdd3-/-小鼠CLP术后血浆中促炎细胞因子水平，机体细菌负荷，肺损伤程度以及术后生存率显著改善。给予Rhbdd3依赖的关键PI3K/Akt通路抑制剂perifosine显著改善CLP小鼠的预后。本研究从糖代谢这一新的角度探讨了Rhbdd3调控DCs功能障碍的主要作用机制，为脓毒症免疫抑制的防治提供新的思路。