代谢对于免疫的调控作用近年来逐渐被人们所重视。甲羟戊酸通路是真核生物中高度保守的代谢途径。甲羟戊酸激酶是该通路中一个重要的酶，其突变会导致该通路功能丧失，使患者出现周期性发热和全身炎症等症状。这提示我们甲羟戊酸通路对免疫应答具有调控作用。然而，甲羟戊酸通路对先天免疫的调控作用以及调控机制至今仍不明确。为了揭示甲羟戊酸通路对于先天免疫的精准调控，本论文以该通路的小分子抑制剂双磷酸和他汀类药物为探针，并结合不同的免疫学相关手段，深入探究了该通路对于促炎细胞因子产生及IL-1β分泌的调控作用。首先，我们发现香叶基香叶基焦磷酸（Geranylgeranyl pyrophosphate，GGPP）的合成受阻可以增强脂多糖（Lipopolysaccharide，LPS）刺激的巨噬细胞产生促炎细胞因子的能力，胆固醇的合成受阻却不影响LPS诱导的促炎细胞因子产生。此外，机制研究表明，GGPP合成受阻可以通过抑制线粒体呼吸以及上调NF-κB信号通路来调控促炎细胞因子的产生。进一步的研究发现，GGPP对于促炎细胞因子的调控具有广泛性。GGPP合成受阻不仅可以增加LPS诱导的促炎细胞因子产生，还可以增加其他先天免疫相关受体（包括TLR2、TLR7、TLR9、TNFR、NOD2、STING、CD40和LTβR）活化后诱导的促炎细胞因子产生。此外，GGPP合成受阻可以促进巨噬细胞向经典活化型极化，而GGPP本身可以促进巨噬细胞向替代活化型极化。最后，我们发现在LPS的刺激下，甲羟戊酸通路抑制剂可以诱导炎症小体的活化，进而导致GSDMD活化。但同时，甲羟戊酸通路抑制剂会阻断GSDMD活化产生的GSDMD-N靶向细胞膜，因此阻断细胞焦亡的发生。另外，甲羟戊酸通路抑制剂介导的IL-1β不是通过GSDMD在细胞膜上打的孔分泌，而是通过ESCRT依赖的外泌体分泌到细胞外。综上，该研究为甲羟戊酸通路对于促炎细胞因子的产生和分泌的调控提供了理论基础和实践支撑，并揭示了GGPP对于先天免疫具有多重调控的潜力，同时也为进一步拓宽双磷酸和他汀类药物的使用范围提供了新的可能。
The regulation of metabolism on immunity has been paid more and more attention in recent years. Mevalonate pathway is a highly conserved metabolic pathway in eukaryotes. Mevalonate kinase is an important enzyme in this pathway. Its mutation will lead to the loss of function of this pathway, which will lead to periodic fever and systemic inflammation in patients. This suggests that mevalonate pathway plays a regulatory role in immune response. However, the regulatory role and mechanism of mevalonate pathway on innate immunity are still unclear.In order to reveal the precise regulation of mevalonate pathway on innate immune response , this paper took bisphosphates and statins of this pathway as probes, and used a variety of immunological methods to deeply explore the regulation of the proinflammatory cytokines production and IL-1β release by mevalonate pathway. We first found that the blockage of geranylgeranyl pyrophosphate (GGPP) enhanced the production of proinflammatory cytokines by lipopolysaccharide (LPS) stimulated macrophages, but the blockage of cholesterol did not. Further mechanism research showed that the blockage of GGPP increased the production of proinflammatory cytokines by inhibiting mitochondrial respiration and up-regulating NF-κB signaling pathway.In addition, we found that GGPP had extensive regulation on proinflammatory cytokines. The blockage of GGPP increased the proinflammatory cytokines produced by activation of other innate immune receptors, including TLR2, TLR7, TLR9, TNFR, NOD2, STING, CD40 and LTβR. Further research found that the reduction of GGPP promoted M(LPS) and M(INF-γ) polarization, while GGPP promoted M(IL-4) polarization.Finally, we found that under the stimulation of LPS, mevalonate pathway inhibitors induced the activation of inflammasome, which in turn led to the activation of GSDMD. The GSDMD-N produced by the activation of GSDMD did not target the plasma membrane, and it did not cause pyroptosis. IL-1β release mediated by mevalonate pathway inhibitors was not dependent on GSDMD pore, but on ESCRT dependent exosomes. This study provides theoretical basis and practical support for the effect of mevalonate pathway on the production and release of proinflammatory cytokines, reveals the potential of GGPP for multiple regulation of innate immunity, and provides a new possibility to further expand the scope of use of bisphosphonates and statins.
