RIPK3通过FoxO1-PDK2信号通路诱导TAMs向M1型极化的分子机制研究

Tumor associated macrophages (TAMs) have been well known for its crucial role in the tumorigenesis. RIPK3, a critical component of necroptosis, have been known for its role in inflammation and tumorigenesis, however, its impact on tumor associated macrophages remains unclear. Studies have reported that in RIPK3-/- tumor-bearing mice the tumor associated macrophage infiltration increased significantly. Our preliminary experiments found that RIPK3 was down-regulated in tumor associated macrophages, and RIPK3 deprivation caused macrophage polarization into M2 phenotype and promote tumor metastasis; high throughput sequencing results showed that in RIPK3 knockout murine macrophages, PDK2, a key regulator in glucose metabolic pathway increased significantly. Bioinformatics analysis suggested that transcription factor FoxO1 may bind to PDK2 promoter. In addition, RIPK3 activation promoted FoxO1 phosphorylation. It is known that PDK2 can promote macrophage M2 polarization, thus we speculate that RIPK3 may inhibit PDK2 expression through phosphorylating FoxO1, reprogramming glucose metabolism in tumor associated macrophages and promotes it polarization into M1 type. This study intends to elucidate the role and regulatory mechanism of RIPK3 in TAMs polarization through the FoxO1-PDK2 signaling pathway, which will provide novel therapeutic targets and ideas of remodeling tumor microenvironment by targeting macrophages metabolism。

肿瘤相关巨噬细胞(TAMs)的极化状态与肿瘤进展密切相关。程序性坏死关键蛋白RIPK3在炎症和坏死中发挥重要作用，然而其对TAMs的影响及机制不明。我们前期研究发现RIPK3在TAMs中下调，且其缺失可促进巨噬细胞向M2型极化促进肿瘤转移；高通量测序结果表明RIPK3敲除小鼠巨噬细胞中糖酵解通路PDK2基因显著上调；生物信息学分析发现PDK2基因上有转录因子FoxO1结合位点；RIPK3激活可促进FoxO1磷酸化。已知PDK2可促进巨噬细胞M2型极化，因此我们推测RIPK3可能通过磷酸化FoxO1抑制PDK2转录表达，重编程TAMs糖代谢使其向M1型极化。本课题拟阐明RIPK3通过FoxO1-PDK2糖代谢通路调控TAMs极化状态的相关机制，为通过靶向糖代谢重塑肿瘤微环境来治疗肿瘤提供新的治疗靶点和思路。

肿瘤相关巨噬细胞是肿瘤微环境中参与肿瘤的发生发展过程的重要免疫细胞之一。它包含了两种可以互相极化的亚型即M1型和M2型。先前研究结果表明RIPK3在肿瘤细胞坏死、炎症发生和巨噬细胞浸润中均发挥了重要作用。我们发现RIPK3在肿瘤肿瘤相关巨噬细胞中下调，且其下调能促使巨噬细胞向M2型极化。然而，RIPK3对TAM的极化的影响及机制尚有待阐明。.本课题研究了RIPK3通过caspase1-PPAR信号通路抑制TAMs脂代谢促进M1型极化抑制肝癌发生的分子机制，其主要结论如下：.1.我们发现在二乙基亚硝胺（DEN）联合胆碱缺失氨基酸(CDAA)饮食诱导的小鼠肝癌模型中， TAMs中RIPK3的表达显著下调，且呈M2型极化，RIPK3 KO的小鼠肝癌明显加重，肝组织损伤更重，TAMs浸润显著增加，且TAMs的M2型标志物CD206和ARG1显著上调。.2.RIPK3敲除小鼠腹腔巨噬细胞、肝脏Kupffer细胞和相较于WT老鼠表现为更多的M2型，RIPK3敲除小鼠TAMs吞噬能力更弱，PD-L1和PD-1表达上调，免疫抑制效应更强。.3.RIPK3 KO的TAMs胞内脂滴积聚更多，FAS、FAO以及脂解相关基因均显著上调；代谢流检测实验亦证实RIPK3 KO组FAO能力增强，表明RIPK3可以调控巨噬细胞脂代谢.4.RIPK3敲除TAMs后ROS表达显著下调，而PPAR通路PPARα、PPARγ及其下游基因Cpt1a表达显著上调，同时RIPK3敲除TAMs中caspase1表达显著下调，表明RIPK3通过上调ROS激活caspase-1切割PPARγ而抑制TAMs脂肪酸氧化使得巨噬细胞向M1型极化。.5.使用脂肪酸氧化抑制剂乙莫克舍（ETO）、脂肪酸合成抑制剂TOFA和脂解抑制剂Orlistat处理则均能抑制TAMs FAO水平，并抑制RIPK3敲除TAMs向M2型极化。进一步体内实验证实，使用脂肪酸氧化抑制剂ETO，或使用抑制剂GW9662干预PPAR信号通路可抑制TAMs FAO所致的M2型极化和肝癌发生。.这些发现为RIPK3作为免疫调节分子通过脂质代谢重编程影响肝癌发生提供了分子基础。