线粒体自噬调控树突状细胞NLRP3炎症小体在肝癌免疫逃逸中的作用机制

The malfunction of dendritic cells (DC) in tumor microenvironment is the major mechanism in hepatocellular carcinoma (HCC) immune escape. However, the regulation mechanism of HCC DC immunosuppression is not well studied. Our previous studies showed NLRP3 inflammasome is significantly down-regulated in the HCC tolerating DCs, but the mitochondria autophagy is up-regulated and strongly associated with the level of IDO. Restoration of NLRP3 can reverse the DC immunosuppression effects. All these suggested in HCC, DC might regulate mitochondria autophagy to inactivate or degrade NLRP3 inflammasome by IDO, and then promote cancer immune escape. In this project we will identify the key roles of NLRP3 in how it regulates HCC DCs differentiation and maturation. Then we observe the relation of phenotype and functions of HCC DCs with NLRP3 activity and mitochondria autophagy based on NLRP3 expression irregularity. We will use co-focal microscopy, fluorescent co-location, mass spectrometry, expression chips and other technologies to elucidate the molecular mechanism and signaling pathways how NLRP3 negatively regulates mitochondria autophagy via IDO. Finally, we will test the mechanisms in vivo by using functional inactivation and activation to observe how the changes in DC intracellular environment influence anti-tumor effects and analyze the relationship between the changes and clinical pathological characteristics. The outcomes will provide new rationales and methods to elucidate the molecular mechanism of HCC immune escape.

肿瘤微环境内树突状细胞（DC）功能异常是肝细胞癌免疫逃逸发生的主要机制，但调控DC免疫抑制的机制未明。我们前期研究发现NLRP3炎症小体在肝癌耐受性DC中显著性下调，线粒体自噬水平增加，且与IDO异常表达密切相关，对NLRP3的重建能够有效逆转DC的免疫抑制效应，提示肝癌DC内可能通过IDO调控线粒体自噬对NLRP3炎症小体的失活或降解促进肿瘤免疫逃逸。本项目拟从NLRP3表达失调为切入点，首先明确NLRP3调控肝癌DC表型功能的重要作用，探讨线粒体自噬对NLRP3负向调控效应与肝癌DC耐受关系；其次通过共聚焦、质谱、表达谱芯片等技术阐明IDO激活的线粒体自噬抑制NLRP3炎症小体的分子机制及主要信号通路；最后采用功能失活、功能激活等策略调控上述关键环节并进行体内验证，观察DC内环境改变对抗肿瘤效应的影响，进而分析与临床病理特征的关系，为阐明肝癌免疫逃逸的分子机制提供新的思路和手段。

（一）结题摘要.肿瘤微环境中固有免疫细胞--树突状细胞（DC）功能异常是肝细胞癌免疫逃逸发生的重要机制之一，但调控DC免疫抑制的机制未明。我们研究发现肝癌耐受性DC功能与吲哚胺2,3-双加氧酶IDO异常表达密切相关，IDO下游代谢物犬尿氨酸KYN能抑制DC成熟分化，使DC ROS累积以及线粒体膜电位下降，促进DC发生线粒体自噬。IDO是免疫检查点分子，而KYN是芳香烃受体AHR的经典配体，我们利用Ahr-/-敲基因小鼠发现KYN通过AHR活化抑制小鼠BMDC的成熟分化及上调PD-L1的表达。线粒体自噬导致mtROS上调能激活NLRP3炎症小体促进肝癌进展，利用Nlrp3-/-敲基因小鼠发现与野生型WT小鼠相比， DC、CTL、NK细胞比例降低而Treg细胞增加，同时DC活化程度及激发T细胞能力减弱。经高通量测序分析及实验验证发现，NLRP3敲除后炎症信号明显下调（如NFκB信号通路、TLR信号通路，MAPK信号通路等），从而对免疫细胞功能进行调控。线粒体膜是细胞内ROS的重要来源，利用Nlrp3fl/fl和AlbcreNlrp3fl/fl条件性敲除鼠，进一步发现Nlrp3 缺失细胞线粒体PGC1α表达降低，线粒体氧化磷酸化功能受到抑制，表明氧化应激的反应性增强促进DC免疫抑制及肝癌进展。