Abstract：
OBJECTIVE: Gut dysbiosis and myeloid-derived suppressor cells (MDSCs) are implicated in primary biliary cholangitis (PBC) pathogenesis. However, it remains unknown whether gut microbiota or their metabolites can modulate MDSCs homeostasis to rectify immune dysregulation in PBC.
METHODS: We measured fecal short-chain fatty acids levels using targeted gas chromatography-mass spectrometry and analyzed circulating MDSCs using flow cytometry in 2 independent PBC cohorts. Human and murine MDSCs were differentiated in vitro in the presence of butyrate, followed by transcriptomic, epigenetic (CUT&Tag-seq and chromatin immunoprecipitation-quantitative polymerase chain reaction), and metabolic (untargeted liquid chromatography-mass spectrometry, mitochondrial stress test, and isotope tracing) analyses. The in vivo role of butyrate-MDSCs was evaluated in a 2-octynoic acid-bovine serum albumin-induced cholangitis murine model.
RESULTS: Decreased butyrate levels and defective MDSC function were found in patients with incomplete response to ursodeoxycholic acid, compared with those with adequate response. Butyrate induced expansion and suppressive activity of MDSCs in a manner dependent on PPARD-driven fatty acid β-oxidation (FAO). Pharmaceutical inhibition or genetic knockdown of the FAO rate-limiting gene CPT1A abolished the effect of butyrate. Furthermore, butyrate inhibited HDAC3 function, leading to enhanced acetylation of lysine 27 on histone H3 at promoter regions of PPARD and FAO genes in MDSCs. Therapeutically, butyrate administration alleviated immune-mediated cholangitis in mice via MDSCs, and adoptive transfer of butyrate-treated MDSCs also displayed protective efficacy. Importantly, reduced expression of FAO genes and impaired mitochondrial physiology were detected in MDSCs from ursodeoxycholic acid nonresponders, and their impaired suppressive function was restored by butyrate.
CONCLUSIONS: We identify a critical role for butyrate in modulation of MDSC homeostasis by orchestrating epigenetic and metabolic crosstalk, proposing a novel therapeutic strategy for treating PBC.
METHODS: We measured fecal short-chain fatty acids levels using targeted gas chromatography-mass spectrometry and analyzed circulating MDSCs using flow cytometry in 2 independent PBC cohorts. Human and murine MDSCs were differentiated in vitro in the presence of butyrate, followed by transcriptomic, epigenetic (CUT&Tag-seq and chromatin immunoprecipitation-quantitative polymerase chain reaction), and metabolic (untargeted liquid chromatography-mass spectrometry, mitochondrial stress test, and isotope tracing) analyses. The in vivo role of butyrate-MDSCs was evaluated in a 2-octynoic acid-bovine serum albumin-induced cholangitis murine model.
RESULTS: Decreased butyrate levels and defective MDSC function were found in patients with incomplete response to ursodeoxycholic acid, compared with those with adequate response. Butyrate induced expansion and suppressive activity of MDSCs in a manner dependent on PPARD-driven fatty acid β-oxidation (FAO). Pharmaceutical inhibition or genetic knockdown of the FAO rate-limiting gene CPT1A abolished the effect of butyrate. Furthermore, butyrate inhibited HDAC3 function, leading to enhanced acetylation of lysine 27 on histone H3 at promoter regions of PPARD and FAO genes in MDSCs. Therapeutically, butyrate administration alleviated immune-mediated cholangitis in mice via MDSCs, and adoptive transfer of butyrate-treated MDSCs also displayed protective efficacy. Importantly, reduced expression of FAO genes and impaired mitochondrial physiology were detected in MDSCs from ursodeoxycholic acid nonresponders, and their impaired suppressive function was restored by butyrate.
CONCLUSIONS: We identify a critical role for butyrate in modulation of MDSC homeostasis by orchestrating epigenetic and metabolic crosstalk, proposing a novel therapeutic strategy for treating PBC.
摘要：
目的：肠道菌群失调和髓源性抑制细胞（MDSCs）与原发性胆汁性胆管炎（PBC）的发病机制有关。然而,目前尚不清楚肠道微生物群或其代谢产物是否可以调节MDSCs稳态以纠正PBC中的免疫失调.
方法：我们通过靶向GC-MS测量了粪便短链脂肪酸（SCFAs）水平，并通过流式细胞术分析了两个独立PBC队列中的循环MDSCs。人和鼠MDSCs在丁酸盐的存在下体外分化，其次是转录组学，表观遗传学(CUT&Tag-seq和ChIP-qPCR)和代谢(非靶向LC-MS，线粒体压力测试,和同位素追踪)分析。在2-辛酸-BSA诱导的胆管炎小鼠模型中评估丁酸盐-MDSC的体内作用。
结果：在对熊去氧胆酸（UDCA）反应不完全的患者中发现丁酸水平降低和MDSCs功能缺陷，与那些有足够反应的人相比。丁酸以依赖于PPARD驱动的脂肪酸β-氧化(FAO)的方式诱导MDSC的扩增和抑制活性。FAO限速基因CPT1A的药物抑制或基因敲除消除了丁酸的作用。此外，丁酸抑制HDAC3功能，导致MDSCs中PPARD和FAO基因启动子区域的H3K27ac修饰增强。治疗学上,丁酸酯通过MDSCs减轻小鼠免疫介导的胆管炎，丁酸酯处理的MDSC的过继转移也显示出保护功效。重要的是,在UDCA无应答者的MDSCs中检测到FAO基因表达降低和线粒体生理学受损，丁酸盐恢复了它们受损的抑制功能。
结论：我们通过协调表观遗传和代谢串扰确定丁酸在调节MDSCs稳态中的关键作用，提出了一种新的治疗PBC的治疗策略。
方法：我们通过靶向GC-MS测量了粪便短链脂肪酸（SCFAs）水平，并通过流式细胞术分析了两个独立PBC队列中的循环MDSCs。人和鼠MDSCs在丁酸盐的存在下体外分化，其次是转录组学，表观遗传学(CUT&Tag-seq和ChIP-qPCR)和代谢(非靶向LC-MS，线粒体压力测试,和同位素追踪)分析。在2-辛酸-BSA诱导的胆管炎小鼠模型中评估丁酸盐-MDSC的体内作用。
结果：在对熊去氧胆酸（UDCA）反应不完全的患者中发现丁酸水平降低和MDSCs功能缺陷，与那些有足够反应的人相比。丁酸以依赖于PPARD驱动的脂肪酸β-氧化(FAO)的方式诱导MDSC的扩增和抑制活性。FAO限速基因CPT1A的药物抑制或基因敲除消除了丁酸的作用。此外，丁酸抑制HDAC3功能，导致MDSCs中PPARD和FAO基因启动子区域的H3K27ac修饰增强。治疗学上,丁酸酯通过MDSCs减轻小鼠免疫介导的胆管炎，丁酸酯处理的MDSC的过继转移也显示出保护功效。重要的是,在UDCA无应答者的MDSCs中检测到FAO基因表达降低和线粒体生理学受损，丁酸盐恢复了它们受损的抑制功能。
结论：我们通过协调表观遗传和代谢串扰确定丁酸在调节MDSCs稳态中的关键作用，提出了一种新的治疗PBC的治疗策略。
