ABSTRACT

Aryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1 was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites.

摘要

芳烃受体 (AhR) 是肠道微生物群与其宿主之间相互作用的关键参与者。然而，调节肠道内的 AhR 的因素，这是一个复杂的代谢组环境，却知之甚少。本研究调查了代谢物组合对 AhR 激活机制的影响。使用荧光素酶报告系统和人类细胞系和人类结肠外植体上 AhR 靶基因的 mRNA 水平评估 AhR 活性。通过放射性配体结合测定、通过免疫荧光对 AhR 的核转位以及 AhR 与 ARNT 的蛋白质共免疫沉淀来研究 AhR 的活化。使用几种测试和抑制剂评估了 AhR 的间接激活。目的基因CYP1A1的启动子通过染色质免疫沉淀和使用组蛋白去乙酰化酶 HDAC 抑制剂 (iHDAC) 进行了研究。短链脂肪酸，尤其是丁酸盐，可增强由内源性色氨酸代谢物介导的 AhR 活性，而不与受体结合。这种效应在人类肠道外植体中得到证实，并且不依赖于 SCFA 靶向受体的激活、AhR 降解的抑制或其配体的清除。丁酸盐通过 iHDAC 活性直接作用于 AhR 靶基因启动子以重塑染色质。我们的研究结果表明，丁酸盐不是 AhR 配体，而是充当 iHDAC，导致在色氨酸衍生的 AhR 激动剂存在下，AhR 向靶基因启动子的募集增加。