The roles of intestinal epithelial AhR in alcohol-induced barrier dysfunction and liver injury

项目总结 在整个新冠肺炎大流行期间，酒类销售、使用和相关住院治疗显著增加 增加，对少数族裔社区的影响不成比例。酒精通过以下途径损害肠道屏障功能 紧密连接蛋白表达的变化。酒精诱导的肠通透性导致肠道内毒素 微生物泄漏并通过门脉循环为肝脏损伤做好准备。芳香烃 受体(AhR)是一种普遍表达的转录因子，参与清除外源物质和 免疫细胞功能的维持，已被认为与肠道屏障功能的维持有关。AHR 在炎症模型中，饮食配体的激活已被证明可以改善肠道屏障功能。 此外，酒精诱导的肠道通透性和肝脏损伤已被证明可部分减弱。 AHR配体。然而，这些有限的研究还没有检查细胞特异性AhR信号在 酒精对肠道屏障功能的维持。我们的初步数据表明， 肠上皮特异性AhR基因敲除与AhR配体β-1治疗的结合 萘黄酮(BNF)对酒精引起的肠道通透性和肝脏损伤具有保护作用。我们 观察到1)乙醇诱导的内毒素血症(肠道通透性标记物)的减少，2) 诱导血清丙氨酸氨基转移酶(ALT；肝损伤标志物)，以及iii)诱导AhR靶基因 在回肠粘膜。以前的研究表明，肠上皮AhR调节膳食AhR的利用率 免疫细胞的配体。因此，我们假设肠上皮AhR的缺失会增加 AhR配体对肠道免疫细胞的可用性，导致免疫AhR增加 信号转导及IL-10和IL-22介导的乙醇诱导的肠屏障通透性改善 以及随后对肝脏损伤的保护。在具体目标1中提出的研究将阐明 肠上皮细胞和免疫细胞特异性AhR在酒精介导的肠通透性中的作用机制 关注小肠和大肠之间的差异-使用肠上皮和肝损伤 免疫细胞特异性AhR基因敲除小鼠。这些研究还将描绘乙醇引起的 微生物代谢物及其在AhR激活中的作用。具体目标2将利用最新的- ART肠上皮-免疫细胞共培养研究肠上皮细胞与免疫细胞之间的串扰 在调节酒精引起的肠道通透性方面。总体而言，成功完成拟议工作可以 对治疗肠道屏障引起的肝损伤，如酒精性肝损伤具有深远的意义 疾病。所有实验都将考虑性别差异可能引起的差异，并注意统计数据 和再现性。拟议的研究将为申请者提供全面的培训，并确定 治疗目标是帮助肠道屏障功能，并允许干预酒精使用障碍。

PROJECT SUMMARY Throughout the COVID-19 pandemic, alcohol sales, use and related hospitalizations have dramatically increased, disproportionately affecting minority communities. Alcohol impairs intestinal barrier function through changes in tight junction protein expression. Alcohol-induced intestinal permeability leads to endotoxin from gut microbes leaking out and traveling through portal circulation to prime the liver for injury. The aryl hydrocarbon receptor (AhR), a ubiquitously expressed transcription factor involved in the clearance of xenobiotics and maintenance of immune cell function, has been implicated in the maintenance of intestinal barrier function. AhR activation with dietary ligands has been shown to improve intestinal barrier function in models of inflammation. Furthermore, alcohol-induced gut permeability and liver injury have been shown to be partially attenuated with AhR ligands. However, these limited studies have not examined the role of cell specific AhR signaling in the maintenance of intestinal barrier function in response to alcohol. Our preliminary data demonstrate that the combination of intestinal epithelial specific (IEC) AhR knockout and treatment with the AhR ligand β- naphthoflavone (BNF) provides protection against alcohol induced gut permeability and liver injury. We observed i) decrease in ethanol-induced endotoxemia (intestinal permeability marker), ii) decrease in the induction of serum alanine aminotransferase (ALT; hepatic injury marker), and iii) induction of AhR target genes in ileal mucosa. Previous studies have shown that intestinal epithelial AhR regulates availability of dietary AhR ligands to immune cells. Therefore, we hypothesize that the absence of intestinal epithelial AhR increases the availability of AhR ligands to intestinal immune cells, leading to an increase in immune AhR signaling, and IL-10 and IL-22 mediated improvement in ethanol induced intestinal barrier permeability and subsequent protection to liver injury. The studies proposed in Specific Aim 1 will elucidate the mechanisms of intestinal epithelial and immune cell specific AhR on alcohol mediated gut permeability—with focus on differences between the small and large intestines—and liver injury using intestinal epithelial and immune cell specific AhR knockout mice. These studies will also delineate the ethanol induced changes in microbial metabolites and their role in AhR activation by reporter assays. Specific Aim 2 will utilize state-of-the- art intestinal epithelial enteroid-immune cell co-cultures to study the crosstalk between IECs and immune cells in mediating alcohol induced gut permeability. Overall, the successful completion of the proposed work could have profound implications for the treatment of intestinal barrier contributing liver injury such as in alcoholic liver disease. All experiments will consider variations that may arise from sex differences, with attention to statistics and reproducibility. The proposed studies will provide comprehensive training for the applicant and identify novel therapeutic targets to aid intestinal barrier function and allow for intervention in alcohol use disorder.