A novel cholinergic circuitry in alcoholic liver disease (NIAAA)

酒精性肝病（NIAAA）中的新型胆碱能回路

基本信息

批准号：
10092344
负责人：
Jun Wu
金额：
$ 41.33万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-15 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10092344
关键词：
Acetaldehyde Acetylcholine Agonist Alcohol consumption Alcohol dehydrogenase Alcoholic Hepatitis Alcoholic Liver Diseases Alcoholic liver damage Alcohols Biogenesis Cell Line Cell membrane Cells Choline O-Acetyltransferase Chronic Chronic Hepatitis Cirrhosis Collaborations Country Data Development Diet Diffuse Enzymes Ethanol Fatty Liver Fibrosis Flow Cytometry Gender Gene Deletion Genetic Heavy Drinking Hematopoietic HepG2 Hepatic Hepatocyte Human Immune Impairment In Vitro Inflammation Inflammatory Injury Intestines Investigation Ion Channel Ion Channel Gating Knockout Mice Kupffer Cells Lead Ligands Liver Liver Cirrhosis Liver Fibrosis Liver diseases Mediating Modeling Molecular Mus National Institute on Alcohol Abuse and Alcoholism Nicotinic Receptors Paracrine Communication Pathogenesis Pathway interactions Pattern Play Primary carcinoma of the liver cells Regimen Regulation Regulatory Pathway Reporter Reporting Risk Role Route Sampling Series Signal Pathway Signal Transduction Site Spatial Distribution Symptoms Testing Therapeutic Intervention Therapeutic Uses Tissues Woman alcohol exposure cholinergic chronic alcohol ingestion chronic liver disease dietary control dysbiosis effective therapy endoplasmic reticulum stress experimental study in vivo insight interdisciplinary approach lipid biosynthesis liver function liver injury liver preservation liver transplantation loss of function macrophage monocyte mortality mouse model new therapeutic target novel pathogen recruit screening sexual dimorphism single-cell RNA sequencing small molecule three-dimensional visualization transcriptome transcriptomics

项目摘要

Project Summary Alcoholic liver disease (ALD) is one of the major chronic liver diseases, encompassing symptoms from fatty liver, alcoholic hepatitis, chronic hepatitis with liver fibrosis and cirrhosis, and potentially hepatocellular carcinoma. Despite extensive investigations, current understanding of the pathogenesis of ALD is still limited and no effective therapies are available for late-stage ALD besides liver transplantation. Our preliminary studies revealed a previously unknown non-neuronal cholinergic signaling pathway between acetylcholine-producing immune cells residing within the liver and hepatocytes expressing the nicotinic acetylcholine receptor, alpha 2 subunit (CHRNA2). This signaling pathway was activated in the livers of mice following alcohol consumption and loss of function mouse models with genetic deletion of either Chrna2 or Chat (choline acetyltransferase, rate limiting enzyme for acetylcholine biogenesis) suffered aggravated liver damage after chronic alcohol consumption. We propose to thoroughly test the hypothesis that this novel hepatic acetylcholine-CHRNA2 signaling pathway plays an adaptive/protective role against alcohol-induced liver damage. Aim 1. We will investigate how ChAT+ hepatic non-parenchymal cells (NPCs), particularly liver-resident Kupffer cells and monocyte-derived macrophages (MDMs), are activated after alcohol consumption. In vivo regulation will be analyzed with flow cytometry using hepatic NPCs isolated from ChATBAC-eGFP mice and a double reporter mice (ChAT-Cre;tdTomato;ChATBAC- eGFP). Mechanistic insights will be investigated with cultured macrophages (BMDMs) and human macrophage cell lines. Spatial distribution of ChAT+ cells in the whole tissue will be visualized in CLARITY-prepared liver samples and single cell RNA-seq will be carried out to characterize the transcriptomic landscape of these hepatic cholinergic NPCs. Aim 2. We will investigate signaling mediated through CHRNA2 in hepatocytes using mouse primary hepatocytes, HepG2 cells and human primary hepatocytes. The composition of the CHRNA2-containing ligand-gated ion channel in hepatocytes will be investigated to enable screening for potential hepatocyte-specific small molecule agonists to activate this signaling pathway. Aim 3. Hepatocyte-specific Chrna2 knockout mice and immune specific ChAT knockout mice will be treated with three regimens of chronic alcohol challenge to reveal the functional significance of this signaling pathway in the pathogenesis of ALD. A team of leading experts in related fields have been recruited to carry out the proposed studies with interdisciplinary approaches. Ultimately, uncovering the mechanisms that underlie this novel hepatic pathway may elucidate new routes of therapeutic intervention for counteracting liver injury arising from excessive alcohol consumption.

项目摘要酒精性肝病（ALD）是主要的慢性肝疾病之一，包括脂肪肝的症状，酒精性肝炎，慢性肝炎，肝纤维化和肝硬化，可能是肝细胞癌。尽管进行了广泛的研究，但目前对ALD发病机理的理解仍然有限，无效除肝移植外，还可以用于晚期ALD的疗法。我们的初步研究表明以前未知的非神经元胆碱能信号传导途径在产生乙酰胆碱的免疫细胞之间驻留在肝脏和肝细胞内，表达烟碱乙酰胆碱受体，α2亚基（CHRNA2）。饮酒后，在小鼠的肝脏中激活了此信号通路具有CHRNA2或CHAT的遗传缺失的功能小鼠模型（胆碱乙酰转移酶，速率限制乙酰胆碱生物发生的酶）在长期饮酒后遭受了严重的肝脏损害。我们建议彻底检验这种新型肝乙酰胆碱-CHRNA2信号通路的假设针对酒精引起的肝脏损害的自适应/保护作用。目标1。我们将研究聊天+肝非副作用细胞（NPC），尤其是肝居民库普弗细胞和单核细胞衍生的巨噬细胞（MDMS），在饮酒后被激活。使用流式细胞仪分析体内调节从Chatbac-EGFP小鼠和双重记者小鼠（Chat-cre; tdtomato; chatbac-）中分离出的肝NPC EGFP）。机械洞察力将使用培养的巨噬细胞（BMDMS）和人类巨噬细胞进行研究细胞系。整个组织中聊天+细胞的空间分布将在清晰的肝脏中可视化将进行样品和单细胞RNA-seq，以表征这些肝的转录组景观胆碱能NPC。 AIM 2。我们将使用小鼠在肝细胞中通过CHRNA2介导的信号传导原发性肝细胞，HEPG2细胞和人类原发性肝细胞。含CHRNA2的组成将研究肝细胞中的配体门控离子通道，以筛选潜在的肝细胞特异性小分子激动剂激活该信号通路。 AIM 3。肝细胞特异性CHRNA 2基因敲除小鼠免疫特定的聊天淘汰小鼠将接受三种慢性酒精挑战方案的治疗揭示了该信号通路在ALD发病机理中的功能意义。一支领先的专家团队在相关领域，已招募了以跨学科方法进行拟议的研究。最终，揭示了这一新颖的肝道途径的基础的机制可能阐明过度饮酒引起的肝损伤的治疗干预措施。