A novel cholinergic circuitry in alcoholic liver disease (NIAAA)

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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.