Mechanism study of the inflammatory injury by glutamate vesicles in hepatocytes and glutamate receptor activation in Kupffer cells in alcohol-associated liver disease

Abstract

Recently, the crosstalk between hepatocytes (HEPs) and hepatic stellate cells via glutamate signaling was reported to play a pivotal role in alcohol-associated steatosis. However, the precise role of hepatic glutamate in the pathogenesis of alcoholic-associated steatohepatitis (ASH) is still largely unexplored. Here, I investigated the accumulative process of alcohol-induced glutamate vesicles (GVs) in HEPs. Furthermore, I elucidated that glutamate excreted from HEPs induces inflammatory liver injury by the activation of metabotropic glutamate receptor 5 (mGluR5) in Kupffer cells (KCs). After chronic alcohol consumption in mice, hepatic expressions of ALDH4A1 and vesicular glutamate transporter 3 (VGLUT3) were upregulated for the glutamate synthesis and storage, respectively, through nuclear factor erythroid-2-related factor 2 (NRF2)-aryl hydrocarbon receptor (AHR) pathway. Simultaneously, alcohol-induced ballooning of HEPs creates pseudo-synaptic clefts between HEPs and KCs to facilitate the interaction of glutamate and mGluR5. After 2-week ethanol feeding, binge drinking rapidly altered the intracellular calcium levels to trigger the exocytosis of VGLUT3$^+$ GVs, and the released glutamate stimulated reactive oxygen species (ROS) production in KCs through mGluR5 activation. Moreover, I found that mGluR5 activation induced NOX2-mediated ROS generation through protein kinase C (PKC) in KCs. Accordingly, the pharmacologic inhibitions of hepatic GV formation or PKC in KCs remarkably reduced alcohol-induced liver injury. KC-specific mGluR5 or NADPH oxidase 2 deficient mice significantly attenuated inflammatory liver injury. In human samples, I consistently demonstrated that glutamate released from vesicles has a positive correlation with the inflammatory markers in ASH patients without liver cirrhosis. In this study, I firstly demonstrated that the alcohol-induced ‘pseudo-synaptic cleft’ between HEPs and KCs mediates the development of alcohol-associated liver disease (ALD) via vesicular glutamate signaling, and provides the novel molecular targets for the ALD treatment.