Background and Aims Mitochondrial double‐stranded RNA (mtdsRNA) and its innate immune responses have been reported previously; however, mtdsRNA generation and its effects on alcohol‐associated liver disease (ALD) remain unclear. Here, we report that hepatic mtdsRNA stimulates toll‐like receptor 3 (TLR3) in Kupffer cells through the exosome (Exo) to enhance interleukin (IL)‐17A (IL‐17A) production in ALD. Approach and Results Following binge ethanol (EtOH) drinking, IL‐17A production primarily increased in γδ T cells of wild‐type (WT) mice, whereas the production of IL‐17A was mainly facilitated by CD4+ T cells in acute‐on‐chronic EtOH consumption. These were not observed in TLR3 knockout (KO) or Kupffer cell–depleted WT mice. The expression of polynucleotide phosphorylase, an mtdsRNA‐restricting enzyme, was significantly decreased in EtOH‐exposed livers and hepatocytes of WT mice. Immunostaining revealed that mtdsRNA colocalized with the mitochondria in EtOH‐treated hepatocytes from WT mice and healthy humans. Bioanalyzer analysis revealed that small‐sized RNAs were enriched in EtOH‐treated Exos (EtOH‐Exos) rather than EtOH‐treated microvesicles in hepatocytes of WT mice and humans. Quantitative real‐time PCR and RNA sequencing analyses indicated that mRNA expression of mitochondrial genes encoded by heavy and light strands was robustly increased in EtOH‐Exos from mice and humans. After direct treatment with EtOH‐Exos, IL‐1β expression was significantly increased in WT Kupffer cells but not in TLR3 KO Kupffer cells, augmenting IL‐17A production of γδ T cells in mice and humans. Conclusions EtOH‐mediated generation of mtdsRNA contributes to TLR3 activation in Kupffer cells through exosomal delivery. Consequently, increased IL‐1β expression in Kupffer cells triggers IL‐17A production in γδ T cells at the early stage that may accelerate IL‐17A expression in CD4+ T cells in the later stage of ALD. Therefore, mtdsRNA and TLR3 may function as therapeutic targets in ALD.

Alcoholic liver disease (ALD) is one of the fastest-growing concerns worldwide. In addition to bacterial endotoxins in the portal circulation, recent lines of evidence have suggested that sterile inflammation caused by a wide range of stimuli induces alcoholic liver injury, in which damage-associated molecular patterns (DAMPs) play critical roles in inducing de novo lipogenesis and inflammation through the activation of cellular pattern recognition receptors such as Toll-like receptors in non-parenchymal cells. Interestingly, alcohol-mediated metabolic, neurological, and immune stresses stimulate the generation of DAMPs that are released not only in the liver, but also in other organs, such as adipose tissue, intestine, and bone marrow. Thus, diverse DAMPs, including retinoic acids, proteins, lipids, microRNAs, mitochondrial DNA, and mitochondrial double-stranded RNA, contribute to a broad spectrum of ALD through the production of multiple pro-inflammatory cytokines, chemokines, and ligands in non-parenchymal cells, such as Kupffer cells, hepatic stellate cells, and various immune cells. Therefore, this review summarizes recent studies on the identification and understanding of DAMPs, their receptors, and cross-talk between the liver and other organs, and highlights successful therapeutic targets and potential strategies in drug development that can be used to combat ALD.

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Chronic alcohol consumption often induces hepatic steatosis but rarely causes severe inflammation in Kupffer cells (KCs) despite the increased hepatic influx of lipopolysaccharide (LPS), suggesting the presence of a veiled tolerance mechanism. In addition to LPS, the liver is affected by several gut-derived neurotransmitters through the portal blood, but the effects of catecholamines on KCs have not been clearly explored in alcohol-associated liver disease (ALD). Hence, we investigated the regulatory roles of catecholamine on inflammatory KCs under chronic alcohol exposure. We discovered that catecholamine levels were significantly elevated in the cecum, portal blood, and liver tissues of chronic ethanol-fed mice. Increased catecholamines induced mitochondrial translocation of cytochrome P450 2E1 in perivenous hepatocytes expressing the β2-adrenergic receptor (ADRB2), leading to the enhanced production of growth differentiation factor 15 (GDF15). Subsequently, GDF15 profoundly increased ADRB2 expression in adjacent inflammatory KCs to facilitate catecholamine/ADRB2-mediated apoptosis. Single-cell RNA sequencing of KCs confirmed the elevated expression of Adrb2 and apoptotic genes after chronic ethanol intake. Genetic ablation of Adrb2 or hepatic Gdf15 robustly decreased the number of apoptotic KCs near perivenous areas, exacerbating alcohol-associated inflammation. Consistently, we found that blood and stool catecholamine levels and perivenous GDF15 expression were increased in patients with early-stage ALD along with an increase in apoptotic KCs. Our findings reveal a novel protective mechanism against ALD, in which the catecholamine/GDF15 axis plays a critical role in KC apoptosis, and identify a unique neuro-metabo-immune axis between the gut and liver that elicits hepatoprotection against alcohol-mediated pathogenic challenges.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) instigates nuclear factor-κB (NF-κB)-mediated inflammatory responses in alcohol-associated liver diseases (ALD). Here, we utilized a novel optogenetically engineered exosome technology called ‘exosomes for protein loading via optically reversible protein–protein interactions (EXPLOR)’ to efficiently deliver the super-repressor IκB-loaded exosomes (Exo-srIκB) to the liver and examined its therapeutic potential in acute-on-chronic alcohol-associated liver injury. We detected enhanced uptake of DiI-labeled Exo-srIκB by LPS-treated inflammatory KCs, which suppressed LPS-induced inflammatory gene expression levels. In animal experiments, a single intravenous injection of Exo-srIκB prior to alcohol binge drinking significantly attenuated alcohol-associated hepatic steatosis and infiltration of neutrophils and macrophages but not a liver injury. Notably, three consecutive days of Exo-srIκB injection remarkably reduced alcohol-associated liver injury, steatosis, apoptosis of hepatocytes, fibrosis-related gene expression levels in hepatic stellate cells, infiltration of neutrophils and macrophages, and inflammatory gene expression levels in hepatocytes and KCs. In particular, the above effects occurred with inhibition of nuclear translocation of NF-κB in liver tissues, and these beneficial effects of Exo-srIκB on ALD were shown regardless of doses. Our results suggest an exosome-based modulation of NF-κB activity in KCs by Exo-srIκB as a novel and efficient therapeutic approach in ALD.