Background and Aims: HSCs contribute to HCC progression by regulating multiple factors. However, the entire immunoregulatory functions of HSCs are still obscure. Here, we aim to investigate whether HSCs impose CX₃CR1⁺ macrophages to protumorigenic properties in the peritumoral area. Approach and Results: In single-cell RNA-sequencing analysis of patients with HCC, a subpopulation of macrophages specifically expressed Arg1 and Cx3cr1 in the peritumoral area and were highly enriched with retinol metabolism–related genes. Flow cytometry analysis showed significantly increased frequencies of CD14⁺CD11b⁺HLA-DR⁻ macrophages with CX₃CR1 in the HCC adjacent region where α-smooth muscle actin–expressing activated hepatic stellate cells (aHSCs) showed colocalized expression of CX₃CL1. Accordingly, in tumor-bearing mice, Cx3cl1 mRNA expression was notably increased in aHSCs within the adjacent HCC, where infiltration of CX₃CR1⁺Ly6C⁺ macrophages was mostly observed with decreased CD8⁺ T cells. In adoptive transfer and in vitro coculture of myeloid cells, we demonstrated that CX₃CR1⁺Ly6C⁺ macrophages migrated and highly expressed arginase-1 by interacting with retinoid-enriched aHSCs in the adjacent HCC. Direct treatment of retinoids or coculturing with retinol-storing mouse aHSCs or human LX-2 cells significantly increased arginase-1 expression in CX₃CR1⁺Ly6C⁺ macrophages and human blood CD14⁺ cells, leading to the suppression of CD8⁺ T-cell proliferation. Moreover, genetic deficiency of CX₃CR1 in myeloid cells or pharmacological inhibition of retinol metabolism remarkably attenuated HCC development. Conclusions: We showed that CX₃CR1⁺Ly6C⁺ macrophages migrate and interact with aHSCs in the peritumoral region where retinoids induce arginase-1 expression in CX₃CR1⁺Ly6C⁺ macrophages, subsequently depriving CD8⁺ T cells of arginine and promoting HCC.

Alcohol-related liver disease (ALD) became an important health issue worldwide. Following chronic alcohol consumption, the development of ALD might be caused by metabolic and immunologic factors, such as reactive oxygen species (ROS) and pro-inflammatory cytokines. For example, hepatic cytochrome P450 2E1 enzyme increases ROS production and stimulates de novo lipogenesis after alcohol exposure. In addition, damage- and pathogen-associated molecular patterns stimulate their specific receptors in non-parenchymal cells, including Kupffer cells, hepatic stellate cells (HSCs), and lymphocytes, which result in hepatocyte death and infiltration of pro-inflammatory cells (e.g., neutrophils and macrophages) in the liver. Moreover, our studies have suggested the novel involvement of neurologic signaling pathways (e.g., endocannabinoid and glutamate) through the metabolic synapse between hepatocytes and HSCs in the development of alcohol-related hepatic steatosis. Additionally, agouti-related protein and beta2-adrenergic receptors aggravate hepatic steatosis. Furthermore, organ-crosstalk has emerged as a critical issue in ALD. Chronic alcohol consumption induces dysbiosis and barrier disruption in the gut, leading to endotoxin leakage into the portal circulation, or lipolysis-mediated transport of triglycerides from the adipose tissue to the liver. In summary, this review addresses multiple pathogeneses of ALD, provides novel neurologic signaling pathways, and emphasizes the importance of organ-crosstalk in the development of ALD.

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.