Hepatic transcriptome profiles of ethanol-treated mice with HMGB1 knockout in hepatocytes and/or macrophages

Background: We previously identified that high-mobility group box-1 (HMGB1) is increased and undergoes post-translational modifications (PTMs) in response to alcohol consumption. Here we hypothesized that specific PTMs, occurring mostly in hepatocytes and myeloid cells, could contribute to the pathogenesis of alcohol-associated liver disease (AALD). Methods: We used the Lieber-DeCarli (LDC) model of early alcohol-induced liver injury, combined with engineered viral vectors and genetic approaches to regulate the expression of HMGB1, its PTMs (reduced [H], oxidized [O], acetylated [Ac], both [O+Ac]), and its receptors (RAGE, TLR4) in a cell-specific manner (hepatocytes and/or myeloid cells). Results: Hmgb1 ablation in hepatocytes or myeloid cells partially protected, while ablation in both prevented steatosis, inflammation, IL1B production, and alcohol-induced liver injury. Hepatocytes were a major source of [H], [O], and [Ac] HMGB1, whereas myeloid cells produced only [H] and [Ac] HMGB1. Neutralization of HMGB1 prevented, whereas injection of [H] HMGB1 increased AALD, which was worsened by injection of [O] HMGB1. While [O] HMGB1 induced liver injury, [Ac] HMGB1 protected and counteracted the effects of [O] HMGB1 in AALD. [O] HMGB1 stimulated macrophage (MF) migration, activation, IL1B production and secretion. Ethanol-fed RageΔMye but not Tlr4ΔMye, RageΔHep, or Tlr4ΔHep mice were protected from AALD, indicating a crucial role of RAGE in myeloid cells for AALD. [O] HMGB1 recruited and activated myeloid cells through RAGE and contributed to steatosis, inflammation, and IL1B production in AALD. Conclusion: These results provide evidence for targeting [O] HMGB1 of hepatocyte origin as a ligand for RAGE signaling in myeloid cells and a driver of steatosis, inflammatory cell infiltration, and IL1B production in AALD. Importantly, we reveal that [Ac] HMGB1 offsets the noxious effects of [O] HMGB1 in AALD. The Lieber-DeCarli (LDC) model was used to provoke early alcohol-associated liver disease (AALD) in mice. An equal number of 10-week-old male and female mice were randomly assigned to either the control or the ethanol group, with 8 mice in each group. The control and ethanol LDC diets (Bio-Serv Inc., Frenchtown, NJ) are equicaloric and have the same composition of fat (42% of calories) and protein (16% of calories). The content of carbohydrates is 42% of total calories (dextrin-maltose) in the control diet and 12% of total calories in the ethanol diet, where up to 30% of carbohydrate calories are replaced by ethanol. To determine the contribution of the cellular source of HMGB1 and the involvement of RAGE and/or TLR4 signaling in AALD, equal number of 10-weeks-old male and female Hmgb1ΔHep, Hmgb1ΔMye, Hmgb1ΔHepΔMye, RageΔHep, RageΔMye, Tlr4ΔHep, Tlr4ΔMye, Hmgb1&RageΔHepΔMye and their corresponding control littermates were subjected to the LDC model of AALD. Upon acclimatization by feeding control LDC diet for three days, mice were fed the ethanol LDC diet with a progressive increase in the percentage of ethanol-derived calories from 10% (1 week) to 20% (1 week), 25% (2 weeks) and 30% (2 weeks). Mice pair-fed with similar calories derived from maltose-dextrin served as control. To further validate the role of HMGB1 in AALD, a separate cohort of WT mice and RageΔMye mice was injected i.p., a pan-HMGB1 neutralizing Ab (ST326052233, Shino-Test, Kanagawa, Japan) at a dose of 0.3 µg/g, [H] HMGB1 (230609, HMGBiotech) or [O] HMGB1 (230817, HMGiotech) at a dose of 0-0.1 µg/g throughout the ethanol feeding protocol.