Uncovering [SO?]HMGB1-Mediated Mechanisms of Alcohol-Associated Liver Disease Through Single-Nucleus Multiomics - PROJECT SUMMARY Alcohol-associated liver disease (AALD) is a major cause of chronic liver failure with rising incidence and few effective therapies. AALD progresses through defined stages - steatosis, alcoholic hepatitis, and fibrosis - driven by complex transcriptional, epigenetic, and intercellular signaling changes across hepatocytes, hepatic stellate cells (HSCs), Kupffer cells, and other immune populations. High-mobility group box-1 (HMGB1), a damage-associated molecular pattern (DAMP), has emerged as a critical modulator of liver injury. Notably, its sulfonated isoform ([SO₃]HMGB1) appears to facilitate resolution of fibrosis and inflammation, yet the underlying gene regulatory and signaling networks remain undefined. To address this gap, we propose a time-resolved, multi-omic single-nucleus approach to identify the transcriptional programs, chromatin states, and cell-cell communication pathways that govern AALD progression and recovery. We hypothesize that peak AALD injury is driven by cell-type-specific gene-regulatory networks and ligand-receptor interactions that become re-programmed during recovery, with [SO₃]HMGB1 acting as a pro-resolving cue that rewires these networks to suppress inflammation, deactivate fibrogenic stellate cells, and restore hepatocyte function. In Specific Aim 1, we will deconvolute bulk RNA-seq datasets from human AALD liver samples using human single-cell RNA-seq references to quantify cell type dynamics across disease stages. This analysis will identify which cellular populations expand or contract with disease severity and establish a human-relevant reference framework to guide murine experiments. In Specific Aim 2, we will perform single-nucleus RNA-seq and ATAC-seq on liver tissues from ethanol-fed mice at control, injury, and recovery stages with and without [SO₃]HMGB1 treatment. These data will be integrated to reconstruct GRNs, identify key transcription factors, and infer ligand-receptor signaling using NicheNet. In Specific Aim 3, we will functionally test a focused set of transcription factors and signaling interactions identified in Aim 2, prioritizing one candidate each from hepatic stellate cells and TREM2⁺/SPP1⁺ macrophages. Functional effects will be assessed using CRISPRi/a perturbation and recombinant ligand treatment in vitro, with in vivo validation using AAV-based delivery during the resolution phase of ethanol-induced liver injury. Importantly, findings from Aim 1 will inform the prioritization of molecular targets for validation by identifying those that are most perturbed in human AALD and conserved in mouse models, thereby enhancing translational relevance. By defining the molecular programs that mediate AALD progression and the pro-reparative effects of [SO₃]HMGB1, this project will uncover novel targets for therapeutic intervention and advance mechanistic understanding of AALD.
