Alcohol-associated liver disease (ALD) is a major cause of alcohol related mortality. The specific mechanisms responsible for ALD development and progression are not fully understood, and there is limited therapy for any stage of ALD. Recent advances in single cell sequencing revealed the complex nature of the disease, particularly the importance of cell-cell communication. Using single cell sequencing and co-culture experiments we found that alcohol promotes pathological changes in cell-cell communication in the liver. These changes are in turn mediated by alcohol-related epigenetic changes in hepatocytes, induced by histone modification enzymes called KDM5B and KDM5C.

Preliminary data suggest that KDM5 demethylases are involved in both ALD development and resolution. During disease development KDM5 demethylases in hepatocytes promote pro-inflammatory and pro-fibrotic signaling in non-parenchymal cells (NPC). During disease resolution KDM5 demethylases control hepatocyte-NPC crosstalk that suppresses pro-resolving macrophages and contributes to poor fibrosis resolution.

Our data suggest that these diverse functions of KDM5 demethylases are mediated by set of transcription factors that control KDM5 expression or/and activity (chromatin binding) in hepatocytes. Some of these factors, such as C/EBPβ, STAT5B and Androgen receptor (AR) are sex-specific, and are involved in sex-specific alcohol response, while others such as LXR are sex-independent.

We plan to determine the role of KDM5 demethylases in the alcohol response and identify the primary targets in males and females. The project will set the stage for further studies to understand the role of cellular communication in liver disease progression and explore the potential targets for ALD therapy.

ACLF

Acute-on-chronic liver failure (ACLF) is a clinical syndrome in which there is rapid development of liver and multi-organ system failure in a patient with previously stable cirrhosis.

Recently we identified that the key mechanism responsible for loss of liver function is linked to endothelial cell dysregulation and HGF-C/EBPβ signaling in hepatocytes. C/EBPβ is a transcription factor that regulates the expression of genes involved in the acute phase response, metabolic functions, and cell cycle progression.  C/EBPβ is transiently activated during liver regeneration. However, sustained activation is harmful because it may prevent re-differentiation. C/EBPβ is activated in ACLF mice and in human ACLF samples. Activated C/EBPβ promoted sustained suppression of metabolic and hepatocyte differentiation genes. We plan to study C/EBPβ-mediated loss of liver function in ACLF using mouse models and human liver transplant explants; and find the link between endothelial dysfunction and C/EBPβ activation.

The results of this project will define if C/EBPβ promotes liver failure in ACLF and will set stage for future studies defining C/EBPβ function and ultimately identifying new therapeutic targets of sepsis associated ACLF.

Retinoic acid signaling in fibrosis resolution after alcohol cessation

We found that retinoic acid metabolism strongly associated with improved fibrosis resolution and retinol metabolites were enriched in liver niches characterized by improved ALD resolution in a mouse model. In agreement with this finding, mice treated after alcohol cessation with the RAR agonist AM580 showed improved fibrosis resolution. Spatial transcriptomic analysis of agonist treated mice and controls suggested that RAR activation dramatically altered liver niches, such as a progenitor-enriched niche and an inflammation-associated niche, in part through macrophage phenotype shifts. We confirmed that RAR activation in macrophages resulted in an accumulation of Marco high Cd163 high Vsig4 high macrophages that showed increased fibrolytic activity in vitro and in vivo. We previously showed that Serum Amyloid A (SAA), a known retinol transporter, plays an important role in generation of fibrolytic niches. RAR inhibition prevented the SAA mediated macrophage phenotype shift, suggesting that an SAA-RAR axis controls macrophage-mediated niche remodeling required for fibrosis resolution in ALD.

Taken together, RAR signaling is necessary and sufficient for improved fibrosis resolution after alcohol cessation. Targeting RAR may be beneficial for disease resolution in patients with ALD.

Acute phase response signaling in ALD resolution

Abstinence is beneficial for patients with alcohol-associated liver disease (ALD), but disease resolution after alcohol cessation occurs slowly and only in a subset of patients. We aimed to study the mechanisms of ALD resolution using spatial transcriptomics.

Using a mouse model of ALD after alcohol cessation we performed spatial transcriptomics and identified a discrete multicellular fibrogenic and fibrolytic niches. Fibrolytic niches contained a unique subpopulation of hepatocytes that express Serum amyloid A (SAA).  SAA expression correlated with fibrolytic genes in mice after alcohol cessation and in human liver samples. In vitro analysis confirmed that Saa1/2high hepatocytes induced matrix metalloproteinase and lysosomal enzyme (Ctsd, Psap) gene expression in liver macrophages in an SAA and FPR2-dependent way. In vivo recombinant SAA or SAA-enriched HDL promoted fibrosis resolution after alcohol cessation in mice.

Taken together, acute phase response activation after alcohol cessation triggers intrahepatic cell-cell communication changes for efficient fibrosis resolution.