Non-parenchymalcells drive the key features of alcohol associated hepatitis (AAH) in a fully humanized mouse liver - Summary Alcohol related liver disease (ALD) and in particular alcohol associated hepatitis (AAH) is a leading cause of liver related deaths worldwide1. AAH involves metabolic alterations in hepatocytes and complex hepatocytes-stromal cell interactions. However, effective treatment options for ALD are very limited due to the lack of suitable in vivo models that recapitulate the full spectrum of ALD. Possible reason for that could be that the humans and rodents liver cells are significantly different in terms of hepatic lipoprotein2, bile acid 3 and in alcohol metabolism rates4. In human AAH, there is severe steatosis, hepatocyte apoptosis, Mallory-Denk hyaline inclusions, cholestasis and peripheral lymphopenia. On the other hand, mouse models can't develop cholestasis with pure alcohol diets and for high degree of steatosis long term of alcohol feeding is required 5. We have already developed a humanized murine system in which mice have been humanized at key loci by knock-in of five human genes (MISTRG6 mouse) and have been further humanized at a cellular level by engraftment of human adult hepatocytes and human CD34+ cord blood (CB) cells. These mice can support human hepatocytes, as well as human immune, endothelial and stellate cell populations (so called human Non-Parenchymal cells, hNPCs) derived from human CD34+ CB cells. Using these mice, we developed an alcoholic model after 10days of alcohol feeding plus one binge of ethanol capturing key features of human disease pathology (severe steatosis, ballooning, Mallory-Denk inclusions, hepatocyte apoptosis, inflammation, mild fibrosis, cholestasis, peripheral lymphopenia). Using this model, we found that when human hepatocytes are engrafted alone and thereby are surrounded only by mouse NPCs disease features in human hepatocytes (steatosis, Mallory denk-bodies, cholestasis) are significantly blocked upon alcohol. These results indicate a species-specific paracrine regulation of hepatocyte alcohol/lipid metabolism and possibly bile acid synthesis/transport that is related to cholestasis. By employing bulk and single-cell RNA sequencing we found NPC ligands-hepatocyte receptors that may be responsible for this phenotype. Our aims are to examine the role of these ligand-receptors interaction in vitro and in vivo by gain and loss of function approaches. In the Aim1 we will examine NPC derived WNT4, WNT10B with hepatocyte FZD6 responsible for the defects in antioxidant defense and bile acid synthesis/transport in hepatocytes. In the Aim2 we will examine NPC derived WNT2, WNT5A with hepatocyte FZD5 responsible for alcohol induced steatosis and mitochondrial dysfunction through alteration in liver cholesterol metabolic fate. In the Aim3 we will examine the role of monocyte/macrophage subsets and their effect on hepatocyte steatosis, cholestasis as well as the role of the monocyte derived Sema3c on hepatocyte Nrp1 for alcohol related steatosis. Our study may reveal important regulators of steatosis and cholestasis in AAH and thereby is in accordance the mission and the scope of NIAAA.
