"CYP1B1+PRRX1+NR4A2+ hepatic stellate cells in alcohol-associated hepatitis" - SUMMARY Hepatic stellate cells participate in liver wound healing but also in progression of liver fibrosis and promotion of liver cancer. Heterogeneous subsets of activated HSC (aHSC) are generated and assumed to have different functionalities in liver disease. Severe alcohol-associated hepatitis (AH) is manifested as acute on chronic liver failure with high mortality on the background of cirrhosis harboring heightened aHSC activity. Yet, the roles of aHSC in AH are understudied. This gap is remarkable considering known functions of aHSC in both liver inflammation and fibrosis, the two key pathologic features of AH. Our scRNA-seq analysis revealed the emergence of a novel subset of aHSC co-expressing the HSC marker Lrat and the portal fibroblast marker Fbln2 in a murine AH model. The Lrat+Fbln2+ aHSC arise from Lrat+ quiescent HSC per genetic lineage tracing and constitute the most profibrotic, myofibroblastic and inflammatory aHSC subset. Ade-convolution analysis of AH patient liver vs. healthy liver RNA-seq, supports an expansion of such subset in AH patients and IHC and FISH confirm the presence of FBLN2+ aHSC expressing inflammatory and immunoregulatory genes in AH patient livers. In a list of differentially overexpressed genes in Lrat+Fbln2+ cells via scRNA-seq, we identified Prrx1 known for myofibroblast (MFB) differentiation, the orphan nuclear receptor Nr4a2, and Cyp1b1. Supervised re-clustering by Prrx1 and Nr4a2 expression and subsequent RNA velocity and copy number variant (CNV) estimate analyses, suggest the roles of these two nuclear proteins in the clonal development of Fbln2+ aHSCs. Further, in vitro loss of function studies on FBLN2+ LX2 cells, reveal CYP181 being a driver of NR4A2 and PRRX1 expression and unique positive cross-regulations among them in the genesis and maintenance of the proinflammatory MFB phenotype of LX2 cells. Based on these results, we propose to test the hypothesis that CYP181 positively controls PRRX1 and NR4A2 which in turn positively feedback-regulate CYP181 to support a clonal development of the FBLN2+ aHSC and contribute to the AH phenotype characterized by heightened fibrosis, inflammation, and immunoregulation. To test this hypothesis, we aim to determine: 1) if and how CYP181, PRRX1 and NR4A2 cross-regulate to generate FBLN2+ aHSC; 2) the phylogenic makeup of the Lrat+Nr4a2+Prrx1+ subpopulation via simultaneous CNV and SNV analysis at single cell level; 3) if CYP181 is required for the genesis of Lrat+Fbln2+ aHSCs in vivo and contributes to the AH pathologic phenotype by using Cyp1b1'1f,-Fbln2-iCre-ERT2 mice; and 4) the translational relevance and spatial characteristics of CYP181+, PRRX1+, NR4A2+ aHSCs in non-AH AALD vs. AH patient livers and insights into their implications in a transition from the former to the latter. Through these efforts, we plan to pursue our ultimate goal of identifying a disease-causing aHSC clone in AH, defining its underlying genetic and molecular mechanisms, and designing innovative AH therapeutic approaches targeting such clone.
