Emergence of highly profibrotic and proinflammatory Lrat+ FbIn2+ HSC subpopulation in alcoholic hepatitis

Background and Aims: Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [FbIn2]) markers and vitamin A (VitA) storage. Approach and Results: Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl4, and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FAGS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-FbIn2- activated HSCs were VitA-depleted (vs. similar to 13% in BDL and CCl4). Supervised clustering identified a subset coexpressing Lrat and FbIn2 (Lrat+FbIn2+). which expanded 44-fold. 17-fold. and 1.3-fold in AH, BDL, and CCl4. Lrat+FbIn2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+FbIn2-cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2). antimicrobial (II-33. Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl4. Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH. Conclusion: A highly profibrotic, inflammatory, and immunoregulatory Lrat+FbIn2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.

Automated summary

This study identified subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in alcoholic hepatitis (AH) and found that a highly profibrotic, inflammatory, and immunoregulatory Lrat+FbIn2+ subpopulation may contribute to the development of AH.