Southern California Research Center for ALPD and Cirrhosis, Department of Pathology , Keck School of Medicine of the University of Southern California , Los Angeles , California , USA.
University of Michigan , Ann Arbor , Michigan , USA.
Hepatology. 2023 Jul 1;78(1):212-224. doi: 10.1002/hep.32793. Epub 2022 Oct 25.
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 [Fbln2]) markers and vitamin A (VitA) storage.
Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- 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 (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl 4 . 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.
A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.
尚不清楚肝干细胞（HSCs）和门脉纤维母细胞在酒精性肝炎（AH）中的相对作用。我们旨在通过单细胞 RNA 测序（scRNA-seq）鉴定小鼠 AH 模型中胶原 1 型α 1（Col1a1）表达细胞的亚群，并通过 HSC（卵磷脂视黄醇酰基转移酶[Lrat]）和门脉纤维母细胞（Thy-1 细胞表面抗原[Thy1]和纤维蛋白 2 [Fbln2]）标志物和维生素 A（VitA）储存来筛选细胞。
Col1a1-green 荧光蛋白（GFP）小鼠接受 AH、CCl4 和胆管结扎（BDL）程序，以获得可比的 F1-F2 肝纤维化。通过 VitA 自发荧光和 GFP 通过 FACS 对 Col1a1 表达细胞进行分选，用于单细胞 RNA 测序。在 AH 中，大约 80%的 Lrat+Thy1-Fbln2-激活 HSCs 耗尽了 VitA（与 BDL 和 CCl4 中的~13%相比）。监督聚类鉴定出一个共同表达 Lrat 和 Fbln2 的亚群（Lrat+Fbln2+），该亚群在 AH、BDL 和 CCl4 中分别扩增了 44 倍、17 倍和 1.3 倍。与 Lrat+Fbln2-细胞相比，Lrat+Fbln2+细胞的促纤维化、肌成纤维细胞和免疫调节基因诱导 3-15 倍，但 HSC 选择性基因诱导 2-4 倍。与 BDL 和 CCl4 相比，AH 激活的 HSCs 上调了炎症（趋化因子[C-X-C 基序]配体 2 [Cxcl2]、趋化因子[C-C 基序]配体 2）、抗菌（IL-33、Zc3h12a）和抗原呈递（H2-Q6、H2-T23）基因。计算 AH 与正常人类批量肝 RNA-seq 数据的去卷积支持 AH 中 LRAT+FBLN2+细胞的扩增；AH 患者肝免疫组织化学显示富含 LRAT+细胞的纤维性隔室中存在 FBLN2 染色；原位杂交证实了 FBLN2 与 CXCL2 和/或人类白细胞抗原 E 在患者 AH 中的共表达。最后，在 Lrat-Cre；Rosa26mTmG 小鼠中追踪 HSC 检测到 GFP+FBLN2+细胞在 AH 中。
AH 中 HSCs 中出现了一种高度促纤维化、炎症和免疫调节的 Lrat+Fbln2+亚群，可能有助于 AH 的炎症和免疫反应性质。
