Introduction: Acute-on-chronic liver failure (ACLF) is an acutely decompensated cirrhosis syndrome with high short-term mortality. Chronic hepatitis B virus (HBV)-related ACLF accounts for >80% of all cases in China, which places a tremendous burden on the health care system. Lymphatic system plays an important role in chronic liver diseases, such as cirrhosis, liver transplantation and hepatocellular carcinoma. However, very little is known about the relationship between the lymphatic system and ACLF. Here, we explored the role of hepatic lymphatic endothelial cells (LyECs) and lymphatic vessels (LVs) in ACLF with the help of single-cell RNA-sequencing (scRNA-seq) technology.

Methods: This study involved 25 human liver samples: 5 from healthy controls (HCs), 10 from cirrhosis patients, and 10 from ACLF patients. Liver damage and inflammation were assessed by H&E staining and plasma markers. After isolating hepatic non-parenchymal cells (NPCs), we analyzed subpopulations of NPCs by scRNA-seq, including KEGG pathway analysis and Ligand/receptor analysis. Intrahepatic LVs were evaluated by immunohistochemical staining. Tube formation assay was done to assess LyECs functions.

Results: ACLF exhibited more severe injury and inflammation to the liver than cirrhosis, as indicated by significant increases in plasma levels of alanine/aspartate aminotransferases and total bilirubin. Compared with cirrhosis cases, the number of intrahepatic LVs was decreased significantly in ACLF patients. scRNA-seq revealed many monocyte/macrophages infiltrating into the liver of ACLF cases. Meanwhile, scRNA-seq revealed a group of apoptotic and dysfunctional LyECs, which were the result of secreted phosphoprotein 1 (SPP1) released from infiltrating monocyte/macrophages. In vitro, SPP1 increased the proportion of dead LyECs significantly and impaired the ability of tube formation of LyECs in a dose- and time-dependent manner.

Conclusions: ACLF is associated with less LVs and LyECs dysfunction at least in part mediated by SPP1 released from infiltrating monocyte/macrophages. Hepatic LVs and LyECs can be a novel therapeutic strategy for ACLF.