Single-cell RNA sequencing Reveals Dysfunction of Liver Lymphatic Endothelial Cells in Hepatitis B-Related Acute-on-Chronic Liver Failure

Aims: Acute-on-chronic liver failure (ACLF) is an acutely decompensated cirrhosis syndrome with high short-term mortality rate. Very little is known about the relationship between the lymphatic system and ACLF. Here, we explored the role of liver lymphatic endothelial cells (LyECs) and lymphatic vessels in ACLF with the help of scRNA-seq technology.Methods: The present study enrolled 25 human liver samples including 5 health control (HC), 10 cirrhosis and 10 ACLF liver samples. Liver damage and inflammation were assessed by H&E staining and plasma markers. After isolating liver non-parenchymal cells (NPCs), we analyzed subpopulations of NPCs by scRNA-seq, including KEGG pathway and Ligand/receptor analysis. Intrahepatic lymphatics were evaluated by IHC and tube formation assay was done to assess the function of LyECs.Results: The present study found that ACLF exhibited more severe liver damage and inflammation than cirrhosis patients, as indicated by significant increases in plasma alanine/aspartate aminotransferase and total bilirubin. scRNA-seq analysis identified that a large of immune cells infiltrated into ACLF liver and were mainly monocytes. We also found thatintrahepatic lymphatic vessels of cirrhosis patients were significantly increased compared with HC patients, however, lymphatic vessels were decreased in ACLF patients. Interestingly, the expression level of lymphangiogenic factor (VEGF-C) was still high in ACLF, which indicated dysfunction or death of LyECs occurred in ACLF liver. With the help of scRNA-seq, we discovered a group of stressed and dysfunctional LyECs, which were result from SPP1 secreted by infiltrated monocytes. Furthermore, we also verified that SPP1 significantly increased the proportion of dead LyECs and damaged the tube formation ability of LyECs in a dose- and time-dependent manner.Conclusions: To our knowledge, this is the first time to illustrated stressed and dysfunctional LyECs in ACLF liver, which deepened our knowledge of the pathophysiological mechanism of ACLF at a cell-specific level and provided a tremendously useful basis for the development of effective and novel therapeutic strategies for ACLF.