Fig 1: scRNA-seq revealed that a large number of monocyte/macrophages infiltrated into ACLF liver. (A) The workflow of scRNA-seq analyses. NPCs isolated from 5 ACLF livers, 3 cirrhosis livers, and 2 HC livers were included in this study. (B) 26,200 cells from HC (n = 2), cirrhosis (n = 3), and ACLF (n = 5) human livers were divided into 20 clusters (0–19), shown by tSNE plotting. (C) The representative marker genes of 20 subpopulations are presented with a heat map. Each column represents a different cell cluster and is arranged in the order of 0–19. (D) The classical marker genes of each cluster: CD14 and CD68 for monocyte/macrophages, CD3D for T cells, CD79A for B cells, NKG7 and KLRD1 for NK cells and T cells, ALB and VWF for epithelial tissue cells (including epithelial cell and endothelial cells), CXCR2 for neutrophils, WDFY4 for dendritic cells, COL3A1 for fibroblasts. (E) We re-annotated the 20 subpopulations according to their marker genes, and 11 classical clusters were annotated to facilitate the next analyses. (F) Cell immunofluorescence of CD14 and CD68 in isolated monocyte/macrophages from ACLF liver (200× field). (G) The proportion of 11 classical clusters among HCs, cirrhosis patients, and ACLF patients. (H) IHC staining for monocyte/macrophages (CD14) in the liver of HCs, cirrhosis patients, and ACLF patients (100× field). (** p < 0.01; bar = 200 μm).
Fig 2: SPP1 secreted by infiltrating monocyte/macrophages induced LyEC death and impaired the ability of tube formation of LyECs in vitro. (A) Cell immunofluorescence of CD14 and SPP1 in isolated monocyte/macrophages from ACLF liver (100× and 200× field). (B) Representative IHC images of SPP1 in liver tissues among HCs, cirrhosis patients, and ACLF patients (100× and 200× field). (C) Flow cytometry showed a higher proportion of 7-AAD-positive LyECs after SPP1 treatment (200 ng/mL and 1000 ng/mL). (D) The tube formation assays of LyECs for CNTL and SPP1 treatment (200 ng/mL and 1000 ng/mL) (100× field). (* p < 0.05, ** p < 0.01; bar = 200 μm).
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