Human liver stem cells attenuate concanavalin A-induced acute liver injury by modulating myeloid-derived suppressor cells and CD4+ T cells in mice

Abstract

Background: Acute liver failure (ALF) is a serious threat to the life of people all over the world. Finding an effective way to manage ALF is important. Human liver stem cells (HLSCs) are early undifferentiated cells that have been implicated in the regeneration and functional reconstruction of the liver. In this study, we aimed to evaluate the protective effects of the HLSC line HYX1 against concanavalin A (ConA)-induced acute liver injury.

Methods: HYX1 cells were characterized by microscopy, functional assays, gene expression, and western blot analyses. We showed that HYX1 cells can differentiate into hepatocytes. We intraperitoneally injected HYX1 cells in mice and administered ConA via caudal vein injection 3, 6, 12, 24, and 48 h later. The effects of HYX1 cell transplantation were evaluated through blood tests, histology, and flow cytometry.

Results: HYX1 cells reduced the levels of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and dramatically decreased the severity of liver injuries. Mechanistically, HYX1 cells promoted myeloid-derived suppressor cell (MDSC) migration into the spleen and liver, while reducing CD4⁺ T cell levels in both tissues. In addition, HYX1 cells suppressed the secretion of proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), but led to increased interleukin-10 (IL-10) production.

Conclusions: These results confirm the efficacy of HLSCs in the prevention of the ConA-induced acute liver injury through modulation of MDSCs and CD4⁺ T cell migration and cytokine secretion.

Keywords: Acute liver injury; CD4+ T cells; Concanavalin A; Human liver stem cells; MDSCs.

Fig. 1

Schematic outline of the experimental groups. a Initial grouping of the mice; mice were sacrificed for sample collection 24 h after ConA treatment. b In the + 1, 2, 3, 4, and 5 groups, mice were sacrificed for sample collection 3, 6, 12, 24, and 48 h, respectively, after ConA injection

Fig. 2

Characterization of HYX1 cells. a During the first 20 days in culture, the cells formed clusters and appeared round. b In subculture, the cells spread out and looked flattened and larger (original magnification × 100). c Representative TEM image of an HYX1 cell. d ICG uptake assay showed pale green ICG-positive cells (as indicated by the yellow arrows). e PAS staining of HYX1 showed positive cells with cytoplasm appeared red (as indicated by the blue arrows). f, g HYX1 cells were treated with DMSO + HGF or PBS (control group) for 5 days and western blot analysis of the indicated proteins. h RT-PCR for the indicated genes. Data are presented as mean ± SEM; compared with control group, *P < 0.05, **P < 0.01, as determined by t test

Fig. 3

Biochemical evaluation of a ALT, b AST, and c TBIL in serum from the M3+ (blue) and T3+ (red) mice. Data are presented as mean ± SEM (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001), as determined by ANOVA tests or t test; ns denotes P > 0.05

Fig. 4

Representative images of haematoxylin and eosin staining of the liver and quantitation of necrosis area in the normal, M, and T groups. a Normal group; b M1 group; c–g T1, T2, T3, T4, and T5 groups; h necrosis area of each group. Data are presented as mean ± SEM (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001), as determined by ANOVA tests; ns denotes P > 0.05. Scale bar = 100 μm

Fig. 5

Representative images of haematoxylin and eosin staining of the liver and quantitation of necrosis area in M3+ and T3+ groups. a M3+ group; b T3+ group; c necrosis area of each group. Data are presented as mean ± SEM (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001), as determined by t test; ns denotes P > 0.05. Scale bar = 100 μm

Fig. 6

Changes in MDSCs (a, b) and CD4⁺ T cell levels (c, d) in the liver (a, c) and spleen (b, d) in M3+ and T3+ groups of mice. Data are presented as mean ± SEM (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001, as determined by ANOVA tests or t test; ns denotes P > 0.05

Fig. 7

The protein levels of TNF-α, IFN-γ, and IL-10 in serum were quantified by ELISA (a–c) in the M3+ and T3+ groups of mice. The mRNA levels of TNF-α, IFN-γ, and IL-10 in the liver were quantified by qPCR (d–f) in groups of M3+ and T3+ mice. Data are presented as mean ± SEM (n = 6); *P < 0.05, **P < 0.01, as determined by ANOVA tests; ns denotes P > 0.05