Transplanted mouse liver stem cells at different stages of differentiation ameliorate concanavalin A-induced acute liver injury by modulating Tregs and Th17 cells in mice

Abstract

Acute liver failure (ALF) is a disease with a considerably high mortality rate that still lacks a safe and effective treatment. Transplantation of liver stem cells (LSCs) has been considered to be a promising therapeutic alternative for ALF since LSCs have been shown to be involved in immunomodulation and functional reconstruction of the liver. Our present study evaluated and compared the protective effects of the two mouse LSC lines, YE and R5, as well as those of adult mouse hepatocyte (HC), on concanavalin A (ConA)-induced acute liver injury. YE and R5 cells were analyzed by microscopy, functional assays, and gene expression. We confirmed that YE and R5 cells were undifferentiated cells that had partial hepatocytic functions and a potential to differentiate into hepatocytes. YE cells has characteristics of LSCs at the early stage of differentiation, whereas the differentiation stage of R5 cells was later than that of YE cells. Subsequently, YE, R5, and HC cells were intraperitoneally transplanted into three groups of mice, followed by injection of ConA through the tail vein of each mouse at 12 h later. Blood tests, histology, flow cytometry, and quantitative PCR were then used to evaluate the therapeutic effects of the cell transplantations at 24 h after ConA injections. Compared with that of the ConA control group, YE, R5, and HC cells reduced the expression of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and alleviated the degree of hepatic necrosis. Moreover, transplantation of these cells induced more regulatory T cells (Tregs) and less T-helper 17 (Th17) cells in the liver and spleen, and also promoted the expression of forkhead box protein 3 (Foxp3) and interleukin (IL)-10; in contrast, these transplantations induced various degrees of inhibition in the expression of retinoic acid-related orphan receptor γt (RORγt), IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α). The protective effects of YE and R5 cells were significantly stronger than those of HC cells, and YE cells at the earlier differentiation stage than that of R5 cells exhibited the strongest protective effects. These results demonstrate that mouse LSCs at different stages of differentiation alleviate ConA-induced acute liver injury in mice by modulating Tregs, Th17 cells, and cytokine secretion.

Keywords: Acute liver failure; Th17 cells; Tregs; cell transplantation; different stages of differentiation; mouse liver stem cells.

Figure 1

Characteristics of cellular morphology of YE, R5, and HC cells. Functional analysis and expressions of marker genes in YE and R5 cells. Representative images under light microscopy (original magnification ×200) of YE cells (A), R5 cells (B), and mature HC cells (C); Representative TEM images of a YE cell (D), R5 cell (E), and HC (F); ICG uptake assay showing pale-green ICG-positive YE cells (G) and R5 cells (H) (as indicated by the yellow arrows). PAS staining of YE cells (I) and R5 cells (J) showing positive cells with cytoplasm appeared red (as indicated by the yellow arrows). (K) RT-PCR showing that YE and R5 cells expressed both hepatocyte marker genes-ALB, CK8, and CK18-and the biliary epithelial cell marker gene, CK19.

Figure 2

Both YE and R5 cells synthesized urea and ALB, but their functions of synthesizing urea and ALB were weaker than those of HC cells. Synthesis levels of urea (A) and albumin (ALB) (B) in the culture supernatants of YE, R5, and HC cells were detected by an automatic analyzer when cultured for 24 h, 36 h, and 48 h. Data are presented as the mean ± SEM (n = 3; *P < 0.05, **P < 0.01).

Figure 3

YE, R5, and HC cells ameliorated the liver functions in ConA-induced ALF mice. The protective effects of YE and R5 cells were significantly stronger than those of the HC cells, and YE cells had the best protective effect among the three cells. Some liver functional parameters including ATL (A), AST (B), and TBIL (C) were determined in Normal, ConA, YE, R5, and HC group mice. Data are presented as the mean ± SEM (n = 8; *P < 0.05, **P < 0.01).

Figure 4

YE, R5, and HC cells ameliorated the hepatic damage in ConA-induced ALF mice. The YE cells had the best protective effect among the three cells. (A) Visual observation of livers in Normal, ConA, YE, R5, and HC groups of mice. (B, C) Representative images of hematoxylin and eosin staining of liver tissues in the five groups of mice with original magnifications of ×40 (B) and ×200 (C). (D) Measurements of necrosis area. Bar chart comparing the quantification of necrosis areas in the liver tissues in the five groups of mice. Data are presented as the mean ± SEM (n = 8; *P < 0.05, **P < 0.01).

Figure 5

YE, R5, and HC cells regulated the Treg/Th17 cell balance in ConA-induced ALF mice. YE and R5 cells exhibited stronger regulation than that of HC cells, and YE cells demonstrated the strongest regulation. A. Density plots of flow cytometry results of CD4⁺/CD25⁺/Foxp3⁺ Tregs in the livers and spleens in Normal, ConA, YE, R5, and HC groups of mice. B. Bar charts comparing the percentages of Tregs in the livers and spleens. C. Density plots of flow cytometry results of CD4⁺/IL-17A⁺ Th17 cells in livers and spleens in the five groups of mice. D. Bar charts comparing the percentages of Th17 cells in the livers and spleens. Data are presented as the mean ± SEM (n = 8; *P < 0.05, **P < 0.01).

Figure 6

YE, R5, and HC cells regulated the mRNA levels of cytokines-IL-10, IL-17A, IL-17F, and TNF-α-and transcription factors-Foxp3, and RORγ-in ConA-induced ALF mice. The regulatory effects of YE and R5 cells were significantly stronger than those of HC cells, with YE cells exhibiting the most optimal regulation. Quantitative PCR (qPCR) was used to detect the mRNA levels of IL-10 (A), IL-17A (B), IL-17F (C), TNF-α (D), Foxp3 (E), and RORγt (F) in the livers of Normal, ConA, YE, R5, and HC groups of mice. Data are presented as the mean ± SEM (n = 8; *P < 0.05, **P < 0.01).