Natural killer T cells play a necessary role in modulating of immune-mediated liver injury by gut microbiota

Abstract

Gut microbiota are implicated in many liver diseases. Concanavalin A (ConA)-induced hepatitis is a well-characterized murine model of fulminant immunological hepatic injury. Oral administration of pathogenic bacteria or gentamycin to the mice before ConA injection, liver injury and lymphocyte distribution in liver and intestine were assessed. Our data show that administration of pathogenic bacteria exacerbated the liver damage. There was more downregulation of activation-induced natural killer T (NKT) cells in the liver of pathogenic bacteria-treated ConA groups. Also, there was a negative correlation between the numbers of hepatic NKT cells and liver injury in our experiments. Moreover, intestinal dendritic cells (DCs) were increased in pathogenic bacteria-treated ConA groups. The activation of DCs in Peyer's patches and the liver was similar to the intestine. However, depletion of gut gram-negative bacteria alleviated ConA-induced liver injury, through suppressed hepatic NKT cells activation and DCs homing in liver and intestine. In vitro experiments revealed that DCs promoted NKT cell cytotoxicity against hepatocyte following stimulation with pathogenic bacteria. Our study suggests that increased intestinal pathogenic bacteria facilitate immune-mediated liver injury, which may be due to the activation of NKT cells that mediated by intestinal bacterial antigens activated DCs.

Figure 1. Exogenous pathogenic bacteria exacerbated ConA-induced liver injury.

Mice received gavage with Streptococcus or Salmonella (5 × 10⁸ CFU/g) followed by injection of ConA (10 mg/kg), and sacrificed 12 h later (n = 8). (a) ALT and AST levels. (b) Hepatic H&E sections and representative images, Left: 40× magnification, right: 200× magnification. (c) Systemic endotoxin concentrations. Data presented are the means ± SD. *p< 0.05, **p< 0.01, one-way ANOVA.

Figure 2. Exogenous pathogenic bacteria promoted DC augmentation and NKT cell activation.

(n = 8). (a) Liver-infiltrating leukocytes were prepared from the four groups; the percentage of DCs NKT, iNKT, NK, T, and CD44⁺T cells were determined using flow cytometry. (b) The percentage of NKT cells in liver was correlated with serum ALT level in ConA-induced liver injury. (c) Intracellular IFN-γ of hepatic NKT cells was detected by flow cytometry. (d) ELISA of TNF-α, IL-12, and IL-4 levels in hepatic tissue from the four groups. Data presented are the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA.

Figure 3. Exogenous pathogenic bacteria exacerbated intestinal tract damage.

(n = 8). (a) H&E sections and representative images of morphological structures of intestine. Left: 40× magnification, right: insets depicting the intestinal tract at 200× magnification. (b) The ratio of γδ T, DC, NKT, and NK cells in the intestine was detected by flow cytometric analysis. (c) ELISA measurement of TNF-α, IL-12, and IL-4 levels. (d) Lymphocytes were isolated from PPs. The percentages of DCs, T and CD44⁺ T cells were assessed after pathogenic bacteria with ConA administration. Data presented are the means ± SD. *P < 0.05, **P < 0.01, ***p < 0.001, one-way ANOVA.

Figure 4. Deletion of gut G^- bacteria alleviated ConA-induced liver and intestine damage.

Treated mice received oral gentamycin (1 × 10⁴ U/g) for one week followed by injection of ConA (10 mg/kg) and were sacrificed 12 h after ConA administration (n = 8). (a) The ALT, AST levels and representative hepatic H&E images (200×). (b) Systemic endotoxin levels. (c) The percentage of hepatic DCs and NKT, NK and T cells was determined by flow cytometry. (d) ELISA of TNF-α and IL-12 expression in hepatic tissue. (e) Representative intestinal H&E images (200×). (f) Intestinal γδ T, DC, NKT, and NK cell ratios were determined using flow cytometric analysis. (g) ELISA assessment of the intestinal TNF-α and IL-12 expression. (h) Lymphocytes were isolated from PPs, The percentages of DCs and T cells were assessed after gentamycin and ConA administration. Data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U-test.

Figure 5. NKT cells was critical in the pathogenic bacteria aggravate the Con A-induced liver injury.

(n = 6). (a–c) CD1d^-/- and WT were gavaged with Salmonella (5 × 10⁸ CFU/mouse) for one week prior to Con A (10 mg/kg) injection, and sacrifice 12 h later. (a) Serum ALT and AST. (b) hepatic T cells, NK cells and DCs. (c) hepatic IL-12 level were showed. (d–f) Two hours before Con A injection, wild type mice received anti-IL-12 neutralizing antibody (250 μg). (d) ALT and AST levels. (e) The percentage of hepatic NKT cells. (f)The intracellular IFN-γlevel of hepatic NKT cells. Data presented are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U-test.

Figure 6. Pathogenic bacterial antigens enhanced NKT cell cytotoxicity via DC activation in vitro.

(a) NKT cells from the liver were stimulated for 48 h in vitro. Cytotoxic assay with target TLR2 cells denoted as the PI-positive rate as analyzed by flow cytometry. The TNF-α level in the supernatant was detected by ELISA. (b) NKT cell cytotoxicity was detected after anti-TNF-α antibody neutralization, co-cultured with CD11c⁺ cells and Salmonella. (c) Schematic representation of immune cell activation in ConA-induced hepatic injury controlled by intestinal microbiota. Data presented are the means ± SD. *P < 0.05 **P < 0.01, one-way ANOVA.