Bacterial translocation aggravates CCl4-induced liver cirrhosis by regulating CD4+ T cells in rats

Abstract

Bacterial translocation (BT) is thought to play an important role in the development of liver cirrhosis, but the mechanisms have not been fully explored. This study aims to investigate the distribution of Treg (CD3⁺CD4⁺CD25⁺Foxp3⁺), Th17 (CD3⁺CD4⁺IL-17⁺), and Th1 (CD3⁺CD4⁺IFN-γ⁺) cells in the intestinal lamina propria, liver and blood and to explore their relationships with BT. Cirrhotic rats with ascites were induced by CCl₄. We found that there were lower levels of total protein and albumin, lower albumin/globulin ratio, lower body weight and higher spleen weight and ascites volume in cirrhotic rats with than without BT. We found that BT may cause increase of Treg cells in the proximal small intestine and decrease of Th17 cells in the whole intestine and blood in cirrhotic rats. It may also aggravate the CCl₄-induced decrease in Th1 cells in the whole intestine, liver, caecum, and blood and the CCl₄-induced increase in Th17 cells in the liver and Tregs in the distal small intestine, colon, and liver. Our data suggest that BT may aggravate liver injury and decrease liver function via an interaction with CD4⁺ T Cells. The results of this study may be helpful for the development of new treatments for liver cirrhosis.

Figure 1. CCl₄-induced cirrhosis increases the incidence of BT in rats.

(a) Results of representative culture experiments using MLNs in cirrhotic rats with or without BT. (b) Correlations between the numbers of colonies isolated from the MLNs and plasma LBP levels were determined using Spearman’s rank test. (c) Plasma LBP concentrations in cirrhotic rats with and without BT and normal rats. (d) Plasma LBP concentration in antibiotic- or placebo-treated cirrhotic rats and normal rats. (e–g) A separate experiment was performed in which cirrhotic rats with ascites were administered 10⁸ RFP-tagged E. coli via gavage. Six hours later, RFP-marked E. coli were observed along the intestinal tract (e), in MLNs and the liver (f), and in the mesentery (g).

Figure 2. BT is associated with liver damage.

Correlations between plasma LBP concentrations and albumin concentration and the albumin/globulin ratio (a), AST, TBil, and DBil levels (b), and spleen weight, body weight and the amount of ascites (c) in cirrhotic rats with BT. Spearman’s rank test was used.

Figure 3. The percentages of Treg, Th17, and Th1 cells in cirrhotic rats without and with BT.

Cells were isolated from the intestinal lamina propria, liver and blood. Cells were gated using the live lymphocyte population. The percentages of Treg (CD3⁺CD4⁺CD25⁺Foxp3⁺), Th17 (CD3⁺CD4⁺IL-17⁺), and Th1 (CD3⁺CD4⁺IFN-γ⁺) cells in the CD4⁺ T cell population were determined in the proximal and distal small intestine, caecum, colon, liver, and blood in the control group (n = 12), the liver cirrhosis without BT group (n = 12), and the liver cirrhosis with BT group (n = 11) using flow cytometry. The data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.

Figure 4. The percentages of Treg, Th17, and Th1 cells in antibiotic- and placebo-treated cirrhotic and normal rats.

Cells were isolated from the intestinal lamina propria, liver and blood. Cells were gated using the live lymphocyte population. The percentages of Treg (CD3⁺CD4⁺CD25⁺Foxp3⁺), Th17 (CD3⁺CD4⁺IL-17⁺), and Th1 (CD3⁺CD4⁺IFN-γ⁺) cells in the CD4⁺ T cell population were determined in the proximal and distal small intestine, caecum, colon, liver, and blood of the antibiotic-treated controls (n = 8), the placebo-treated controls (n = 8), the antibiotic-treated liver cirrhosis group (n = 10), and the placebo-treated liver cirrhosis group (n = 10) using flow cytometry. The data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired Student’s t test with Bonferroni’s correction was used.

Figure 5. BT is associated with changes in the cellular immunity response.

(a) Correlations between the concentration of LBP and the percentage of Treg cells in the proximal small intestine, distal small intestine, and liver. (b) The correlation between the concentration of LBP and the percentage of Th17 cells in the proximal small intestine, distal small intestine, and caecum. (c) The correlation between the concentration of LBP and the percentage of Th1 cells in the proximal small intestine, distal small intestine, and liver. Spearman’s rank test was used.