Levels of hepatic Th17 cells and regulatory T cells upregulated by hepatic stellate cells in advanced HBV-related liver fibrosis

Abstract

Background: Liver fibrosis which mainly occurs upon chronic hepatitis virus infection potentially leads to portal hypertension, hepatic failure and hepatocellular carcinoma. However, the immune status of Th17 and Treg cells in liver fibrosis is controversial and the exact mechanisms remain largely elusive.

Methods: Liver tissues and peripheral blood were obtained simultaneously from 32 hepatitis B virus infected patients undergoing surgery for hepatocellular carcinoma at the medical center of Sun Yat-sen University. Liver tissues at least 3 cm away from the tumor site were used for the analyses. Levels of Th17 cells and regulatory T cells were detected by flow cytometry analysis and immunohistochemistry. In vitro experiment, we adopted magnetic cell sorting to investigate how hepatic stellate cells regulate the levels of Th17 cells and regulatory T cells.

Results: We found that hepatic Th17 cells and regulatory T cells were increased in patients with advanced stage HBV-related liver fibrosis. Hepatic stellate cells upregulated the levels of Th17 cells and regulatory T cells via PGE2/EP2 and EP4 pathway.

Conclusions: We found that the increased levels of Th17 cells and regulatory T cells were upregulated by hepatic stellate cells. These results may provide insight into the role of hepatic stellate cells and Th17 cells and regulatory T cells in the persistence of fibrosis and into the occurrence of hepatocellular carcinoma following cirrhosis.

Keywords: Hepatic stellate cells; Liver fibrosis, HCC; Regulatory T cells; Th17 cells.

Fig. 1

The levels of Th17 cells and Tregs in patients with different stages of HBV-LF. a The different stages of fibrosis were assessed by Sirius Red staining of liver tissues. The following scores were assigned to the different stages of fibrosis by the Laennec system: F1 portal fibrosis without septa, F2 portal fibrosis with rare septa, F3 numerous septa with bridging fibrosis without cirrhosis, and F4 cirrhosis. Patients with F1 through 2 were classified as Group 1 and patients with F3 or 4 were classified as Group 2. b CD4+ T cells gating strategy. Lymphocytes were derived from total live PBMCs/hepatic mononuclear cells gated by forward and side scatter. CD4+ T cells were defined by double positive of CD3 and CD4. c, e Flow cytometry analysis of the percentages of Th17 cells (c) and Tregs (e) in freshly isolated CD4+ T cells from peripheral blood and tissues. The values in the quadrants represent the percentage of Th17 cells and Tregs. The data shown are representative dot plots of at least 10 individuals from more than three independent experiments. d, f Comparision of the percentages of Th17 cells and Tregs between two groups. The percentages of both Th17 cells (d) and Tregs (f) increased significantly in liver tissues but not in peripheral blood in Group 2 (black filled profiles) compared with Group 1 (open profiles). e, f Liver tissues from different stages of liver fibrosis were immunostained with antibodies against IL-17 and Foxp3 in representative samples. The numbers of IL-17+ cells (g) and Foxp3+ cells (h) were significantly higher in Group 2than in Group 1. Positive cells are highlighted by black arrow. One of 10 representative micrographs is shown. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Supernatants from HSC increased the percentages of Th17 cells and Tregs. a Phenotypes of primary HSC extracted from HBV-related fibrotic liver tissues in Group 2. Sections were immunostained with desmin, FAP, FSP, vimentin, fibronectin and a-SMA antibodies. One of 20 representative micrographs is shown. b, d Purified CD4+ T cells were cultured alone (Blank) or with 30% indicated supernatants. The values in the quadrants represent the percentages of Th17 cells and Tregs. The data shown are representative dot plots from more than three independent experiments. c, e The statistical analysis of the effect of LX-2 and pHSC supernatant on the percentages of Th17 cells (c) and Tregs (e). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

HSC increased the levels of Th17 cells and Tregs via the PGE2/EP2 and EP4 pathway. a, e Purified CD4+ T cells were cultured alone or with 30% indicated supernatants with or without NS398 or with 0.1 µM PGE2, Butaprost or Misoprostol. After 5 days, the percentage of Th17 cells and Tregs was analysed by flow cytometry. The values in the quadrants represent the percentages of Th17 cells and Tregs. The data shown are representative dot plots from more than three independent experiments. b, f The statistical analysis of the percentages of Th17 cells and Tregs in cultured purified CD4+ T cells with LX-2 supernatant or pretreated with NS398. c, g The statistical analysis of the percentages of Th17 cells and Tregs in cultured purified CD4+ T cells with pHSC supernatant or pretreated with NS398. d, h The statistical analysis of the percentages of Th17 cells and Tregs in cultured purified CD4+ T cells with PGE2, Butaprost or Misoprostol. Supernatant from LX-2 and pHSC significantly increased the percentage of Th17 cells and Tregs, and when with pretreated with NS398, the percentages of Th17 cells and Tregs declined significantly. PGE2, and its receptor agonists Butaprost and Misoprostol increased significantly the percentages of Th17 cells and Tregs. (NS398 indicates LX-2 supernatant pretreated with NS398; NS398# indicates pHSC supernatant pretreated with NS398). *p < 0.05, **p < 0.01, ***p < 0.001