Activated IL-23/IL-17 pathway closely correlates with increased Foxp3 expression in livers of chronic hepatitis B patients

Abstract

Background: Foxp3 protein plays a critical role in mediating the inflammatory response and can inhibit the proinflammatory IL-23/IL-17 pathway. However, the molecular interplay of Foxp3 and the IL-23/IL-17 pathway in patients with chronic hepatitis B (CHB) remains unclear. To this end, we analyzed the expression patterns of Foxp3- and IL-23/IL-17 pathway-related proinflammatory cytokines in 39 patients with acute-on-chronic liver failure, 71 patients with CHB and 32 healthy controls.

Results: Foxp3 expression was found to be elevated in and mainly expressed by the CD4+ T cell sub-population of peripheral blood mononuclear cells and liver tissues of patients with hepatitis B. The intrahepatic expression of Foxp3 strongly correlated with the copies of HBV DNA and the concentration of surface antigen, HBsAg. IL-23/IL-17 pathway-related proinflammatory cytokines were also found to be significantly increased in patients' liver tissues, as compared to healthy controls. Moreover, Foxp3 expression was strikingly correlated with the production of these cytokines in liver tissues of CHB patients.

Conclusions: The closely-correlated increase of Foxp3 and IL-23/IL-17 pathway activity in HBV-infected livers suggests that the proinflammatory IL-23/IL-17 pathway had not been effectively suppressed by the host immune machinery, such as Treg (Foxp3) cells. Constitutive activation of the IL-23/17 pathway, thus, may support the chronic hepatitis B state.

Figure 1

Foxp3 expression is significantly higher in peripheral blood of patients with CHB. PBMC from healthy controls and patients with CHB or ACLF were used to detect the expression levels of Foxp3 by qPCR (A) and flow cytometry (B and C). (A) Relative mRNA expression of Foxp3 in PBMC. (B) Pooled data indicating the percentages of Foxp3⁺ cells in total CD4⁺ T cells. (C) Representative dotplots of Foxp3⁺ cells in CD4⁺ T cells. The values in the quadrants indicate the percentage of each CD4⁺ T cell subset. Error bars indicate SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2

Foxp3 is significantly augmented in liver tissue of patients infected with HBV. Liver biopsy samples from healthy controls and patients with CHB or ACLF were used for the detection of Foxp3 mRNA by qPCR (A) and visualization of Foxp3 by immunohistochemistry (B) and laser scanning confocal microscopy (C, D). (A) Relative mRNA expression of Foxp3 in liver tissues. Error bars indicate SD; *, P < 0.05; **, P < 0.01. (B) In situ expression of Foxp3 in liver tissues. The Foxp3 expressing cells are stained brown. Representative results are shown from three independent experiments (100 × magnification). (C, D) Co-localization of Foxp3 with CD4 or CD8 in liver tissues from CHB patients. Anti- Foxp3 (red); anti- CD4 (green); anti- CD8 (green); and DAPI (blue). Representative results are shown from three independent experiments (200 × magnification). The right panels represent the enlarged portion of the indicated (white hatched box) area in the left panels.

Figure 3

Increased Foxp3 expressions is positively correlated with HBV persistence in host liver. Liver biopsy samples from 38 CHB patients were used for the detection of Foxp3 mRNA by qPCR. Sera and plasma samples from CHB patients were measured for ALT level, log10 HBsAg (IU/ml), or log10 HBV loads (copies/ml). The relationships between Foxp3 and clinical symptoms of these patients were analyzed. P < 0.05 was considered significant.

Figure 4

HBV-infected liver tissues express higher levels of IL-23/IL-17 pathway-related cytokines than non-infected tissues. Liver biopsy samples from healthy controls and CHB patients were used to detect mRNA level for IL-23, IL-17, TNF-α and IL-8 by qPCR (A), and protein expression level of Foxp3, IL-23 and IL-17 by Western-blot assay (B). Error bars indicate SD; **, P < 0.01 vs HC; ***, P < 0.001 vs HC.

Figure 5

Foxp3 expression is positively correlated with IL-23/IL-17 pathway in host liver. Liver biopsy samples from 38 CHB patients were used to detect mRNA for Foxp3, IL-23, IL-17, TNF-α and IL-8 by qPCR, and then the relationship between Foxp3 and IL-23, IL-17, TNF-α or IL-8 was analyzed. P < 0.05 was considered significant.