IL-12/Th1 and IL-23/Th17 biliary microenvironment in primary biliary cirrhosis: implications for therapy

Abstract

The interleukin (IL)-12/IL-23-mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, interferon-gamma (IFN-γ), IL-12RB2, IL-23p40, IL-23p19, IL-17, and IL-23R using liver from PBC (n = 51) and non-PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN-γ expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN-γ production that contribute to PBC pathology.

Conclusion: Our data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.

Figure 1

Liver H&E of two representative portal sections from PBC subjects. Lymphocytic infiltration was observed in all of the PBC subjects. Epithelioid granulomas were also observed near damaged bile duct and/or hepatic parenchyma.

Figure 2

Liver immunohistochemical staining of Th1 cytokines, including IL-12p35, IL-12p70, IFN-γ, and IL-12 receptor subunit IL-12RB2 in PBC and disease controls. Representative staining images from patients with PBC (n = 51), AIH (n = 41), and HBV (n = 26) are shown.

Figure 3

Liver immunohistochemical staining for Th17 cytokines, including IL-23p19, IL-23p40, IL-17, and IL-23 receptor subunit IL-23R in PBC and disease controls. Representative staining from patients with PBC (n = 51), AIH (n = 41), and HBV (n = 26) are shown.

Figure 4

The Th1/Th17 ratio in PBC (n =51), AIH (n = 41), HBV (n = 26), and HCV (n = 13). The Th1/Th17 ratio is very close to the base line zero in PBC and AIH, while the Th1/Th17 ratio is higher than zero in HBV and lower than zero in HCV. The Th1/Th17 ratio in HBV is higher than that in PBC (**p < .01), while the ratio in HCV is lower than that in PBC (***p < .001).

Figure 5

The distribution of the Th1/Th17 ratio in (A) total PBC (n = 51), (b) early PBC (n = 22), and (C) advanced PBC (n = 29). The intensity of Th1/Th17 staining in the majority of total and early PBC patients was Th1 and Th17 balanced. However, the intensity skewed from Th1 to Th17 predominantly in advanced PBC. The Th1/Th17 ratio was negatively correlated (Spearman rs = 0.2959) with disease stage in PBC (p < .05).

Figure 6

(A) The expression of IL-12RB2 and the presence of IFN-γ positive mononuclear cells (black arrows) around damaged bile ducts (white arrows) in PBC. (B) The expression of IFN-γ in the biliary lumen of degenerated biliary epithelial cells in PBC. (C) The expression of IL-23R and the infiltration of IL-17 positive cells around damaged biliary epithelial cells in PBC. Liver sections were stained with anti-IL-23R individually, as well as anti-IL-23p19 (red) combined with anti-IL-17 (brown) or IFN-γ (red) combined with IL-12RB2 (brown) concurrently. Representative staining images from patients with PBC (n = 51) are shown.

Figure 7

Liver immunohistochemical staining for IL-23p19 in PBC and disease controls. Representative staining images from patients with PBC (n = 51), AIH (n = 41), and HBV (n = 26) are shown. The cholangiocytes, oval cells, and small hepatocytes are strongly positive especially in PBC.