Effect of Low Testosterone Levels on the Expression of Proliferator-Activated Receptor Alpha in Female Patients with Primary Biliary Cholangitis

Abstract

Sex-dependent patterns in chronic immune-mediated cholangiopathies, like primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), remain poorly understood. Peroxisome proliferator-activated receptor alpha (PPAR-α), expressed in immune cells, plays a key role in innate defence. In this study, the relationship between PPAR-α expression in peripheral blood mononuclear cells (PBMCs), serum androgen levels, IFNγ production, and sex-dependent tendencies during the development of PBC and PSC was investigated. We confirmed that normal cholangiocytes respond to PPAR-α and inhibit the lipopolysaccharide-induced expression of IL-6, IL-1b, and TNFα. Compared with PSC patients, PPAR-α was downregulated, while IFNγ was upregulated, in the PBMCs of PBC patients. When the analysis was conducted on females only, there was no difference in PPAR-α, but IFNγ was elevated in females with PBC compared with those with PSC. Serum testosterone concentrations in females with PBC were below the normal range (regardless of age) and correlated positively with PPAR-α and negatively with IFNγ. While PPAR-α has been reported to be a target of miR-155 and miR-21, no correlations with these microRNAs were observed in the PBMCs. However, a positive correlation between miR-21 and IFNγ was observed. Our results showed suppressed PPAR-α expression accompanied by reduced testosterone levels in women with PBC, which should elicit interest in the role of testosterone in PBC development.

Keywords: immune-mediated cholangitis; liver injury testosterone; peroxisome proliferator-activated receptor alpha.

Figure 1

The effect of lipopolysaccharide (LPS) and fenofibrate (FB) stimulation on peroxisome proliferator-activated receptor α (PPAR-α), interleukin 6 (IL-6), interleukin-1 beta (1L-1b), and tumour necrosis factor-α (TNFα) expression in normal human cholangiocytes (NHCs). Incubation with LPS (100 µM) reduced PPAR-α and upregulated IL-6 and IL-1b after 24 h (A), whereas pre-treatment with FB (200 µM) for 2 h prior to LPS stimulation upregulated PPAR-α and suppressed IL-6, IL-1b, and TNFα (B). At least three independent experiments were conducted. Levels of gene expression were normalized to the endogenous reference 18S RNA. Bars indicate the mean ± SEM.

Figure 2

The expression of PPAR-α, interferon-gamma (IFNγ), and interleukin 17a (IL-17a) mRNA in the peripheral blood mononuclear cells (PBMCs) of PSC and PBC patients. Compared with PSC patients, the expression of PPAR-α was reduced in PBC patients (A), whereas IFNγ was upregulated (B). No difference was observed in IL-17a mRNA expression between PBC and PSC patients (C). Levels of gene expression are presented as fold changes and were normalised to the endogenous reference 18S RNA. Bars indicate the mean ± SEM.

Figure 3

The expression of PPAR-α and IFNγ mRNA in patients with PBC or PSC. There was no difference between female PBC and PSC patients in their expression of PPAR-α (A). A higher expression of IFNγ was observed in females with PBC compared with those with PSC (B). In all PSC patients, females had lower levels of PPAR-α than males (C), but no difference in the relative expression of IFNγ was observed (D). Levels of gene expression are presented as fold changes and were normalised to the endogenous reference 18S RNA. Bars indicate the mean ± SEM.

Figure 4

The serum concentration of testosterone in females with PBC and PSC. Bar charts show considerably lower concentrations of testosterone in the sera of female patients with PBC than in females with PSC (A). Testosterone levels were below the normal range in females with PBC, and there was no difference between younger (PBC < 46 years old) and older women (PBC > 46 years old). The dotted lines represent the normal range for females (B). The serum concentration of testosterone correlated positively with PPAR-α mRNA (C) and negatively with IFNγ mRNA (D). Dots illustrate each patient.

Figure 5

The expressions of miR-155 and miR-21 in PBMCs. The relative expression of miR-21 was lower in PSC patients compared with both controls and PBC patients (A). The difference between the diseases was similar when the analysis was made with only female patients (B). The expression of miR-155 was higher in PSC patients compared with PBC patients (C). The disease-specific variation was also maintained when only female patients were evaluated (D). Levels of miRNA expression were normalized to the endogenous reference miR-16. Bars indicate the mean ± SEM.