MELATONIN THERAPY REDUCES BILIARY SENESCENCE, LIVER INFLAMMATION AND FIBROSIS IN A MURINE MODEL OF PRIMARY BILIARY CHOLANGITIS (PBC).

Aims: The mechanism of gut microbiota involved in the etiopathogenesis of primary cholangitis (PBC) remains unclear. Here, we investigated the role of gut microbiota in liver immune response in PBC.
Methods: Female C57BL6/J mice (n=8 per group) were gavaged with placebo, feces from healthy controls or PBC patients after antibiotic cocktails. Alpha-naphthylisothiocyanate (ANIT) was given to induce cholestasis. The liver, mesenteric lymph node (MLN) and spleen of mice were cultured under anaerobic and aerobic conditions. Serum, intestinal and liver tissues were isolated for ELISA, histology, real-time PCR and flow cytometry analysis. Fecal and liver biopsies from PBC patients (n = 14) and healthy controls (n = 5) were collected to confirm the distribution of Klebsiella pneumonia and T helper 17 (Th17) infiltration. The liver biopsies from early and advanced PBC patients (n = 13) were isolated for RNA-seq. Immortalized Human Intrahepatic Biliary Epithelial Cells (HIBEpiCs) were used for investigation the effect of K. pneumonia on the chemotaxis of Th17.
Results: More severe liver injuries and Th17 cell infiltration were found in mice gavaged by feces from PBC patients than control. Denser Th17 cells was observed in liver of PBC group after ANIT treatment (P < 0.05). K. pneumonia was isolated from the liver, spleen and MLN of the PBC group mice. Interestingly, administration of feces from PBC patients increased serum endotoxin and impairment of the intestinal epithelial barrier (P < 0.05). Antibiotic treatment significantly decreased Th17 infiltration, intestinal epithelial barrier and liver injury induced by PBC-derived microbiota. Besides, K. pneumoniae monocolonization can induce K. pneumoniae translocation and inflammatory cell infiltration in liver. Furthermore, PBC patients showed increased of CCL20 expression and infiltration Th17 cells in liver, which was positively correlated with disease progression. Notably, the abundance of K. pneumoniae was increased in the liver and fecal of PBC patients. In vitro, the bacterial lysate of K. pneumoniae upregulated cholangiocyte senescence and CCL20 in HIBEpiCs in a concentration-dependent manner.
Conclusions: We demonstrate that K. pneumoniae translocation to liver increased CCL20 secretion promote Th17 immune response in PBC.

Background: Primary biliary cholangitis (PBC) is a cholestatic liver disease that targets cholangiocytes and is characterized by enhanced biliary senescence, ductular reaction (DR), autoimmune response, liver inflammation and fibrosis that is triggered by the biliary release of senescence-associated secretory phenotype (e.g., TGFβ1). Heterozygous 162 nt AU-rich element (ARE) region deletion in the 3’ untranslated region of the interferon-gamma gene (ARE-Del^+/-) in mice at 20 wk of age display distinct pathological features that are characteristic of human PBC. We have previously demonstrated that melatonin therapy ameliorates damage in mouse models of primary sclerosing cholangitis via decreased expression of the MT1 melatonin receptor and selected circadian genes including CLOCK. Thus, we aimed to evaluate the therapeutic effects of melatonin in a mouse model of PBC.
Methods: We measured biliary senescence (by SA-β-gal staining), and the biliary immunoreactivity of MT1/CK19 and CLOCK/CK19 by double immunofluorescence (IF) in liver sections from healthy controls (2 female and 2 male) and late-stage PBC patients (1 male and 3 female). In vivo, male, and female wild-type (WT) and ARE-Del^+/- mice at 20 wk of age had free access to drinking water containing melatonin (0.03%) for 1 wk (daily intake 1.2 mg). We evaluated (i) liver damage by H&E staining; (ii) DR by immunohistochemistry (IHC) for CK19; (iii) biliary senescence by double IF for p16/CK19 and SA-β-gal staining; (iv) liver inflammation by IHC for CD68; and (v) liver fibrosis by Sirius Red staining in liver sections from WT and ARE-Del^+/- mice treated with/without melatonin. Liver autoimmune response was evaluated in mouse liver sections by IHC for CD3⁺ T cell and CD20⁺ B cell infiltration. The biliary immunoreactivity of MT1 and CLOCK was measured by IF in liver sections co-stained with CK19.
Results: Enhanced biliary senescence as well as increased immunoreactivity of MT1 and CLOCK were detected in liver sections from late-stage PBC patients compared to healthy controls. There was enhanced expression of MT1 and CLOCK and increased liver damage, DR, and biliary senescence as well as autoimmune response, liver inflammation and liver fibrosis in ARE-Del^+/- compared to WT mice, phenotypes that were decreased in melatonin-treated ARE-Del^+/- mice compared to vehicle-treated mice.
Conclusion: Melatonin administration to ARE-Del^+/- mice ameliorates PBC phenotypes through reduced MT1 and CLOCK expression. Restoration of melatonin-dependent signaling via melatonin treatment may be therapeutic for patients with PBC.