MAST CELLS (MCS) PROMOTE PRIMARY SCLEROSING CHOLANGITIS (PSC) PHENOTYPES IN MICE L-HISTIDINE DECARBOXYLASE KNOCKOUT (HDC^-/-) MICE VIA MC-NFKB AND HEPATIC IL-33 SIGNALING

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.

Background: PSC is devasting cholestatic liver disease without curative treatment. In PSC patients and cholestatic mouse models, MC presence and activation significantly increases. MC injection induces PSC phenotypes increasing ductular reaction (DR), biliary senescence, and hepatic fibrosis in wild-type (WT) or MC-deficient mice. NFkB promotes inflammation, and, in MCs, IL-33 induces NFkB translocation causing MC activation and subsequent inflammatory cytokine release. In rodents, inhibition of NFkB by caffeic acid phenethyl ester (CAPE) decreases cholestatic phenotypes. Aim: To determine the effects of inhibition of MC-specific NFkB on PSC phenotypes. Methods: 12 wk male HDC^-/- mice (balb/c (WT)) were injected via tail vein with cultured mouse MCs (mMCs) pretreated with vehicle or CAPE (20 mM) 1x prior to sacrifice 3 days later. Serum, liver, and cholangiocyte supernatants were collected. Liver damage was assessed by serum AST. Serum HA content and serum/cholangiocyte IL-33 secretion was measured by EIA. Downstream of IL-33, we measured IRAK4 gene expression in total liver by qPCR. DR and senescence were determined by semiquantitative CK-19 immunohistochemistry and qPCR for p21 in total liver mRNA, respectively. Hepatic fibrosis and hepatic stellate cell (HSC) activation were evaluated by semiquantitative Fast Green/Sirius Red staining, immunofluorescence for desmin (co-stained with CK-19) and qPCR for collagen-1a and a-SMA in total liver. mMCs were evaluated for NFkB signaling components, p105 and p65 (RelA) by immunostaining and immunofluorescence, respectively. 3D human organoids were generated from normal primary isolated human cholangiocytes, HSCs, and human MCs (hMCs) pretreated with vehicle or CAPE (20 mM); organoids developed for 72 hrs. HA content was measured by EIA and chymase (Cma1) gene expression by qPCR. Results: mMCs express p105 and RelA and injection of vehicle-mMCs into HDC^-/- mice increased AST, senescence, and fibrosis compared to WT or HDC^-/- mice. These parameters were reduced in HDC^-/- mice injected with mMC-CAPE. No significant changes were found in DR across all groups. Serum HA and IL-33 increased in HDC^-/- mice treated with vehicle-mMCs that decreased in HDC^-/- mice injected with mMC-CAPE. Biliary IL-33 increased in vehicle-mMC injected HDC^-/- mice, whereas inhibition of mMC-NFkB reduced IL-33/IRAK signaling. Normal 3D human organoids generated with vehicle hMCs had increased HA content and chymase gene expression that were reduced when hMCs were treated with CAPE prior to organoid formation. Conclusion: Injected mMCs (i) promote hepatic damage resembling PSC phenotypes and (ii) increase hepatic IL-33 signaling in HDC^-/- mice. Inhibition of MC-NFkB signaling reduces damaging phenotypes by decreasing biliary and hepatic IL-33 signaling. Modulation of inflammatory MC mediators may be key to reduce PSC phenotypes.