Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice

Introduction Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. Methods FVB/NJ and Mdr2−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. Results Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. Conclusion Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression

Biliary damage and liver fibrosis are ameliorated in a novel mouse model lacking l-histidine decarboxylase/histamine signaling

Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2−/−) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC−/− mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC−/− in Mdr2−/− mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2−/− mice and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-β1 and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-β1 and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression

Recent Advances in Understanding Cholangiocarcinoma [version 1; referees: 2 approved]

Cholangiocarcinoma (CCA) is an aggressive malignancy that arises from damaged epithelial cells, cholangiocytes, and possibly de-differentiated hepatocytes. CCA has a poor overall survival rate and limited therapeutic options. Based on this data, it is imperative that new diagnostic and therapeutic interventions be developed. Recent work has attempted to understand the pathological mechanisms driving CCA progression. Specifically, recent publications have delved into the role of cancer stem cells (CSCs), mesenchymal stem cells (MSCs), and microRNAs (miRNAs) during CCA pathology. CSCs are a specific subset of cells within the tumor environment that are derived from a cell with stem-like properties and have been shown to influence recurrence and chemoresistance during CCA. MSCs are known for their anti-inflammatory activity and have been postulated to influence malignancy during CCA, but little is known about their exact functions. miRNAs exert various functions via gene regulation at both the transcriptional and the translational levels, giving miRNAs diverse roles in CCA progression. Additionally, current miRNA-based therapeutic approaches are in clinical trials for various liver diseases, giving hope for similar approaches for CCA. However, the interactions among these three factors in the context of CCA are unknown. In this review, we focus on recently published data (within the last 3 years) that discuss the role of CSCs, MSCs, and miRNAs and their possible interactions during CCA pathogenesis

Alcoholic liver disease and mast cells: What's your gut got to do with it?

Alcoholic liver disease (ALD) remains one of the leading causes of liver injury and death when left untreated. The gut microbiota has been recognized as a key regulator of a number of pathologies, including ALD. The role of mast cells (MCs) during liver disease progression has been demonstrated in a number of animal models and in human liver diseases. The interaction between the gut microbiota and MCs has been investigated, and links between the gut and these immune cells are being uncovered. The interplay between the gut microbiota and MCs during ALD has been evaluated and studies suggest that there could be an important link between MCs, their mediators and gut inflammation during the progression of ALD. Keywords: Alcoholic liver disease (ALD), Alcoholic steatohepatitis (ASH), Cirrhosis, Steatosis, Mast cells (MCs), Microbiota, Gu

Amelioration of Large Bile Duct Damage by Histamine-2 Receptor Vivo-Morpholino Treatment

Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated β-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-β1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-β1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC

Mast Cells Regulate Ductular Reaction and Intestinal Inflammation in Cholestasis Through Farnesoid X Receptor Signaling

Background & Aim Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF15). Primary sclerosing cholangitis (PSC) patients typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile duct ligated (BDL) MC-deficient mice (KitW-sh), ductular reaction (DR) and liver fibrosis decrease compared to BDL WT; and MC injection exacerbates liver damage in normal mice. Approach & Results In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared to control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in PSC and PSC-IBD patients compared to healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence associated secretory phenotype (SASP), fibrosis and histamine in KitW-sh mice. Inhibition of MC-FXR prior to injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15 and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and Mdr2-/- mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. Conclusion Our studies demonstrate the novel findings that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling