BaCKgRoUND aND aIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. appRoaCH aND ReSUltS: Wild-type (WT) and Kit W-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and Kit W-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulinlike growth factor 1 expression that promotes MC migration/ activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in Kit W-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WDinduced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in Kit W-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in Kit W-sh WD mice and was found to target ALDH1A3.CoNClUSIoNS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment. (Hepatology 2021;74:164-182). N onalcoholic fatty liver disease (NAFLD) can develop into nonalcoholic steatohepatitis (NASH). (1) High mortality rates are seen in patients with NAFLD, and NASH is the third most common indication for liver transplantation in

Biliary senescence and hepatic fibrosis are hallmarks of cholangiopathies including primary sclerosing cholangitis (PSC). Senescent cholangiocytes display senescence-associated secretory phenotypes (SASPs, e. g., TGF-β1) that further increase biliary senescence (by an autocrine loop) and trigger liver fibrosis by paracrine mechanisms. To determine the effect of p16 inhibition and role of the TGF-β1/miR-34a/SIRT1 axis in biliary damage and liver fibrosis in the Mdr2-/- mouse model of PSC. We treated (i) in vivo male wild-type (WT) and Mdr2-/- mice with p16 Vivo-Morpholino or controls before measuring biliary mass (IBDM) and senescence, biliary SASPs levels and liver fibrosis; and (ii) in vitro intrahepatic murine cholangiocyte lines (IMCLs) with siRNA against p16 before measuring the mRNA expression of proliferation, senescence and fibrosis markers. p16 and miR-34a increased but SIRT1 decreased in Mdr2-/- mice and PSC human liver samples compared to controls. P16 immunoreactivity and biliary senescence and SASPs levels increased in Mdr2-/- mice but decreased in Mdr2-/- mice treated with p16 Vivo-Morpholino. The increase in IBDM and hepatic fibrosis (observed in Mdr2-/- mice) returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. TGF-β1 immunoreactivity and biliary SASPs levels were higher in Mdr2-/- compared to those of WT mice, but returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. The expression of fibrosis/senescence markers decreased in cholangiocytes from Mdr2-/- mice treated with p16 Vivo-Morpholino (compared to Mdr2-/- mice) and in IMCLs (after p16 silencing) compared to controls. Modulation of the TGF-β1/miR-34a/SIRT1 axis may be important in the management of PSC phenotype.

Background and Aims Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF‐β1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF‐β1. Approach and Results Wild‐type, KitW‐sh (MC‐deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l‐histidine decarboxylase were injected with vehicle or PKH26‐tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF‐β1 receptor inhibitor (TGF‐βRi), LY2109761 (10 μM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK‐19) immunohistochemistry and F4/80 staining coupled with real‐time quantitative PCR (qPCR) for interleukin (IL)‐1β, IL‐33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP‐9), desmin, and alpha smooth muscle actin (α‐SMA). TGF‐β1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC‐TGF‐β1 expression/secretion were measured after TGF‐βRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC‐conditioned medium, and biliary proliferation/senescence was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium and qPCR; HSC activation evaluated for α‐SMA, SYP‐9, and collagen type‐1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF‐β1 secretion, and angiogenesis. Injection of MC‐TGF‐βRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF‐β1. Conclusions Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC‐derived TGF‐β1 may be a target in chronic cholestatic liver disease.

iver diseases are a major health concern and affect a large proportion of people worldwide. There are over 100 types of liver disorders, including cirrhosis, cholangiocarcinoma (CCA), hepatocellular carcinoma, and hepatitis. Despite the relevant number of people who are affected by liver diseases, and the increased awareness with regard to these disorders, the number of deaths corresponding to liver injury is expected to increase in the foreseeable future. One of the possible reasons for this is that a complete comprehension of the mechanisms of hepatic damage involving specific liver anatomical districts is lacking, and, as a consequence, current treatments available are suboptimal. A major burden in the clinical setting are chronic cholestatic liver diseases (e.g., primary biliary cholangitis [PBC], primary sclerosing cholangitis [PSC], biliary atresia), which target the biliary epithelium and are characterized by cholestasis. (1,2) Because the secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes in the liver) (3,4) is the major regulator of ductal bile secretion, (5,6) it is intuitive that this axis plays a key role in the maintenance of biliary homeostasis during the progression of cholangiopathies. For instance, PBC is characterized by reduced bicarbonate secretion, a phenomenon possibly impeding the formation of an HCO − 3 canalicular film ("bicarbonate umbrella") on bile ducts, which has protective properties against highly concentrated bile acids (BAs). (1,7,8) In this review, we examined the molecular mechanisms by which the Sct/SR axis regulates biliary function and the homeostasis of the biliary epithelium in normal and pathophysiological conditions. Sct/SR aXIS: tHe BaSICS Sct, a neuroendocrine, gastrointestinal peptide of 27 amino acids is secreted primarily by the intestinal S cells located in the Lieberkühn crypts. There is evidence that Sct is also secreted by other epithelia in different organs including the pancreas, intestine and liver, thus exerting various biological effects in

BaCKgRoUND aND aIMS:Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) 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 (B6. Cg-Kit W-sh /HNihrJaeBsmJ [Kit W-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. appRoaCH aND ReSUltS:In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in Kit W-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and Kit W-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL Kit W-sh and Kit W-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in Kit W-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ ATP-binding cassette family 2 member 4 knockout (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. CoNClUSIoNS:Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/ FGF15 signaling. (Hepatology 2021;74:2684-2698. Cholangiocytes are the target cells of cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), which is characterized by