Jennifer Demieville

Inhibition of mast cell‐secreted histamine decreases biliary proliferation and fibrosis in primary sclerosing cholangitis Mdr2−/− mice

Hepatic fibrosis is marked by activation of hepatic stellate cells (HSCs). Cholestatic injury precedes liver fibrosis, and cholangiocytes interact with HSCs promoting fibrosis. Mast cells (MCs) infiltrate following liver injury and release histamine, increasing biliary proliferation. We evaluated if inhibition of MC‐derived histamine decreases biliary proliferation and fibrosis. Wild‐type and multidrug resistance 2 knockout mice (9‐11 weeks) were treated with cromolyn sodium for 1 week to block MC‐derived histamine. Biliary mass and proliferation were evaluated by immunohistochemistry for cytokeratin 19 and Ki‐67. Bile flow, bicarbonate excretion, and total bile acids were measured in all mice. Fibrosis was evaluated by sirius red/fast green staining and by quantitative polymerase chain reaction for alpha‐smooth muscle actin, fibronectin, collagen type 1a, and transforming growth factor‐beta 1. HSC activation was evaluated by quantitative polymerase chain reaction in total liver and immunofluorescent staining in tissues for synaptophysin 9. Histamine serum secretion was measured by enzymatic immunoassay. Mouse liver and human liver samples from control or primary sclerosing cholangitis patients were evaluated for MC markers by quantitative polymerase chain reaction and immunohistochemistry. In vitro, cultured MCs were transfected with histidine decarboxylase short hairpin RNA to decrease histamine secretion and subsequently cocultured with cholangiocytes or HSCs prior to measuring fibrosis markers, proliferation, and transforming growth factor‐beta 1 secretion. Treatment with cromolyn sodium decreased biliary proliferation, fibrosis, histamine secretion, and bile flow in multidrug resistance 2 knockout mice. Primary sclerosing cholangitis mice and patients have increased MCs. Knockdown of MC histidine decarboxylase decreased cholangiocyte and HSC proliferation/activation. Conclusion: MCs are recruited to proliferating cholangiocytes and promote fibrosis. Inhibition of MC‐derived histamine decreases fibrosis, and regulation of MC mediators may be therapeutic for primary sclerosing cholangitis. (Hepatology 2016;64:1202‐1216)

Bile duct ligation–induced biliary hyperplasia, hepatic injury, and fibrosis are reduced in mast cell–deficient KitW‐sh mice

Activated mast cells (MCs) release histamine (HA) and MCs infiltrate the liver following bile duct ligation (BDL), increasing intrahepatic bile duct mass (IBDM) and fibrosis. We evaluated the effects of BDL in MC‐deficient (KitW‐sh) mice. Wild‐type (WT) and KitW‐sh mice were subjected to sham or BDL for up to 7 days and KitW‐sh mice were injected with cultured mast cells or 1× phosphate‐buffered saline (PBS) before collecting serum, liver, and cholangiocytes. Liver damage was assessed by hematoxylin and eosin and alanine aminotransferase levels. IBDM was detected by cytokeratin‐19 expression and proliferation by Ki‐67 immunohistochemistry (IHC). Fibrosis was detected by IHC, hydroxyproline content, and by qPCR for fibrotic markers. Hepatic stellate cell (HSC) activation and transforming growth factor‐beta 1 (TGF‐β1) expression/secretion were evaluated. Histidine decarboxylase (HDC) and histamine receptor (HR) expression were detected by qPCR and HA secretion by enzymatic immunoassay. To evaluate vascular cells, von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)‐C expression were measured. In vitro, cultured HSCs were stimulated with cholangiocyte supernatants and alpha‐smooth muscle actin levels were measured. BDL‐induced liver damage was reduced in BDL KitW‐sh mice, whereas injection of MCs did not mimic BDL‐induced damage. In BDL KitW‐sh mice, IBDM, proliferation, HSC activation/fibrosis, and TGF‐β1 expression/secretion were decreased. The HDC/HA/HR axis was ablated in sham and BDL KitW‐sh mice. vWF and VEGF‐C expression decreased in BDL KitW‐sh mice. In KitW‐sh mice injected with MCs, IBDM, proliferation, fibrosis, and vascular cell activation increased. Stimulation with cholangiocyte supernatants from BDL WT or KitW‐sh mice injected with MCs increased HSC activation, which decreased with supernatants from BDL KitW‐sh mice. Conclusion: MCs promote hyperplasia, fibrosis, and vascular cell activation. Knockout of MCs decreases BDL‐induced damage. Modulation of MCs may be important in developing therapeutics for cholangiopathies. (Hepatology 2017;65:1991‐2004).

BDL‐induced biliary hyperplasia, hepatic injury and fibrosis are reduced in mast cell deficient Kitw‐sh mice

Activated mast cells (MCs) release histamine (HA) and MCs infiltrate the liver following bile duct ligation (BDL), increasing intrahepatic bile duct mass (IBDM) and fibrosis. We evaluated the effects of BDL in MC‐deficient (KitW‐sh) mice. Wild‐type (WT) and KitW‐sh mice were subjected to sham or BDL for up to 7 days and KitW‐sh mice were injected with cultured mast cells or 1× phosphate‐buffered saline (PBS) before collecting serum, liver, and cholangiocytes. Liver damage was assessed by hematoxylin and eosin and alanine aminotransferase levels. IBDM was detected by cytokeratin‐19 expression and proliferation by Ki‐67 immunohistochemistry (IHC). Fibrosis was detected by IHC, hydroxyproline content, and by qPCR for fibrotic markers. Hepatic stellate cell (HSC) activation and transforming growth factor‐beta 1 (TGF‐β1) expression/secretion were evaluated. Histidine decarboxylase (HDC) and histamine receptor (HR) expression were detected by qPCR and HA secretion by enzymatic immunoassay. To evaluate vascular cells, von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)‐C expression were measured. In vitro, cultured HSCs were stimulated with cholangiocyte supernatants and alpha‐smooth muscle actin levels were measured. BDL‐induced liver damage was reduced in BDL KitW‐sh mice, whereas injection of MCs did not mimic BDL‐induced damage. In BDL KitW‐sh mice, IBDM, proliferation, HSC activation/fibrosis, and TGF‐β1 expression/secretion were decreased. The HDC/HA/HR axis was ablated in sham and BDL KitW‐sh mice. vWF and VEGF‐C expression decreased in BDL KitW‐sh mice. In KitW‐sh mice injected with MCs, IBDM, proliferation, fibrosis, and vascular cell activation increased. Stimulation with cholangiocyte supernatants from BDL WT or KitW‐sh mice injected with MCs increased HSC activation, which decreased with supernatants from BDL KitW‐sh mice. Conclusion: MCs promote hyperplasia, fibrosis, and vascular cell activation. Knockout of MCs decreases BDL‐induced damage. Modulation of MCs may be important in developing therapeutics for cholangiopathies. (Hepatology 2017;65:1991‐2004).

Isolation and characterization of hepatic mast cells from cholestatic rats

Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decreases biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 h and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse-coated Dynal beads. MCs were stained with toluidine blue, and in isolated MCs the expression of FCɛRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF receptors, and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1, and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCɛRI, chymase, tryptase, RMCP-I, and RMCP-II, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats+NaCl and these parameters decreased in cells treated with MC supernatants from BDL+cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary/liver pathologies.

Recent Advances in Understanding Cholangiocarcinoma

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.

Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2–/– mice and human cholangiocarcinoma tumorigenesis

Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild‐type and multidrug‐resistant knockout (Mdr2–/–) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L‐histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz‐ChA‐1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial–mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 μM) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)–treated Mdr2–/– mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC‐associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018).

The emerging role of mast cells in liver disease

The depth of our knowledge regarding mast cells has widened exponentially in the last 20 years. Once thought to be only important for allergy-mediated events, mast cells are now recognized to be important regulators of a number of pathological processes. The revelation that mast cells can influence organs, tissues, and cells has increased interest in mast cell research during liver disease. The purpose of this review is to refresh the readers knowledge of the development, type, and location of mast cells and to review recent work that demonstrates the role of hepatic mast cells during diseased states. This review focuses primarily on liver diseases and mast cells during autoimmune disease, hepatitis, fatty liver disease, liver cancer, and aging in the liver. Overall, these studies demonstrate the potential role of mast cells in disease progression.

Knockout of l-Histidine Decarboxylase Prevents Cholangiocyte Damage and Hepatic Fibrosis in Mice Subjected to High-Fat Diet Feeding via Disrupted Histamine/Leptin Signaling

Feeding a high-fat diet (HFD) coupled with sugar, mimicking a Western diet, causes fatty liver disease in mice. Histamine induces biliary proliferation and fibrosis and regulates leptin signaling. Wild-type (WT) and l-histidine decarboxylase (Hdc-/-) mice were fed a control diet or an HFD coupled with a high fructose corn syrup equivalent. Hematoxylin and eosin and Oil Red O staining were performed to determine steatosis. Biliary mass and cholangiocyte proliferation were evaluated by immunohistochemistry. Senescence and fibrosis were measured by quantitative PCR and immunohistochemistry. Hepatic stellate cell activation was detected by immunofluorescence. Histamine and leptin levels were measured by enzyme immunoassay. Leptin receptor (Ob-R) was evaluated by quantitative PCR. The HDC/histamine/histamine receptor axis, ductular reaction, and biliary senescence were evaluated in patients with nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, or end-stage liver disease. Hdc-/- HFD mice had increased steatosis compared with WT HFD mice. WT HFD mice had increased biliary mass, biliary proliferation, senescence, fibrosis, and hepatic stellate cell activation, which were reduced in Hdc-/- HFD mice. In Hdc-/- HFD mice, serum leptin levels increased, whereas biliary Ob-R expression decreased. Nonalcoholic steatohepatitis patients had increased HDC/histamine/histamine receptor signaling. Hdc-/- HFD mice are susceptible to obesity via dysregulated leptin/Ob-R signaling, whereas the lack of HDC protects from HFD-induced fibrosis and cholangiocyte damage. HDC/histamine/leptin signaling may be important in managing obesity-induced biliary damage.

Ursodeoxycholate inhibits mast cell activation and reverses biliary injury and fibrosis in Mdr2 −/− mice and human primary sclerosing cholangitis

Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2−/− mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2−/− mice and human PSC. Wild-type and Mdr2−/− mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15 mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40 μM) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2−/− mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXRβ. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.The bile acid rsodeoxycholic acid (UDCA) is the choice of treatment for primary biliary cholangitis patients with abnormal liver enzymes; however, its mechanism is not clear, and is the focus of this investigation. Bile acids alter mast cell (MC) histamine release. Following liver injury, such as that seen in primary sclerosing cholangitis, MCs infiltrate the liver. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.

Cellular crosstalk during cholestatic liver injury

The functions of the liver are very diverse. From detoxifying blood to storing glucose in the form of glycogen and producing bile to facilitate fat digestion, the liver is a very active and important organ. The liver is comprised of many varied cell types whose functions are equally diverse. Cholangiocytes line the biliary tree and aid in transporting and adjusting the composition of bile as it travels to the gallbladder. Hepatic stellate cells and portal fibroblasts are located in different areas within the liver architecture, but both contribute to the development of fibrosis upon activation after liver injury. Vascular cells, including those that constitute the peribiliary vascular plexus, are involved in functions other than blood delivery to and from the liver, such as supporting the growth of the biliary tree during development. Mast cells are normally found in healthy livers but in very low numbers. However, after injury, mast cell numbers greatly increase as they infiltrate and release factors that exacerbate the fibrotic response. While not an all-inclusive list, these cells have individual roles within the liver, but they are also able to communicate with each other by cellular crosstalk. In this review, we examine some of these pathways that can lead to an increase in the homeostatic dysfunction seen in liver injury.