Background and Aim 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. Methods WT and Mdr2−/− 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 CK-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 qPCR for α-SMA, fibronectin, collagen type 1a and TGF-β1. HSC activation was evaluated by qPCR in total liver and immunofluorescent staining in tissues for synaptophysin 9. Histamine serum secretion was measured by EIA. Mouse liver and human liver samples from control or PSC patients were evaluated for MC markers by qPCR and immunohistochemistry. In vitro, cultured MCs were transfected with HDC shRNA to decrease histamine secretion and subsequently co-cultured with cholangiocytes or HSCs prior to measuring fibrosis markers, proliferation and TGF-β1 secretion. Results Treatment with cromolyn sodium decreased biliary proliferation, fibrosis, histamine secretion, and bile flow in Mdr2−/− mice. PSC mice and patients have increased MCs. Knockdown of MC HDC 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 a therapeutic for PSC.

The tumor microenvironment of cholangiocarcinoma (CCA) is composed of numerous cells, including mast cells (MCs). MCs release histamine, which increases CCA progression and angiogenesis. Cholangiocytes secrete stem cell factor, which functions via the MC growth factor receptor c-Kit. Here, we show that cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth. MC recruitment in the tumor microenvironment increased CCA growth. MC infiltration and MC markers were detected by toluidine blue staining and real-time PCR in human biopsies and in tumors from athymic mice treated with saline, histamine, histidine decarboxylase inhibitor, or cromolyn sodium. Tumor growth, angiogenesis, and epithelial-mesenchymal transition (EMT)/extracellular matrix (ECM) markers were measured in mice treated with cromolyn sodium. In vitro, human CCA cells were treated with MC supernatant fluids before evaluating angiogenesis and EMT/ECM expression. Migration assays were performed with CCA cells treated with the stem cell factor inhibitor. MC supernatant fluids increased CCA histidine decarboxylase, vascular endothelial growth factor, and MC/EMT/ECM expression that decreased with pretreatment of cromolyn sodium. MCs were found in human biopsies. In mice treated with cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of CCA stem cell factor blocked MC migration and MC/EMT/ECM in CCA. MCs migrate into CCA tumor microenvironment via c-Kit/stem cell factor and increase tumor progression, angiogenesis, EMT switch, and ECM degradation. Cholangiocarcinoma (CCA) cancers are primary tumors that arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts of the liver.1 CCA is the second most prevalent liver tumor after hepatocellular carcinoma and accounts for 10% to 20% of deaths caused by hepatobiliary malignancies.2 Increased risk of developing CCA is associated with primary sclerosing cholangitis, liver fluke infestation, hepatitis C virus infection, and other diseases that lead to chronic biliary obstruction and inflammation.3 CAA is a metastatic cancer, and studies have found its potential to migrate outside of the biliary tract. 4 We have found that histamine via the H4 histamine receptor (HR)

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 decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 hrs 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 FCεRI 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 FCεRI, chymase, tryptase, RMCPI and RMCPII, 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.