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

Abstract

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.

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

“…When BDL rats or Mdr2 –/– mice are treated with the MC stabilizer, cromolyn sodium (blocks MC HA release), biliary mass and proliferation are significantly reduced . Thus, we suspect that MCs are an important player in the regulation of biliary hyperplasia following BDL.…”

“…We evaluated the effects of MC deficiency on intrahepatic bile duct mass (IBDM) and proliferation in both WT and Kit W‐sh mice with sham or BDL surgery. IHC, real‐time PCR, and western blotting were performed for the biliary marker, CK‐19, to measure IBDM, and cholangiocyte proliferation was determined by Ki‐67 staining …”

“…In total liver from all groups, we measured the following fibrotic markers by real‐time PCR: α‐smooth muscle actin (α‐SMA), collagen type‐1a, and fibronectin‐1 . Because TGF‐β1 is a known regulator of fibrosis, we evaluated expression of TGF‐β1 in total liver by real‐time PCR and secretion of TGF‐β1 by EIA in serum from these mice . We also evaluated the effects of MC deficiency on HSC activation by IF for synaptophysin‐9 (SYP‐9) .…”

“…In addition to HSCs and cholangiocytes, hepatic mast cells (MCs) are also involved in fibrosis . While traditionally noted for their role in allergic responses, MCs are being more recognized in a number of pathologies, including liver disease, and our recent studies have demonstrated that MCs play critical and detrimental roles in BDL‐induced liver damage, PSC, and cholangiocarcinoma (CCA) . MC infiltration occurs following mechanical, chemical, or toxic liver injury, and MCs migrate into CCA tumors where they promote tumor growth .…”

Mechanisms of cholangiocyte responses to injury

“…A recent study has shown that inhibition of mast cell-derived histamine secretion attenuates bile duct hyperplasia during BDL in vivo [69]. Another study has shown that serum levels of histamine are increased in Mdr2 −/− mice, and inhibition of mast cell-derived histamine secretion decreases serum histamine levels as well as bile duct hyperplasia and fibrosis in these mice [70]. Mast cell-deficient mice show less liver damage, bile duct mass, and fibrosis during BDL compared to wild-type [71].…”