Mast Cells Contribute to Hepatic Neurokinin1 Receptor Signaling, Subsequent Biliary Damage and Peribiliary Fibrosis Via TGF‐β1 Signaling in MDR2‐/‐ Mouse Model of Primary Scelrosing Cholangitis

Abstract

BackgroundNeuropeptides modulate the pathogenesis of primary sclerosing cholangitis (PSC) by affecting i) biliary senescence/ductular reaction (DR), and ii) liver inflammation and fibrosis. Damaged cholangiocytes develop a neuroendocrine phenotype and respond to neurokinin 1 (NK1, i.e. substance P [SP]) signaling via NK1 receptor (NK1R). SP serum levels increase in PSC patients and depletion of NK1R in Mdr2‐/‐ mice (NK1R/Mdr2 double knockout (DKO)) ameliorates DR and fibrosis compared to Mdr2‐/‐ mice. Mast cells (MCs) (i) respond to sensory neuropeptide signaling; (ii) infiltrate the liver following injury; (iii) reside near bile ducts and (iv) promote PSC phenotypes in Mdr2‐/‐ mice, and injection of MCs into WT mice increases DR, inflammation and hepatic fibrosis. We hypothesize that MCs contribute to DR and liver fibrosis via SP/NK1R signaling in cholestasis.MethodsMale WT, NK1R‐/‐, Mdr2‐/‐ and DKO mice at 12 wks were used. NK1R‐/‐ and DKO mice received a single tail vein injection of cultured, tagged murine MCs (ATCC MC/9, 105 cells/injection) or saline. Liver damage was evaluated by H&E staining. MC presence was confirmed by tryptase β2 immunohistochemistry (IHC). MC activation was determined by serum histamine (HA) levels using enzyme‐linked immunoassay (EIA). DR was measured by cytokeratin‐19 (CK‐19) IHC and biliary senescence by co‐immunofluorescence (co‐IF) for p16/CK‐19. Inflammation was measured by F4/80 IHC and hepatic fibrosis by Sirius Red staining. Serum SP levels were measured by EIA. αSMA immunoreactivity was measured by co‐IF with CK‐19 in liver sections. Ingenuity Pathway Analysis (IPA) showed that SP modulates TGF‐β1 expression; therefore, serum TGF‐β1 levels were measured by EIA. In vitro, primary cholangiocytes from PSC patients were treated with an NK1R antagonist, Aprepitant (10 μM, 24 hrs) or vehicle in the bottom of Boyden chambers. MCs were added to the upper chamber and MC migration was evaluated by toluidine blue staining.ResultsInjected MCs localized in peribiliary regions of NK1R‐/‐ and DKO mice and increased hepatic damage, DR, biliary senescence, inflammation and fibrosis in both NK1R‐/‐ and DKO mice compared to their respective controls and WT. Serum HA and SP levels were reduced in NK1R‐/‐ and DKO mice compared to Mdr2‐/‐ mice, which increased following MC injection. Serum TGF‐β1 levels were reduced in NK1R‐/‐ and DKO mice compared to Mdr2‐/‐ and increased following MC injection. By Pearson’s correlation analysis, serum TGF‐β1 positively correlated with HA in all groups. In vitro, NK1R antagonist treatment reduced MC migration towards isolated PSC cholangiocytes compared to control.ConclusionMCs contribute to hepatic SP/NK1R signaling, biliary damage and peribiliary fibrosis via TGF‐β1 signaling. MC activation of NK1R signaling is critical for MC‐mediated damage in PSC.