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Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

ABSTRACT: Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.

SUBMITTER: Popov Y

PROVIDER: S-EPMC2838516 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

Popov Yury Y Sverdlov Deanna Y DY Bhaskar K Ramakrishnan KR Sharma Anisha K AK Millonig Gunda G Patsenker Eleonora E Krahenbuhl Stephan S Krahenbuhl Lukas L Schuppan Detlef D

American journal of physiology. Gastrointestinal and liver physiology 20100107 3

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase ...[more]

PMID: 20056896

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Project description:During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor β (TGF-β). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-β signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-β. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-β response. EZH2 overexpression suppressed TGF-β-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-β. TGF-β also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-β. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-β-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2-/- mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-β regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.

Project description:ERα, one of the classical receptors of estrogen, has been found to be abnormally up-regulated in patients with primary biliary cholangitis (PBC), which is an important factor leading to ductopenia. ERα-mediated signaling pathways are involved in proliferation of human intrahepatic biliary epithelial cells (HiBECs) and portal inflammation. Our previous studies have shown that the expression levels of ERα in the liver tissues of PBC patients are positively correlated with the levels of serum pro-inflammatory cytokines. The present study was designed to assess the relationship between abnormal ERα expression in small bile ducts and the progression of PBC. We examined the levels of multiple cytokines and analyzed their relationship with clinical parameters of livers functions in a cohort of 43 PBC patients and 45 healthy controls (HC). The levels of ERα expression and the relation with the levels of cytokines were further assessed. The localization of cytokines and ERα-mediated signaling pathways in liver were examined using immunohistochemistry. The possible underlying mechanisms of these alterations in PBC were explored in vitro. Our results demonstrated that the levels of IL-6, IL-8, and TNF-α were increased in PBC patients, and positively correlated with the serum AKP levels and ERα expression levels. Moreover, the expression of these cytokines were up-regulated in HiBECs that were stimulated with 17β-estradiol and PPT (an ERα agonist) and they also were positive in intrahepatic bile duct of PBC patients. The ERα-mediated expression of pro-inflammatory cytokines was induced by JNK, P38, and STAT3 phosphorylation in HiBECs. In addition, the CD54 expression was increased in HiBECs after ERα activation, which induced peripheral blood monouclear cells (PBMCs) recruitment. In conclusion, the present study highlighted a key role of abnormal ERα expression in inducing an inflammatory phenotype of HiBECs, which was critical in the development of inflammation and damage in small bile duct.

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Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

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Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

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