A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis - PubMed (original) (raw)
A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis
Peter Fickert et al. Am J Pathol. 2007 Aug.
Abstract
Xenobiotics and drugs may lead to cholangiopathies and biliary fibrosis, but the underlying mechanisms are largely unknown. Therefore, we aimed to characterize the cause and consequences of hepatobiliary injury and biliary fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a novel model of xenobiotic-induced cholangiopathy. Liver morphology, markers of inflammation, cell proliferation, fibrosis, bile formation, biliary porphyrin secretion, and hepatobiliary transporter expression were studied longitudinally in DDC- and control diet-fed Swiss albino mice. DDC feeding led to increased biliary porphyrin secretion and induction of vascular cell adhesion molecule, osteopontin, and tumor necrosis factor-alpha expression in bile duct epithelial cells. This was associated with a pronounced pericholangitis with a significantly increased number of CD11b-positive cells, ductular reaction, and activation of periductal myofibroblasts, leading to large duct disease and a biliary type of liver fibrosis. After 4 weeks, we constantly observed intraductal porphyrin pigment plugs. Glutathione and phospholipid excretion significantly decreased over time. Expression of Ntcp, Oatp4, and Mrp2 was significantly reduced, whereas Bsep expression remained unchanged and adaptive Mrp3 and Mrp4 expression was significantly induced. We demonstrate that DDC feeding in mice leads to i) a reactive phenotype of cholangiocytes and bile duct injury, ii) pericholangitis, periductal fibrosis, ductular reaction, and consequently portal-portal bridging, iii) down-regulation of Mrp2 and impaired glutathione excretion, and iv) segmental bile duct obstruction. This model may be valuable to investigate the mechanisms of xenobiotic-induced chronic cholangiopathies and its sequels including biliary fibrosis.
Figures
Figure 1
DDC feeding leads to ductular proliferation and cholangitis with onion skin type-like periductal fibrosis in mice. Livers of control mice (A and E) and mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (B and F), 4 weeks (C and G), and 8 weeks (D and H) are shown. Liver histology of DDC-fed mice reveals increasing ductular proliferation over time (outlined by arrowheads) (B–D). At 4 weeks, an increasing number of porphyrin plugs occluding the lumina of small bile ducts is observed (C and D). F–H: Increasing severity of cholangitis and periductal fibrosis of larger bile ducts over time. Hematoxylin and eosin staining: pv, portal vein; bd, bile duct. Original magnification, ×20 (A–H).
Figure 2
DDC feeding induces ductular reaction in mice. Immunohistochemistry for keratin 19 (K19) in control liver (A) and mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (B), 4 weeks (C), and 8 weeks (D) is shown. In contrast to control liver (A), there is a significantly increased number of K19-postive cholangiocytes/portal field in DDC-fed animals (B–D). E: For morphometric analysis, the bile duct mass was measured by a semiautomatic system and normalized to the size of portal veins showing significantly increased K19-positive area/portal field in DDC-fed mice. *P < 0.05 (controls versus 1- to 4-week old DDC-fed mice). pv, portal vein. Original magnification, ×10 (A–D).
Figure 3
DDC feeding induces segmental subtotal mechanical bile duct obstruction via porphyrin plugs and large duct disease in mice. Plastination of the bile duct system followed by maceration of the liver was performed in (A) standard diet-fed and (E) 4-week old DDC-fed mice. A: The bile duct system was filled with plastogen G after cannulation of the gallbladder (GB). In contrast to the delicately structured bile duct system in the control diet-fed mouse (A), the DDC-fed mouse (E) shows slight dilatation of bile ducts (highlighted by white arrows), showing dilatation of the bile duct and some porphyrin plugs (black dots) within the plastogen. B: Histological examination of the DHC shows a slim duct from the liver hilus (L) down to the pancreas (P) in a control diet-fed mouse. F: In contrast, in 8-week-old DDC-fed mice, the DHC wall is markedly thickened. G and H: The bile duct epithelium shows reactive changes and substantial neutrophil infiltration of the DHC wall. GB, gall bladder, L, liver; P, pancreas. Original magnification: ×4 (B and F); ×20 (C and G); ×40 (D and H).
Figure 4
DDC feeding induces a reactive phenotype of cholangiocytes and pericholangitis in mice. Immunohistochemistry for VCAM (A–D) and neutrophil marker CD11b (E–H) in control liver (A and E) and mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (B and F), 4 weeks (C and G), and 8 weeks (D and H). In contrast to control liver (A), proliferating bile ducts of DDC-fed mice (B–D) express VCAM. F–G: VCAM overexpression of bile ducts is paralleled by the occurrence of a dense neutrophil infiltrate. I: Protein quantification of VCAM by Western blotting reveals significantly increased VCAM hepatic protein level in DDC-fed animals. Densitometric values are the means from three to four animals in each group. *P < 0.05 compared with standard diet-fed controls. pv, portal vein. Original magnification, ×20.
Figure 5
DDC feeding induces TNF-α mRNA expression in cholangtiocytes. In situ hybridization for TNF-α mRNA in control liver (A), lipopolysaccharide-injected mouse (positive control) (B), and 1-week DDC-fed mouse livers (C). Note the significant increase of the TNF-α signal in Kupffer cells in lipopolysaccharide-treated mouse liver (B), serving as a positive control. C: In contrast, in DDC-fed mice cholangiocytes of bile duct proliferates represent the main source for TNF-α. pv, portal vein; bd, bile duct. Original magnification, ×40.
Figure 6
Biliary fibrosis DDC-fed mice. Sirius red staining (A–H) in control liver (A and E) and livers of mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (B and F), 4 weeks (C and G), and 8 weeks (D and H). B–D: Ductular proliferation leading to portal-portal bridges is paralleled by an increase in extracellular matrix deposition (ie, Sirius-red stain-positive collagen fibers indicated by arrowheads) in DDC-fed mice over time. F–G: Increasing periductal onion skin-like fibrosis of larger ducts in DDC-fed mice. H: Note that there is also subtotal obstruction of the bile duct by a porphyrin plug. pv, portal vein, bd, bile duct. Original magnification, ×20.
Figure 7
Proliferation of periductal myofibroblasts in DDC-fed mouse liver. Immunohistochemistry of α-SMA-positive cells (A) in control liver and (B–C) livers of mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (B), 4 weeks (C), and 8 weeks (D). A: In the control liver, only smooth muscle cells of hepatic artery (ha) and portal vein (pv) branches are α-SMA-positive. B–D: In contrast, there is an increasing number of α-SMA-positive myofibroblasts surrounding the bile ducts (bd) in DDC-fed mice. Note that hepatic arteries and portal veins of the control liver and DDC-fed mice are of the same size. pv, portal vein; bd, bile duct. Original magnification, ×20.
Figure 8
DDC feeding increases hepatic hydroxyproline content in mice. Hepatic hydroxyproline content was determined in liver homogenates from control diet-fed mice (Control) and mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (1w DDC), 4 weeks (4w DDC), and 8 weeks (8w DDC). Hepatic hydroxyproline content was significantly increased in 4- and 8-week-DDC-fed mice compared with controls. Values are mean ± SD from five animals per group. *P < 0.05.
Figure 9
DDC feeding induces hepatic osteopontin expression. Immunohistochemistry for osteopontin in (A) control liver and in livers of mice fed 0.1% (w/w) DDC-supplemented diet for (B, E, and F) 1 week, (C) 4 weeks, and (D and H) 8 weeks. A: In the control liver, bile ducts show some osteopontin expression (arrowheads). B–C: In contrast, in DDC-fed mice osteopontin expression is induced in hepatocytes (as indicated by the white arrowhead) in acinar zone 1 (zone of cholate stasis) and proliferating reactive cholangiocytes (indicated by black arrowheads). E: Note the immunoreactivity of hepatocytes along margin of the liver lobule (highlighted in red) with osteopontin preceding the development of portal-portal fibrous bridges. For better orientation, portal fields are framed in green. F: Higher magnification of E demonstrating the cytoplasmic osteopontin-staining pattern of hepatocytes along the margin of the liver acinus margin. pv, portal vein. Original magnifications: ×20 (A–D), ×10 (E), ×60 (F).
Figure 10
Effects of DDC feeding on hepatic Ntcp, Mrp2, Bsep, and Mrp3 protein levels. Liver membranes were isolated from control diet-fed mice (control) and mice fed 0.1% (w/w) DDC-supplemented diet for 1 week (1w DDC), 4 weeks (4w DDC), and 8 weeks (8w DDC) and analyzed by Western blotting. Densitometry data are expressed as the fold change relative to control diet-fed animals. Values are the means from three to four animals in each group. There is a significant decrease in Ntcp and Mrp2 concomitant with a significant increase in Mrp3 and Bsep protein levels in DDC-fed mice, whereas β-actin expression remained unchanged. *P < 0.05.
Figure 11
Suggested pathobiology of DDC-induced cholestatic liver disease. (1) DDC leads to a reactive phenotype of BECs with overexpression of proinflammatory and profibrogenetic cytokines and adhesion molecules, and consequently to (2) infiltration of the portal field with neutrophils (ie, pericholangitis). Activation and injury of BECs results in ductular reaction triggering (3) the formation of portal-portal septa. (4) Activation and proliferation of periductal myofibroblasts leads to increased production of extracellular matrix components and consequently to sclerosing cholangitis. (5) The formation of intraductal porphyrin plugs further promotes the development of sclerosing cholangitis and biliary fibrosis. (6) At the hepatocellular level, down-regulation of bile acid uptake systems (Ntcp, Oatp4) and up-regulation of export pumps (Mrp3, Mrp4) may represent an adaptive response of hepatocytes and, at least in part, explain pronounced increase of serum bile acid levels.
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