Fig 1.
DDC diet induces biliary damage in mice.
(A) Bilirubin and ALT serum levels were measured from control animals and after 7 and 14 days of DDC-diet with a clinical chemistry module. (B, C) Liver sections of control animals and mice subjected to DDC-Diet for 7 and 14 days were stained with HE, anti-F4/80 Ab, anti-CK19 Ab, Sirius Red and anti- Laminin Ab as described in material and methods. All images were taken in 40x original magnification. The percentage of F4/80-, CK19-, PicroSirius- and Laminin -positive area per field was assessed as described in detail in materials and methods section. All data are presented as mean ± SEM for n = 5/group, *p<0.05 and ***p<0.001.
Fig 2.
Macrophage depletion attenuates activation of myofibroblast like cells and preserves a type I ductular reaction in DDC fed mice.
(A) Bilirubin and ALT serum levels were measured from control animals and mice subjected to a 14 day DDC diet in co-treatment with either CLOLipo or PBSLipo at experimental day 7. (B) Liver sections were stained with HE, anti-F4/80 Ab, anti-CK19 Ab, Sirius Red, anti-Laminin Ab and anti-alpha-Sma Ab as described in material and methods. All single stained images were taken in 20x, original magnification. Orange arrow indicate irregular CK19+ cell clusters migrating from the portal tract into parenchyma or porto-portal (Type II DR). The percentage of F4/80-, CK19-, Sirius red-, Laminin- and αSma- positive area per field was assessed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Overall amount of laminin matrix deposition and maximum migration distance of CK19 positive cell clusters is positively correlated.
(A) Liver sections of control animals and mice subjected to a 14 day DDC-Diet in co-treatment with either CLOLipo or PBSLipo at experimental day 7, were co-stained with CK19/Laminin to assess the correlation between CK19+ cell clusters and ECM. (B) Positive correlation between overall amount of laminin matrix deposition. (C) Maximum migration distance of CK19+ cell clusters from the portal vicinity into the lobule. All stainings and quantifications were performed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group, *p<0.05 and ***p<0.001.
Fig 4.
Under macrophage depletion proliferation of CK19 positive ductules in the portal region is inhibited.
(A) Liver sections of the same mouse collective were co-stained with CK19/Ki67 to visualize the proliferation index. All images were taken in 40x original magnification. PV = Portal vein. White arrow indicating porphyrin deposition. (B) To quantify the proliferation index, the quantity of Ki 67-positive cells/field was determined. (C) To obtain the extent of apoptosis, TUNEL stainings were carried out (S8 Fig) and the amount of TUNEL pos. cells per field was quantified. All stainings and quantifications were performed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group, ***p<0.001.
Fig 5.
Schematic representation of the effect of macrophage depletion during DDC diet administration.
DDC diet promotes a ductular reaction (DR) budding from resident bile ducts (rBD) chaperoned by laminin rich matrix. In PBSLipo co-treated mice (from day 7 onwards), a DR type II can be observed with CK19+ cells either forming amorphous bile ducts, clusters with pseudoluminae or rosettes, sprouting into the lobule. CLOLipo co-treatment (from day 7 onwards) preserves a type I DR, comprising confined ductular proliferates in proximity to the portal vein (PV). The newly budding CK19+ cells create more differentiated, bile ductular structures with regular concentric luminae.