DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-induced model of biliary fibrosis

DDC feeding induces bile duct injury, cholangitis, ductular reaction, and portal-portal bridging fibrosis [3], recapitulating clinical features of human biliary fibrosis. Mice at the age of 8 weeks were fed a control diet or a DCC-supplemented diet (0.1%) for 3 weeks to induce advanced biliary fibrosis as described [23]. The differences between DDC-fed mice and healthy control animals (receiving control diet) were calculated for each group. The alterations in values between WT or Cd39^fl/fl and CD39 deficient animals were contrasted, by using the WT/Cd39^fl/fl delta as reference.

Immunohistochemistry and immunofluorescence were performed as previously reported (24, 25). Primary antibodies were F4/80 (#MCA497; Bio-Rad, Hercules, CA, USA) and CD39 for IF (C9F, kind gift from Jean Sévigny, Centre de Recherche du CHU de Québec, Université Laval, Quebec, Canada), as well as CD39 (#AF-4398; R&D Systems, Minneapolis, MN, USA) and pan-Cytokeratin for IHC (#Z0622; Dako, Agilent Technologies, Santa Clara, CA, USA). In DDC-fed mice, staining was developed using ImmPACT VIP Peroxidase (#SK-4605, Vector Laboratories, Burlingame, CA), which results in a purple reaction product distinct from the brownish porphyrine plugs present in DDC-fed livers. In all other animals, ImmPACT DAB Peroxidase (#SK-4100, Vector Laboratories) for brown color development was used. All slides were examined and recorded on a Nikon microscope. Immunofluorescence was performed on acetone-fixed frozen liver sections with DAPI as a nuclear counterstain. Images were captured using Zeiss Axioimager M1 (Carl Zeiss, Oberkochen, Germany).

Hepatic collagen was quantified using a biochemical assay that measures the hydroxyproline content. The assay was performed using two liver sections from the median and left lobe (250–300 mg in total, representing at least 10% of total liver volume), as previously described [24]. Hepatic collagen content was expressed as relative hepatic hydroxyproline levels (per 100 mg of wet liver).

Serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (TBIL) were measured on automated Catalyst Dx Chemistry Analyzer (IDEXX Laboratories, Inc., Westbrook, ME) according to the manufacturer’s instructions.

Peritoneal macrophages were harvested from the peritoneal cavity of adult Cd39^fl/fl, Cd39^−/−, or LysMCreCd39^fl/fl mice by lavaging the cavity with cold PBS. Peritoneal cells were washed and thereafter analyzed by flow cytometry.

Peritoneal macrophages or splenocytes were stained with PB CD11b (clone M1/70, #101223), FITC CD19 (clone 6D5, #115506), APC CD45R/B220 (clone RA3-6B2, #103211) (BioLegend, San Diego, CA) FITC F4/80 (clone BM8, #11-4801-82), PE CD39 (clone 24DMS1, #12-0391-82) (Thermo Fisher, Burlingame, CA). Cells were incubated at 4 °C in the dark for 30 min and washed with phosphate-buffered saline, supplemented with 1% fetal calf serum, before analysis was made. For surface marker analysis, macrophages were gated based on the F4/80 + CD11b + population, whereas splenic B-cells were identified based on CD19 and B220 (CD45R) expression. Samples were analyzed using GalliosTM flow cytometer (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter).

At harvest, liver tissue was snap frozen in liquid nitrogen and stored at − 80 °C until further processing. Total RNA was isolated from frozen liver tissue using TRIzol® reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcription of total RNA was done using the iScriptTM cDNA Synthesis kit (Bio-Rad, Hercules, CA). Real-time quantitative polymerase chain reaction (qRT-PCR) was performed on a Stratagene Mx3005P (Agilent Technologies, Santa Clara, CA) with the TaqMan and SYBR Green methodology. Sequences of primers and probes are summarized in Supplementary Table 1.

Data are expressed as mean ± standard error of mean and were analyzed using Graph-Pad Prism version 5.0 (GraphPad Software, San Diego, CA). Statistical comparisons between two groups were performed by the Student’s t test. P values < 0.05 were considered significant.