Lineage fate of ductular reactions in liver injury and carcinogenesis

Abstract

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.

Figure 4. DDC-induced DRs do not escape their biliary fate after genetic ablation of NOTCH and/or activation of WNT/β-catenin signaling.

(A) For HNF1β compartment-specific genetic deletion of the NOTCH-adaptor protein RBP-Jκ and/or stable expression of the WNT effector β-catenin, R26^Tom Rbpj^fl/fl Hnf1b-CreER (n = 8), R26^Tom Catnb^Δex3 Hnf1b-CreER (n = 5), and R26^Tom Catnb^Δex3 Rbpj^fl/fl Hnf1b-CreER (n = 4) animals were injected with tamoxifen (i.p., 100 μg/g BW at week 5) and subjected to 2 weeks of DDC diet (week 6–8). Equally treated R26^Tom Hnf1b-CreER animals (n = 8) served as controls. Representative co-IF images for tdTom, tdTom/CK19, tdTom/Ki67, and tdTom/acetylated tubulin are shown that demonstrate reduced proliferative expansion and impaired biliary maturation of tdTom⁺ DRCs after both NOTCH inactivation and/or WNT/ β-catenin activation (arrows indicate tdTom⁺ DRCs that coexpress Ki67 or acetylated tubulin). In all models, DRCs failed to undergo lineage conversion to hepatocytes, as assessed by tdTom/HNF4α co-IF analysis. (B) Quantification of co-IF data (mean value ± SEM expressed in percentage; *P < 0.05, **P < 0.01, ***P < 0.001 [1-way ANOVA followed by Tukey’s post-test]). Scale bar: 100 μm (first column); 20 μm (second through fifth columns).

Figure 5. DRCs do not give rise to tumors in selected mouse HCC models.

(A) R26^Tom Hnf1b-CreER animals (tamoxifen at P10) were injected with DEN (25 mg/kg BW) and analyzed at 10 to 12 months after DEN treatment (n = 6). Representative IHC micrographs of serial sections obtained from one DEN-induced tumor nodule stained for collagen IV (Col IV), Ki67, GP73, or AFP. Representative macroscopic transmitted light and fluorescent whole-liver images from one DEN-treated animal show all nodular lesions (encircled by dashed lines) negative for tdTom. Co-IF analysis for tdTom and collagen IV revealed sporadic tdTom⁺ DRCs (asterisks) at the tumor margins and within the tumor nodules, but tumor cells never costained for tdTom (tumor nodules obtained from different animals are encircled by dashed lines; insets are shown at higher magnification below). (B) Likewise, characterization of tumors observed in Mdr2^–/– R26^Tom Hnf1b-CreER animals (tamoxifen at P21) was performed by IHC for collagen IV, Ki67, GP73, and AFP in 12-month-old animals; nodule formation was observed in 4 out of n = 6 animals. Representative serial cryosections of tumor nodules processed from different mice for H&E and co-IF for tdTom and collagen IV indicate tumor cells negative for tdTom (encircled by dashed lines). tdTom⁺ DRCs (asterisks) are typically observed outside tumor margins and rarely found within. Tu, tumor. Scale bar: 1,000 μm (low-magnification H&E, top panel, B); 500 μm (H&E mouse 1–3, B); 100 μm (low-magnification H&E, IHC, and top row IF, A; high-magnification H&E, IHC, and top row IF, B); 20 μm (high-magnification H&E and bottom row IF, A; bottom row IF, B).

Figure 3. Minimal contribution of DRCs to hepatocyte neogenesis after long-term liver injury.

(A) We traced the fate of lineage-labeled DRs in R26^Tom Hnf1b-CreER animals after prolonged DDC diet (up to 8 months) or CDE diet (4 months) as well as lineage-labeled DRs in Mdr2^–/– R26^Tom Hnf1b-CreER animals (up to 12 months). Despite dramatic expansions of A6⁺ (IHC) or tdTom⁺/CK19⁺ (IF) DRs in all long-term injury models, only mice fed CDE diet for 4 months displayed a small fraction of HNF4α⁺ hepatocytes coexpressing tdTom (IF, yellow arrows). Representative pictures are shown of n = 4 to 6 animals analyzed per group. (B) Proportion of HNF4α⁺ hepatocytes that coexpress tdTom. tdTom⁺/HNF4α⁺ hepatocytes (mean value ± SEM) were quantified from n = 4 to 6 animals per group. (C) In the DDC model, IHC analysis for A6 and SOX9 shows hepatocytes and cells of intermediate morphology that express biliary markers (arrows and arrowheads in IHC panels), with increasing time of injury surrounding typical A6⁺ DRs. Co-IF analysis for tdTom, A6, and SOX9 revealed no coexpression of tdTom in A6⁺ or SOX9⁺ hepatobiliary cells (arrows and asterisks), indicating their hepatocyte origin (for quantification, refer to Supplemental Figure 5). Scale bar: 100 μm (top 2 rows, A); 20 μm (bottom 2 rows, A, and C).

Figure 2. DRs arise from the biliary HNF1β⁺ compartment in a broad range of liver injury models.

(A) Seven days after tamoxifen injection, R26^Tom Hnf1b-CreER mice were subjected to 2 to 3 weeks of various short-term liver injury models (DDC diet, BDL, MDA, CDE diet). For fate mapping of DRs in MDR2-deficient mice, Mdr2^–/– R26^Tom Hnf1b-CreER animals received tamoxifen at week 3 to 4 and were analyzed at week 8. (B) In all models, characteristic DRs were assessed by IHC for CK19. Regardless of the mode of injury co-IF analysis revealed tdTom expression in most CK19⁺ cells, while expression of tdTom in HNF4α⁺ hepatocytes was consistently only observed in the CDE model (yellow arrows). (C) Colocalization (mean value ± SEM) of tdTom⁺/CK19⁺ and tdTom⁺/HNF4α⁺ cells expressed as percentage colocalization (DDC diet, n = 9; BDL, n = 4; MDA, n = 4; Mdr2^–/–, n = 4; CDE diet, n = 5). n.d., not detected. Scale bar: 100 μm (first and third rows); 20 μm (second, fourth, and fifth rows).

Figure 1. Efficient and specific inducible lineage labeling of the biliary compartment in R26^Tom Hnf1b-CreER animals.

(A) In the adult mouse, HNF1β was specifically expressed in bile ducts and biliary ductules/canals of Hering (arrows). HNF1β⁺ cells were identical with SOX9⁺ and CK19⁺ cells, as assessed by co-IF (HNF1β⁺/SOX9⁺: 97.04% ± 0.52%, n = 3; HNF1β⁺/CK19⁺: 99.89% ± 0.10%, n = 5), while HNF1β expression in HNF4α⁺ hepatocytes was virtually absent (0.008% ± 0.004%, n = 8). (B) Cre-induced expression of the fluorescent dye tdTom was determined in 5- to 6-week-old R26^Tom Hnf1b-CreER reporter mice 7 days after a single tamoxifen injection. tdTom expression was restricted to HNF1β⁺/SOX9⁺/CK19⁺ bile ducts and periportal ductules (n = 5), whereas labeling of HNF4α⁺ hepatocytes was a rare event (0.012% ± 0.003%, n = 10). All periportal areas revealed a high tdTom labeling efficacy of the HNF1β⁺ compartment (>30 portal tracts per mouse were quantified at ×600 magnification from 3 discontinuous sections; mean labeling efficiency: 84.3% ± 0.6%; n = 5 animals). BD, bile duct; PV, portal vein; Tam, tamoxifen. Scale bar: 20 μm.