Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease

Abstract

During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.

Figure 1. Spatial regulation of the HPC niche is dependent on adult disease pattern

(a) Upper photomicrographs: Healthy adult liver with panCK positive mHPCs, F4/80 positive macrophages, αSMA positive myofibroblasts with collagen–1 surrounding the vasculature. In biliary regeneration (middle photomicrographs) αSMA positive myofibroblasts, as well as collagen–I surround mHPCs, F4/80 macrophages around the PT but not associated with the mHPCs. During hepatocellular (lower photomicrographs) damage mHPCs are associated with both F4/80 positive macrophages and diffuse αSMA positive myofibroblasts, with little collagen deposition around the mHPCs. (b) 3D reconstruction of the mHPC niche. mHPCs (red, denoted with white arrows) expand as pseudo–ducts or chords of mHPCs in biliary and hepatocyte regeneration respectively. In biliary regeneration mHPCs are closely associated with myofibroblasts (green) close to the portal tract between the mHPCs and macrophages (blue). During hepatocyte regeneration there is little association of myofibroblasts (green) and mHPCs (red), macrophages (blue) are closely associated with mHPCs. (c) Addition of collagen–I to the 3–D constructs reveals that there is an extracellular barrier between mHPCs and macrophages that is more prominent during biliary injury and regeneration than hepatocellular injury and regeneration.

Figure 2. Modulation of the Notch pathway in vitro and in vivo affects biliary regeneration

(a) mRNA expression of the NOTCH signalling pathway components in isolated ductular reactions of both hepatocellular and biliary patterns of disease. Immunohistochemistry for NOTCH2 and JAGGED1 with positivity during biliary disease (upper photomicrographs), however in hepatocellular regeneration only NOTCH2 protein is detectable (lower photomicrographs). (b) Notch pathway expression in murine models of biliary (upper photomicrographs) and hepatocellular (lower photomicrographs) regeneration; Notch1 protein (red) in the EpCAM positive biliary tree (green) and Jagged1 (red) in the surrounding ductular stromal αSMA positive fibroblasts (green). (c) mRNA expression of Notch targets and liver enriched transcription factors after inhibition of the Notch pathway with DAPT in direct co–cultures of mHPCs and myofibroblasts in vitro (d) Quantification of absolute mHPC numbers in animals treated with DAPT in vivo vs. vehicle alone control. (e) Expression of Notch pathway targets and liver enriched transcription factors in mHPCs isolated from DAPT treated animals vs. vehicle alone controls. Data is expressed as means ± S.E.M; *P < 0.05, ** P < 0.01, ***P <0.001. Human: PBC/PSC n = 10 HCV n = 6. Murine CDE n = 4 DDC n = 4. Scale bar = 50μm

Figure 3. Numb represses the Notch signalling pathway and allows hepatocyte differentiation

(a) NUMB mRNA expression and presence (white arrows) or absence (black arrows) of NUMB protein in human ductular reactions isolated from HCV or PSC/PBC disease patterns. (b) Expression of murine Numb protein in healthy liver and during hepatocyte regeneration (black arrows) the loss of Numb in mHPCs during biliary regeneration is highlighted (red arrows); these changes are reflected in Numb mRNA expression levels from isolated mHPCs. (c) Expression levels of Hnf4α and Numb in isolated mHPCs when stimulated with Wnt3a in vitro. The Numb protein (green) can be identified in the mHPC BMOL cell line using immunohistochemistry. (d) Expression of Numb, Notch pathway targets and liver enriched transcription factors in response to Numb RNAi, a similar affect is seen using a second independent Numb sequence (Supplementary Fig 3b). Data is expressed as means ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001. Human: PBC/PSC n = 10 HCV n = 6. Murine CDE n = 4 DDC n = 4. Scale bar = 50μm. In vitro n = 12.

Figure 4. The Wnt pathway drives exit of mHPCs from a biliary fate into a hepatocyte phenotype

(a) Photomicrographs demonstrating immunohistochemistry for nuclear Ctnnb1 in mHPCs during hepatocellular and biliary regeneration. Relative expression of canonical Wnt targets Axin2, Myc, Sox9 and Twist1 in mHPCs from hepatocellular vs. biliary regeneration. (b) Immunohistochemistry for stabilized Ctnnb1 (black arrows) in Krt19–Cre expressing cells harbouring the Ctnnb1^ΔEx3 or Ctnnb1^WT locus . Central photomicrographs showing nuclear Ctnnb1 in hepatocytes of Ctnnb1^ΔEx3 mutants or Ctnnb1^WT (inset image) after biliary damage; Quantification of hepatocytes with nuclear Ctnnb1 in animals harbouring the Ctnnb1^ΔEx3 vs. Ctnnb1^WT. Data is expressed as means ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001.CDE n = 4 DDC n = 4, Wnt3a treated HPCs n = 6, Fed BMDMs n = 6, LRP5/6 n = 4 – 6. Scale bar = 50μm

Figure 5. Macrophages are a source of canonical Wnt ligand in the regenerating adult liver

(a) Expression of the canonical ligand Wnt3a in F4/80 positive macrophages isolated from healthy animals versus animals undergoing biliary or hepatocyte regeneration. The Wnt3a protein (red) localised around mononuclear cells adjacent to mHPCs (green) during hepatocyte regeneration (b) Quantification of Wnt3a and Wnt7a expression by macrophages cultured with hepatocyte debris, latex beads or liposomes in vitro. (c) Gene expression analysis of Wnt pathway targets and liver enriched transcription factors in co–culture of post–phagocytic macrophages and mHPCs in the presence of rhWIF1 or vehicle alone. Data is expressed as means ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001.CDE n = 4 DDC n = 4, Fed BMDMs n = 6, co–cultures n = 12. Scale bar = 25μm

Figure 6. Ablation of Macrophages in vivo results in the re–specification of mHPCs

(a) Upper panels panCK positive mHPCs during hepatocyte regeneration in the presence or absence of macrophages; lower panels, localisation of Ctnnb1 in mHPCs during hepatocyte regeneration in the presence or absence of macrophages. (b) Transcript expression of Wnt pathway targets and liver enriched transcription factors in mHPCs isolated from animals depleted for macrophages compared to PBS controls (c) Photomicrographs of Hnf1β (upper panels) and Hnf4α (lower panels) in mHPCs of livers depleted of macrophages or controls. This positivity is quantified as absolute number of Hnf1β or Hnf4α positive cells (upper and lower histogram respectively) in control vs. macrophage depleted animals. Data is expressed as means ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001. liposomal clodronate and control n = 10. Scale bar = 100μm