Congenital Hepatic Fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure, however the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1del4/del4) mouse, which is orthologous of CHF, we show that Pkhd1del4/del4 cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including CXCL1, CXCL10 and CXCL12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1del4/del4 cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local TGFβ1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. Conclusion our results show that fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment and collagen deposition. These findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis and macrophage polarization over time.

Background and Aims Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. Approach and Results Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine‐rich repeat–containing G‐protein‐coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional‐specific markers corresponding to their location of origin. Of particular interest, down‐regulation of biliary markers and up‐regulation of cell‐cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. Conclusions Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

Background & Aims Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling. Methods Treatment with DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild type mice and in mice with a liver specific defect in the Notch-2 receptor (Notch-2-cKO) or in RPB-Jk. Hepatic progenitor cells, ductular reaction, and mature ductules were quantified using K19 and SOX-9. Results In DDC treated wild type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2-cKO mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jk-cKO mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild type mice. The mouse progenitor cell line BMOL cultured on matrigel, formed a tubular network allowing the study of tubule formation in vitro; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1, Notch-2 or Jagged-1 significantly reduced both the length and number of tubular branches. Conclusions These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro. Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.

Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning human hepatic cell development. Here, we addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We utilised this information to derive bipotential hepatoblast organoids and then exploited this novel model system to validate the importance of key signalling pathways and developmental cues. Furthermore, these insights into hepatic maturation enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a new platform to investigate the basic mechanisms of human liver development and to produce cell types for clinical applications.

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Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2 defective mice, cAMP/PKA-dependent activation of ERK/mTOR signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca2+ and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO mice (Pkd2flox/-:pCxCreER™ – hence called Pkd2KO) and compared to cholangiocytes from Wild Type mice (WT). Our results show that, compared to wild type cells, in PC2 -defective cholangiocytes (Pkd2KO) cytoplasmic and ER-Ca2+ (measured with Fura-2 and Mag-Fluo4) levels are decreased, store-operated Ca2+ entry (SOCE) is inhibited, while the expression of Ca2+-sensor STIM1 and of store-operated Ca2+ channels (Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca2+ depletion increases [cAMP] and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase 6 (AC6). Conclusion these data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca2+ depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, while coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD due to PC2-deficiency represents the first example of human disease linked to inappropriate activation of “Store-operated cAMP production” (SOcAMP).

Mutations in polycystins (PC1 or PC2) are a cause of polycystic liver disease (PLD-ADPKD). In PC2-defective mice, cAMP/PKA-dependent activation of the Ras/Raf/MEK-ERK1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf-inhibitor used for the treatment of liver and kidney cancers, inhibits liver cysts growth in PC-2 defective mice, we treated PC2 (i.e., Pkd2flox/−:pCxCreERTM, abbreviated as Pkd2cKO) mice with sorafenib-tosylate for 8 weeks (20–60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67) and expression of pERK compared to Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation (MTS and BrdU) at doses between 0.001 and 1 µM; but, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 µM. Raf kinase activity assay showed that, while B-Raf is inhibited by sorafenib in both WT and Pkd2cKO cells, Raf-1 is inhibited in WT cells, but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO-cells pretreated with a PKA inhibitor (PKI 1µM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished and cyst growth was inhibited. Conclusions In PC2-defective cells, sorafenib inhibits B-Raf, but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation and cyst growth in vivo. These effects are consistent with the ability of Raf-inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib.