Guicciardi, Maria Eugenia

Cholangiopathies comprise a heterogeneous group of chronic liver diseases characterised by progressive bile duct injury, ductular reaction and fibrotic remodelling. Despite distinct aetiologies, these disorders share common cellular stressors that ultimately converge on cholangiocyte dysfunction and loss. In this context, programmed cell death has emerged as a central mechanism linking epithelial stress, immune activation and the development of biliary fibrosis. A range of regulated cell death pathways, including apoptosis, necroptosis, pyroptosis, ferroptosis and autophagy-dependent cell death, can be triggered in cholangiocytes in a disease-dependent and context-dependent manner. Growing evidence suggests that these pathways rarely act in isolation. Instead, they intersect through shared molecular nodes, forming integrated cell death programmes such as PANoptosis. Importantly, it is becoming clear that not only overt cell death but also sublethal activation of death-related signalling can perpetuate cholangiocyte dysfunction and fuel chronic disease progression. Current therapeutic strategies, such as ursodeoxycholic acid and peroxisome proliferator-activated receptor agonists, mainly aim to modify bile composition and improve biochemical parameters. However, they do not directly target the molecular networks governing cholangiocyte death, which may partly explain their limited impact on fibrosis and long-term outcomes. In this review, we synthesise current knowledge on regulated cell death mechanisms in cholangiopathies, highlight key conceptual and technical challenges that complicate their interpretation and therapeutic targeting, and discuss how sublethal death signalling may connect with senescence to sustain cholangiopathy progression and shape therapeutic perspectives.

Cholangiocyte pathophysiological activation was examined in a model of murine cholestasis. CX3CR1 + macrophages are recruited to the periportal region, likely mediated by cholangiocyte expression of CX3CL1. Cholangiocyte transcriptomics from cholestatic mice display activation of a “pathogen-induced cytokine storm” pathway, and exhibit activation of the transcription factor NF-Y. In human cholangiocytes, NF-Y promotes expression of the profibrogenic ligand TGFβ1. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.

Abstract Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor‐mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor‐induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. © 2013 American Physiological Society. Compr Physiol 3:977‐1010, 2013.

Cholangiopathies comprise a spectrum of chronic intrahepatic and extrahepatic biliary tract disorders culminating in progressive cholestatic liver injury, fibrosis, and often cirrhosis and its sequela. Treatment for these diseases is limited, and collectively, they are one of the therapeutic “black boxes” in clinical hepatology. The etiopathogenesis of the cholangiopathies likely includes disease-specific mediators but also common cellular and molecular events driving disease progression (eg, cholestatic fibrogenesis, inflammation, and duct damage). The common pathways involve cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts, which are central to the pathogenesis of these disorders. Current information suggests that cholangiocytes function as a signaling “hub” in biliary tract-associated injury. Herein, we review the pivotal role of cholangiocytes in cholestatic fibrogenesis, focusing on the crosstalk between cholangiocytes and portal fibroblasts and HSCs. The proclivity of these cells to undergo a senescence-associated secretory phenotype, which is proinflammatory and profibrogenic, and the intrinsic intracellular activation pathways resulting in the secretion of cytokines and chemokines are reviewed. The crosstalk between cholangiocytes and cells of the innate (neutrophils and macrophages) and adaptive (T cells and B cells) immune systems is also examined in detail. The information will help consolidate information on this topic and guide further research and potential therapeutic strategies for these diseases.

Background: The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study, we aimed to investigate and characterize the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Methods: Cholangiocytes obtained from patients with PSC and without PSC by endoscopic retrograde cholangiography were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing. Results: Unsupervised clustering of all integrated single-cell RNA sequencing data identified 8 cholangiocyte clusters that did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, as noted by an increased number of differentially expressed genes by transcriptomics and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, genome sequencing identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. Conclusions: PSC and non-PSC patient-derived ECO respond differently to IL-17 stimulation, implicating this pathway in the pathogenesis of PSC.

Abstract The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Background and Aims In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug‐resistant 2 (Mdr2−/− ) double knockout (DKO) mice lacking tumor necrosis factor–related apoptosis‐inducing ligand receptor (Tr−/− ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2−/− mice. This observation suggests that the magnitude of the DR‐cell population may be regulated by apoptosis. Approach and Results To examine this concept, we cultured epithelial cell adhesion molecule–positive reactive cholangioids (ERCs) obtained from wild‐type (WT), Tr−/− , Mdr2−/− and DKO mice. Single‐cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR‐cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3‐mimetic therapy, significantly decreased DKO and Mdr2−/− ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR‐cell population and markers of inflammation and liver fibrosis in Mdr2−/− and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high‐definition mass cytometry by time‐of‐flight. Coculture of bone marrow–derived macrophages with ERCs from DKO mouse livers up‐regulated expression of the B cell–directed chemokine (C‐C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl‐2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. Conclusions DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR‐cell and B‐cell populations and hepatic fibrosis.

Cholangiocytes are the target of a group of chronic liver diseases termed the “cholangiopathies,” in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.