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Expanded biliary structures in the ductular reaction function as bile-excreting channels a CK19-CreERT;R26R-tdTomato mice were used to visualize biliary epithelial cells (BECs) with a red fluorescent protein. b Experimental design. BECs were labeled with tdTomato at 8 weeks of age and were then subjected to chronic liver injury (TAA or DDC). After 8 weeks of liver injury, the mice were analyzed by intravital imaging. Cholyl-lysyl-fluorescein (CLF)was injected immediately before intravital imaging. c, d Representative images of intravital observation of the biliary channel structure (CLF, green) and BECs (tdTomato, red) in the ductular reaction induced in the TAA (c) and DDC (d) models (n = 8 and 5 mice, respectively). The left panels show 3D reconstructed images, and center and right panels show pictures of a 2D optical section. Right panels are magnified views of the center images. Scale bars, 100 μm. e, f Representative images of intravital observation of chloromethyl fluorescein diacetate (CMFDA, green), which is metabolized in hepatocytes to form a fluorescent metabolite, and BECs (tdTomato, red) in the ductular reaction induced in the TAA (e) and DDC (f) models (n = 4 mice for each). Right panels are magnified views of the center images. Scale bars, 100 μm.
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Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells th...
Citations
... In this work, we established a mouse model, where cholangiocyte-specific RAGE ablation 420 strongly reduces CDE-diet induced DR but does not attenuate the degree of hepatocyte injury. CDE-diet induced DR in WT and RAGE HET mice showed luminal contiguity with the canalicular network in vivo, confirming previous reports 6,7 . However, the DR also induces a general dedifferentiation pressure on hepatocytes as evidenced by downregulation of basolateral bile transporters and compromised canalicular network formation. ...
... So far, our data correspond to the findings of Kamimoto et al. 445 (2020). However, different techniques were used by Kamimoto et al. (2020) and in the present work to study the transport from sinusoidal blood into hepatocytes and further into canaliculi, which require a differentiated interpretation. Kamimoto and colleagues used (non-fluorescent) CMFDA that is cleaved to (green fluorescent) 5-CMF in the cytoplasm of hepatocytes, which was secreted into the canalicular network, demonstrating that apical membrane transport in 450 hepatocytes was intact. ...
Ductular reactions inevitably co-occur with liver tissue injury along with inflammation, cholestasis, and fibrosis. However, it remains unknown whether they represent an adaptive response facilitating bile drainage, or if they exacerbate dysfunction. By using a reporter mouse with cholangiocyte-specific deletion of the pattern recognition receptor RAGE, specific effects of the DR could be differentiated from the consequences of the cytotoxicity of the CDE-diet. While CDE-diet induced hepatoxicity triggers the DR via RAGE-dependent DAMP-sensing on cholangiocytes, it does not itself lead to loss of liver function. Instead, the presence of DR causes downregulation of basolateral bile acid transporters. Thus, although the DR forms a contiguous network capable of draining bile, it remains unutilized due to interrupted bile acid transport from blood to hepatocytes. The de-differentiating influence of the DR on hepatocytes thereby results in cholestasis and eventual fibrosis, that are more insidious to liver function than its initial cytotoxic trigger.
... While these cells are vital for healthy hepatic function, approaches utilized by researchers to characterize cholangiocytes and biliary tree architecture during development and disease progression are not standardized to quantify cell number and biliary tract structure in an unbiased researcher-independent way. Current methods for the analysis of cholangiocytes include time-consuming and error-prone observer-driven manual or automated-cell counting (5,6), nonquantitative retrograde imaging of the biliary tract using India ink injection (7,8), resin cast (9,10), or various tissue-clearing methods (11,12). The latter three approaches provide visualization of disease-associated perturbations in branch architecture within the biliary tree yet lack the ability to quantify biliary cells or structures. ...
The progress of research focused on cholangiocytes and the biliary tree during development and following injury is hindered by limited available quantitative methodologies. Current techniques include two-dimensional standard histological cell-counting approaches, which are rapidly performed error-prone and lack architectural context; or three-dimensional analysis of the biliary tree in opacified livers, which introduce technical issues along with minimal quantitation. The present study aims to fill these quantitative gaps with a supervised machine learning model (BiliQML) able to quantify biliary forms in the liver of anti-Keratin 19 antibody-stained whole slide images. Training utilized 5,019 researcher-labeled biliary forms, which following feature selection, and algorithm optimization, generated an F-score of 0.87. Application of BiliQML on seven separate cholangiopathy models; genetic ( Afp-CRE; Pkd1l1 null/Fl , Alb-CRE; Rbp-jk fl/fl , Albumin-CRE; ROSA NICD ), surgical (bile duct ligation), toxicological (3,5-diethoxycarbonyl-1,4-dihydrocollidine), and therapeutic ( Cyp2c70 -/- with ileal bile acid transporter inhibition), allowed for a means to validate the capabilities, and utility of this platform. The results from BiliQML quantification revealed biological and pathological differences across these seven diverse models indicate a highly sensitive, robust, and scalable methodology for the quantification of distinct biliary forms. BiliQML is the first comprehensive machine-learning platform for biliary form analysis, adding much needed morphologic context to standard immunofluorescence - based histology, and provides clinical and basic-science researchers a novel tool for the characterization of cholangiopathies.
... 42 The expanded and remodelled BECs in DR perform as effective bile excretory conduits and constitute functional and complementary network of bile excretory ducts in damaged tissues where hepatic bile canaliculi were missing. 43 According to reports, cell proliferation in DR provides excellent relief from cholestasis-induced injury by acquiring a cholangiocyte-like phenotype through hepatocellular metaplasia. 12 We found that the deletion of Tgm2 affected the development and maturation of BECs in DDC-induced DR. ...
Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2‐knockout mice (Tgm2−/−) and Tgm2‐CreERT2‐Rosa26‐mTmG flox/flox (Tgm2CreERT2‐R26T/Gf/f) mice and performed a three‐dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC‐induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC‐induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.
... 36 Others found that thioacetamide (TAA) and DDC fed mice have a collapse of bile canaliculi that precedes DR; however, during recovery, reduced DR is paralleled with reconstruction of the bile canaliculi. 37 Therefore, DR may have a beneficial role regarding regeneration. In 2 models of HPC activation, portal HPC expansion was associated with myofibroblast activation and laminin deposition; however, the source of laminin was not evaluated. ...
Biliary fibrosis is seen in cholangiopathies, including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In PBC and PSC, biliary fibrosis is associated with worse outcomes and histologic scores. Within the liver, both hepatic stellate cells (HSCs) and portal fibroblasts (PFs) contribute to biliary fibrosis, but their roles can differ. PFs reside near the bile ducts and may be the first responders to biliary damage, whereas HSCs may be recruited later and initiate bridging fibrosis. Indeed, different models of biliary fibrosis can activate PFs and HSCs to varying degrees. The portal niche can be composed of cholangiocytes, HSCs, PFs, endothelial cells, and various immune cells, and interactions between these cell types drive biliary fibrosis. In this review, we discuss the mechanisms of biliary fibrosis and the roles of PFs and HSCs in this process. We will also evaluate cellular interactions and mechanisms that contribute to biliary fibrosis in different models and highlight future perspectives and potential therapeutics.
... Histologic analysis is routinely applied daily in clinical and research settings and is considered essential for visually assessing the structure and pathology of the tissue under study. Advances in cell-based immunohistochemical and immunofluorescent detection protocols have led to multi-faceted progress in most biological fields (Kamimoto et al., 2020;Ramachandran et al., 2020;Tan et al., 2020;Taylor, 2020;Guilliams et al., 2022). Historically, light microscopy has been utilized by pathologists to determine disease etiology as well as determining the extent or degree of damage. ...
Current means to quantify cells, gene expression, and fibrosis of liver histological slides are not standardized in the research community and typically rely upon data acquired from a selection of random regions identified in each slide. As such, analyses are subject to selection bias as well as limited subsets of available data elements throughout the slide. A whole-slide analysis of cells and fibrosis would provide for a more accurate and complete quantitative analysis, along with minimization of intra- and inter-experimental variables. Herein, we present LiverQuant, a method for quantifying whole-slide scans of digitized histologic images to render a more comprehensive analysis of presented data elements. After loading images and preparing the project in the QuPath program, researchers are provided with one to two scripts per analysis that generate an average intensity threshold for their staining, automated tissue annotation, and downstream detection of their anticipated cellular matrices. When compared with two standard methodologies for histological quantification, LiverQuant had two significant advantages: increased speed and a 50-fold greater tissue area coverage. Using publicly available open-source code (GitHub), LiverQuant improves the reliability and reproducibility of experimental results while reducing the time scientists require to perform bulk analysis of liver histology. This analytical process is readily adaptable by most laboratories, requires minimal optimization, and its principles and code can be optimized for use in other organs.
... They defined a DR as the aggregation of inflammatory cells in the liver with organized and/or disorganized proliferation of the biliary epithelium. 2 According to modern understanding, DRs are a histopathological phenomenon, which implies an increased number of ductular profiles (DPs) lined by cells of the ductal phenotype on the histological images of the liver. 3, 4 Besides, the proliferation of clusters of cells with the ductular phenotype, or even individual cells, is also considered as a DR. 1 The interest of researchers in this topic is due to the fact that DR is considered as a phenomenon closely related to carcinogenesis and regeneration. 1, 5 DRs are widely studied both in the clinic and in experimental models, especially in rodents with mass liver injury, hepatocellular carcinoma, liver resection, or common bile duct occlusion (CBDO). ...
In this paper, the features of ductular reaction (DR) and remodeling of the biliary tract in experimental models are discussed in
total and selective biliary occlusion. It has been shown that the intensity of DR, as well as the shape, number, and topography
of ductular profiles following common bile duct occlusion (CBDO) are closely related to the duration of the biliary obstruction.
In addition, the formation of new ductular profiles can occur by the widening of existing bile ducts/ductules as a result of
cholangiocyte proliferation, hepatocyte transdifferentiation, and/or activation and differentiation of stem/progenitor cells. It
has been concluded that DR induced by CBDO consists of the components of all types of DRs, including I, II (A and B), and III,
thus increasing the interest in further studies of this model. In the DR following CBDO, the consequent “preproliferative” and
“proliferative” phases developed in parallel with cells differentiation and transdifferentiation (the “para-proliferative” phase)
should be distinguished. The dynamics of these phases are important to consider for further detailed classification of DRs. During
selective biliary obstruction, the full range of DR characteristics for CBDO has not been determined (mainly the events of
biliary proliferation and fibrosis are noted). However, the great compensatory potential of the biliary bed has been confirmed,
as reflected by the formation of new collaterals between congested and noncongested bile ducts.
... 7e9 Three-dimensional confocal imaging and retrograde ink injection of noninvasive DRs exhibit defined lumens. 10,11 Invasive DR generally results from extensive hepatocyte injury and is characterized the by expansion of IHBDs into the hepatic parenchyma ( Figure 1C). In humans, invasive DR is seen in patients with nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis C. 12 Rodent models for invasive DR include choline-deficient ethionine (CDE)esupplemented diet and thioacetamide (TAA) treatment. ...
... In humans, invasive DR is seen in patients with nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis C. 12 Rodent models for invasive DR include choline-deficient ethionine (CDE)esupplemented diet and thioacetamide (TAA) treatment. 6,11 Invasive DR is hypothesized as being important to form de novo connections between bile ducts and hepatocyte canaliculi to promote bile removal and reduce bile-induced damage to hepatocytes. 13,14 Earlier characterization of human liver disease denoted noninvasive DR as typical DR and invasive DR as atypical DR. 15 However, this nomenclature has fallen out of use because it is considered somewhat subjective and implies distinct pathologic characteristics of atypical DR, such as premalignancy. ...
... For example, some mouse models of DR proceed primarily via self-replication of existing BECs, whereas others exhibit higher rates of hepatobiliary metaplasia. 11,19 Furthermore, the interaction between different cellular responses to BEC stress and injury is likely critical for certain cholangiopathies, such as the interaction between inflammation-induced cell death and senescence observed in PSC. 72 Although this list of common cellular responses is not exhaustive, it is a conceptual starting point for approaching the complex cell biology of BEC injury and disease. ...
In the liver, biliary epithelial cells (BECs) line intrahepatic bile ducts (IHBDs) and are primarily responsible for modifying and transporting hepatocyte-produced bile to the digestive tract. BECs comprise only 3-5% of the liver by cell number but are critical for maintaining choleresis through homeostasis and disease. To this end, BECs drive an extensive morphological remodeling of the IHBD network termed ductular reaction (DR) in response to direct injury or injury to the hepatic parenchyma. BECs are also the target of a broad and heterogenous class of diseases termed cholangiopathies, which can present with phenotypes ranging from defective IHBD development in pediatric patients to progressive periductal fibrosis and cancer. DR is observed in many cholangiopathies, highlighting overlapping similarities between cell and tissue-level responses by BECs across a spectrum of injury and disease. Here, we propose a core set of cell biological BEC responses to stress and injury that may moderate, initiate, or exacerbate liver pathophysiology in a context-dependent manner: cell death, proliferation, transdifferentiation, senescence, and acquisition of neuroendocrine phenotype. By reviewing how IHBDs respond to stress, we seek to highlight fundamental processes with potentially adaptive or maladaptive consequences. A deeper understanding of how these common responses contribute to DR and cholangiopathies may identify novel therapeutic targets in liver disease.
... This revealed, in addition to the known intracellular canaliculi (Fig. 4A,B), a substantial proportion of intercellular canaliculi, shared between the membranes of neighbouring hepatocytes (Fig. 4A,C,D; Movie 3), demonstrating plasticity of canalicular topology in zebrafish. Altered canalicular topologies are also observed upon chronic liver injury in mammals (Clerbaux et al., 2019;Kamimoto et al., 2020), further suggesting that canaliculi topology is generally more plastic than previously known and that common mechanisms of canaliculi formation exist between zebrafish and mammals. ...
Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.
... BECs represent only 3-5% of the liver by mass but can produce up to 40% of daily bile output volume [9]. While normally quiescent, BECs demonstrate extensive remodeling and proliferation following liver injury, in a process called "ductular reaction" [10][11][12]. Though BECs of the intrahepatic and extrahepatic biliary tree share many biomarkers and make up a continuous ductal network, these cells have distinct embryological origins [13,14]. ...
Epithelial tissues comprise heterogeneous cellular subpopulations, which often compartmentalize specialized functions like absorption and secretion to distinct cell types. In the liver, hepatocytes and biliary epithelial cells (BECs; also called cholangiocytes) are the two major epithelial lineages and play distinct roles in (1) metabolism, protein synthesis, detoxification, and (2) bile transport and modification, respectively. Recent technological advances, including single cell transcriptomic assays, have shed new light on well-established heterogeneity among hepatocytes, endothelial cells, and immune cells in the liver. However, a “ground truth” understanding of molecular heterogeneity in BECs has remained elusive, and the field currently lacks a set of consensus biomarkers for identifying BEC subpopulations. Here, we review long-standing definitions of BEC heterogeneity as well as emerging studies that aim to characterize BEC subpopulations using next generation single cell assays. Understanding cellular heterogeneity in the intrahepatic bile ducts holds promise for expanding our foundational mechanistic knowledge of BECs during homeostasis and disease.
... The assumption about the participation of DR in regeneration, adaptation, and carcinogenesis attracts much interest to the study of DR in various experimental models to clarify the zones and origins of the development of ductular profiles (DP), their relationship with existing cholangiocytes, stem cells, or transdifferentiated hepatocytes; the question of whether all DP are associated with the bile drainage pathway has not been resolved [7,8]. ...
Ductular reaction develops during liver regeneration, fibrosis, and carcinogenesis. However, the types, stages of formation, and topography of ductular profiles in various pathologies remain insufficiently studied. Using the model of common bile duct occlusion, we showed that the number and topography of ductular profiles are closely related to the duration of biliary obstruction. The ductular profiles can be located inside the portal tract, along the existing bile ducts, and/or intramurally, around the portal vein, periportally, inside the lobules, in the portocaval fibrous connections, in the adventitia of the hepatic veins, in the septs connecting the portal tracts, and also in the "portal plate" of the liver. The ductular profiles can be formed as a result of expansion of existing bile ducts, cholangiocyte proliferation, as well as transdifferentiation of hepatocytes and activation of mesenchymal stem cells.
