Low-dose biliatresone treatment of pregnant mice causes subclinical biliary disease in their offspring: Evidence for a spectrum of neonatal injury.

Biliary atresia is a neonatal disease characterized by damage, inflammation, and fibrosis of the liver and bile ducts and by abnormal bile metabolism. It likely results from a prenatal environmental exposure that spares the mother and affects the fetus. Our aim was to develop a model of fetal injury by exposing pregnant mice to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock, and then to determine whether there was a hepatobiliary phenotype in their pups. Pregnant mice were treated orally with 15 mg/kg/d biliatresone for 2 days. Histology of the liver and bile ducts, serum bile acids, and liver immune cells of pups from treated mothers were analyzed at P5 and P21. Pups had no evidence of histological liver or bile duct injury or fibrosis at either timepoint. In addition, growth was normal. However, serum levels of glycocholic acid were elevated at P5, suggesting altered bile metabolism, and the serum bile acid profile became increasingly abnormal through P21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at P21. These results suggest that prenatal exposure to low doses of an environmental toxin can cause subclinical disease including liver inflammation and aberrant bile metabolism even in the absence of histological changes. This finding suggests a wide potential spectrum of disease after fetal biliary injury.

Human vascularized bile duct-on-a chip: a multi-cellular micro-physiological system for studying cholestatic liver disease.

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity ofin vitromodels that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured inex vivosystems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Glisson's capsule matrix structure and function is altered in patients with cirrhosis irrespective of aetiology.

Background & Aims: Glisson's capsule is the interstitial connective tissue that surrounds the liver. As part of its normal physiology, it withstands significant daily changes in liver size. The pathophysiology of the capsule in disease is not well understood. The aim of this study was to characterise the changes in capsule matrix, cellular composition, and mechanical properties that occur in liver disease and to determine whether these correlate with disease severity or aetiology. Methods: Samples from ten control patients, and six with steatosis, seven with moderate fibrosis, and 37 with cirrhosis were collected from autopsies, intraoperative biopsies, and liver explants. Matrix proteins and cell markers were assessed by staining and second harmonic generation imaging. Mechanical tensile testing was performed on a test frame. Results: Capsule thickness was significantly increased in cirrhotic samples compared with normal controls irrespective of disease aetiology (70.12 ± 14.16 µm and 231.58 ± 21.82 µm, respectively), whereas steatosis and moderate fibrosis had no effect on thickness (90.91 ± 11.40 µm). Changes in cirrhosis included an increase in cell number (fibroblasts, vascular cells, infiltrating immune cells, and biliary epithelial cells). Key matrix components (collagens 1 and 3, hyaluronan, versican, and elastin) were all deposited in the lower capsule, although only the relative amounts per area of hyaluronan and versican were increased. Organisational features, including crimping and alignment of collagen fibres, were also altered in cirrhosis. Unexpectedly, capsules from cirrhotic livers had decreased resistance to loading compared with controls. Conclusions: The liver capsule, similar to the parenchyma, is an active site of disease, demonstrating changes in matrix and cell composition as well as mechanical properties. Impact and implications: We assessed the changes in composition and response to stretching of the liver outer sheath, the capsule, in human liver disease. We found an increase in key structural components and numbers of cells as well as a change in matrix organisation of the capsule during the later stages of disease. This allows the diseased capsule to stretch more under any given force, suggesting that it is less stiff than healthy tissue.

Circadian Disruption Primes Myofibroblasts for Accelerated Activation as a Mechanism Underpinning Fibrotic Progression in Non-Alcoholic Fatty Liver Disease.

Circadian rhythm governs many aspects of liver physiology and its disruption exacerbates chronic disease. CLOCKΔ19 mice disrupted circadian rhythm and spontaneously developed obesity and metabolic syndrome, a phenotype that parallels the progression of non-alcoholic fatty liver disease (NAFLD). NAFLD represents an increasing health burden with an estimated incidence of around 25% and is associated with an increased risk of progression towards inflammation, fibrosis and carcinomas. Excessive extracellular matrix deposition (fibrosis) is the key driver of chronic disease progression. However, little attention was paid to the impact of disrupted circadian rhythm in hepatic stellate cells (HSCs) which are the primary mediator of fibrotic ECM deposition. Here, we showed in vitro and in vivo that liver fibrosis is significantly increased when circadian rhythm is disrupted by CLOCK mutation. Quiescent HSCs from CLOCKΔ19 mice showed higher expression of RhoGDI pathway components and accelerated activation. Genes altered in this primed CLOCKΔ19 qHSC state may provide biomarkers for early liver disease detection, and include AOC3, which correlated with disease severity in patient serum samples. Integration of CLOCKΔ19 microarray data with ATAC-seq data from WT qHSCs suggested a potential CLOCK regulome promoting a quiescent state and downregulating genes involved in cell projection assembly. CLOCKΔ19 mice showed higher baseline COL1 deposition and significantly worse fibrotic injury after CCl4 treatment. Our data demonstrate that disruption to circadian rhythm primes HSCs towards an accelerated fibrotic response which worsens liver disease.

Evidence for continuity of interstitial spaces across tissue and organ boundaries in humans.

Bodies have continuous reticular networks, comprising collagens, elastin, glycosaminoglycans, and other extracellular matrix components, through all tissues and organs. Fibrous coverings of nerves and blood vessels create structural continuity beyond organ boundaries. We recently validated fluid flow through human fibrous tissues, though whether these interstitial spaces are continuous through the body or discontinuous, confined within individual organs, remains unclear. Here we show evidence for continuity of interstitial spaces using two approaches. Non-biological particles (tattoo pigment, colloidal silver) were tracked within colon and skin interstitial spaces and into adjacent fascia. Hyaluronic acid, a macromolecular component of interstitial spaces, was also visualized. Both techniques demonstrate interstitial continuity within and between organs including within perineurium and vascular adventitia traversing organs and the spaces between them. We suggest that there is a body-wide network of fluid-filled interstitial spaces that has significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

A Bile Duct-on-a-Chip With Organ-Level Functions.

BACKGROUND AND AIMS: Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. APPROACH AND RESULTS: We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. CONCLUSIONS: This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

Publisher Correction: SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.