The role of macrophages in the development of biliary injury in a lipopolysaccharide-aggravated hepatic ischaemia-reperfusion model

Introduction

Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence.

The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine

Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 μg/L, 7.5 μg/L, 15 μg/L, or 30 μg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver.     

Role of the bicarbonate-responsive soluble adenylyl cyclase in cholangiocyte apoptosis in primary biliary cholangitis; a new hypothesis

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.     

Drug-induced bile duct injury

Drug-induced liver injury includes a spectrum of pathologies, some related to the mode of injury, some to the cell type primarily damaged. Among these, drug-induced bile duct injury is characterized by the destruction of the biliary epithelium following exposure to a drug. Most of the drugs associated with bile duct injury cause immune-mediated lesions to the epithelium of interlobular ducts. These share common histopathological features with primary biliary cholangitis, such as inflammation and necrosis at the expense of cholangiocytes and, if the insult persists, bile duct loss and biliary cirrhosis. Some drugs selectively target larger ducts. Such injury is often dose-dependent and thought to be the result of intrinsic drug toxicity. The histological changes resemble those seen in primary sclerosing cholangitis. This overview focuses on the clinical and pathological features of bile duct injury associated with drug treatment and on the immunological and biochemical effects that drugs exert on the biliary epithelium. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Advances in the generation of bioengineered bile ducts

The generation of bioengineered biliary tissue could contribute to the management of some of the most impactful cholangiopathies associated with liver transplantation, such as biliary atresia or ischemic cholangiopathy. Recent advances in tissue engineering and in vitro cholangiocyte culture have made the achievement of this goal possible. Here we provide an overview of these developments and review the progress towards the generation and transplantation of bioengineered bile ducts. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.     

Mechanisms of cholangiocyte responses to injury

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Development of liver microtissues with functional biliary ductular network

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.     

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.     

Liver Ischemia and Ischemia–Reperfusion Induces and Trafficks the Multi-specific Metal Transporter Atp7b to Bile Duct Canaliculi: Possible Preferential Transport of Iron into Bile

Both Atp7b (Wilson disease gene) and Atp7a (Menkes disease gene) have been reported to be trafficked by copper. Atp7b is trafficked to the bile duct canaliculi and Atp7a to the plasma membrane. Whether or not liver ischemia or ischemia–reperfusion modulates Atp7b expression and trafficking has not been reported. In this study, we report for the first time that the multi-specific metal transporter Atp7b is significantly induced and trafficked by both liver ischemia alone and liver ischemia–reperfusion, as judged by immunohistochemistry and Western blot analyses. Although hepatocytes also stained for Atp7b, localized intense staining of Atp7b was found on bile duct canaliculi. Inductive coupled plasma-mass spectrometry analysis of bile copper, iron, zinc, and manganese found a corresponding significant increase in biliary iron. In our attempt to determine if the increased biliary iron transport observed may be a result of altered bile flow, lysosomal trafficking, or glutathione biliary transport, we measured bile flow, bile acid phosphatase activity, and glutathione content. No significant difference was found in bile flow, bile acid phosphatase activity, and glutathione, between control livers and livers subjected to ischemia–reperfusion. Thus, we conclude that liver ischemia and ischemia–reperfusion induction and trafficking Atp7b to the bile duct canaliculi may contribute to preferential iron transport into bile.     

Pathophysiological basis of liver disease in cystic fibrosis employing a DeltaF508 mouse model

The molecular pathogenesis of cystic fibrosis (CF) liver disease is unknown. This study investigates its earliest pathophysiological manifestations employing a mouse model carrying DeltaF508, the commonest human CF mutation. We hypothesized that, if increased bile salt spillage into the colon occurs as in the human disease, then this should lead to a hydrophobic bile salt profile and to "hyperbilirubinbilia" because of induced enterohepatic cycling of unconjugated bilirubin. Hyperbilirubinbilia may then lead to an increased bile salt-to-phospholipid ratio in bile and, following hydrolysis, precipitation of divalent metal salts of unconjugated bilirubin. We document in CF mice elevated fecal bile acid excretion and biliary secretion of more hydrophobic bile salts compared with control wild-type mice. Biliary secretion rates of bilirubin monoglucuronosides, bile salts, phospholipids, and cholesterol are increased significantly with an augmented bile salt-to-phospholipid ratio. Quantitative histopathology of CF livers displays mild early cholangiopathy in approximately 53% of mice and multifocal divalent metal salt deposition in cholangiocytes. We conclude that increased fecal bile acid loss leads to more hydrophobic bile salts in hepatic bile and to hyperbilirubinbilia, a major contributor in augmenting the bile salt-to-phospholipid ratio and endogenous beta-glucuronidase hydrolysis of bilirubin glucuronosides. The confluence of these perturbations damages intrahepatic bile ducts and facilitates entrance of unconjugated bilirubin into cholangiocytes. This study of the earliest stages of CF liver disease provides a framework for investigating the molecular pathophysiology of more advanced disease in murine models and in humans with CF.     

Repopulating the biliary tree from the peribiliary glands

The larger ducts of the biliary tree contain numerous tubulo-alveolar adnexal glands that are lined with biliary epithelial cells and connected to the bile duct lumen via small glandular canals. Although these peribiliary glands (PBG) were already described in the 19th century, their exact function and role in the pathophysiology and development of cholangiopathies have not become evident until recently. While secretion of serous and mucinous components into the bile was long considered as the main function of PBG, recent studies have identified PBG as an important source for biliary epithelial cell proliferation and renewal. Activation, dilatation, and proliferation of PBG (or the lack thereof) have been associated with various cholangiopathies. Moreover, PBG have been identified as niches of multipotent stem/progenitor cells with endodermal lineage traits. This has sparked research interest in the role of PBG in the pathogenesis of various cholangiopathies as well as bile duct malignancies. Deeper understanding of the regenerative capacity of the PBG may contribute to the development of novel regenerative therapeutics for previously untreatable hepatobiliary diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Interleukin-5 inhibition of biliary cell chloride currents and bile flow

Recent studies have detected significant elevations of interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver transplantation. In both of these disorders, intrahepatic biliary epithelial cells (BECs) are the targets of injury. We hypothesized that BECs may themselves express IL-5 receptors that may modulate key biliary functions. RNAs coding for IL-5alpha and -beta receptors were amplified by RT/PCR from a biliary cell line derived from a human cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5 receptor distribution was detected immunocytochemically on Mz-ChA-1 cells, immortalized murine BEC, bile duct-ligated rat liver, and isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated chloride currents. Additional functional studies showed that IL-5 inhibits secretin-induced bile flow. We conclude that BECs express IL-5 receptors and that IL-5 modulates BEC chloride currents and fluid secretion. Since IL-5 has previously been associated with cholestatic liver disease, we speculate that IL-5 may contribute to liver injury through its effects on biliary secretion.     

塑料胆道支架引流术治疗复杂性胆总管结石的临床研究

目的:探讨内镜下逆行胰胆管造影术(ERCP)下塑料胆道支架引流术治疗复杂性胆总管结石的临床疗效和安全性。方法:回顾性分析2011年9月至2013年9月在我院经ERCP下胆道支架引流术治疗的32例复杂性胆总管结石患者的临床病例资料。结果:32例患者塑料胆道支架引流术全部成功,平均手术时间15-30分钟。术后,2例发生高淀粉酶血症,经禁食72小时后恢复正常,无穿孔、消化道大出血等ERCP严重并发症发生。术后1周,患者腹痛、发热消失,转氨酶及胆红素水平明显下降,平均住院时间6-15天。3个月复查B超,发现结石缩小19例,结石碎裂1例,支架脱落1例。术后7天、术后3个月的肝功能指标与术前比较均显著改善,差异均有统计学意义(P0.05)。结论:ERCP下塑料胆道支架引流术是一种复杂性胆总管结石安全有效的治疗方法,具有创伤小、风险较低、操作时间短、患者易耐受及手术成功率高等优点。     

Overexpression of insulin like growth factor binding protein 5 reduces liver fibrosis in chronic cholangiopathy

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4(-/-)) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4(-/-) mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12 weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4(-/-) mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 overexpressing mice even 12 weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy.     

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ～80% compared to ～50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.     

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3⁺ and CD8⁺ T cells correlated with the advancement of emperipolesis (R² = 0.318, P < .001; R² = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R² = 0.236, P < .001; R² = 0.267, P < .001). We conclude that emperipolesis mediated by CD8⁺ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.     

Gut regulatory peptides bombesin and neurotensin reduce hepatic oxidative stress and histological alterations in bile duct ligated rats

Gut regulatory peptides bombesin (BBS) and neurotensin (NT) exert a wide spectrum of biological actions on gastrointestinal tissues and we have previously shown that they improve intestinal barrier function and oxidative stress in experimentally jaundiced rats. In the present study, we explored their potential action on liver histology and oxidative status in bile duct ligated rats. Seventy male Wistar rats were randomly divided into five groups: controls, sham operated, bile duct ligated (BDL), BDL+BBS (10 μg/kg, s.c. ×3), BDL+NT (300 μg/kg, i.p.). At the end of the experiment, on day 10, serum total bilirubin and alanine aminotransferase (ALT) levels were determined and endotoxin was measured in portal and aortic blood. Liver tissue samples were examined histologically for evaluation of the ratio of portal tracts presenting changes of obstructive cholangiopathy and neutrophils' number in portal tracts. In addition, hepatic oxidative status was estimated on liver homogenates by measurements of lipid peroxidation (malondialdehyde), protein oxidation (protein carbonyl groups) and thiol redox state [reduced glutathione (GSH), oxidized glutathione (GSSG), total non-protein mixed disulfides (NPSSR) and protein thiols (PSH)]. Administration of BBS or NT significantly reduced portal and aortic endotoxaemia observed in obstructive jaundice. Both agents significantly ameliorated liver injury, as demonstrated by improvement of obstructive cholangiopathy and reduction of ALT. This effect was accompanied by prevention of lipid peroxidation, protein oxidation and decrease of the oxidized forms GSSG and NPSSR. Moreover, neutrophil accumulation in portal tracts was significantly decreased. In conclusion, this study shows that gut regulatory peptides BBS and NT reduce cholestatic liver injury, exerting protective effects on portal tract architecture, neutrophil infiltration and hepatic oxidative stress in bile duct ligated rats.     

胆总管内注射无水乙醇致胆道闭锁动物模型的建立

目的应用胆总管内注射无水乙醇建立SD大鼠胆道闭锁模型。方法将SD雄性大鼠随机分为实验组和对照组,在实验组中经静脉留置针插入胆总管注入无水乙醇,对照组注入生理盐水。观察SD大鼠的生化及病理结果。结果在实验组中SD大鼠根据病理及生化检测分为肝功能持续恶化组和肝功能修复组,肝功能持续恶化组在8周以后生化明显高于对照组及肝功能修复组。常规HE染色及SMA、Masson染色也出现明显变化。结论胆总管无水乙醇注射诱导胆道闭锁模型是一种可靠的动物模型,此动物模型会帮助人们进一步研究胆道闭锁提供更多的研究手段。     

胆道手术患者胆汁的菌群分析

本文对59例胆道手术患者的胆汁标本进行了菌群分析。共检出各类细菌156株,其中厌氧菌70株,以类杆菌最为常见(44株);兼性需氧菌86株,以大肠杆菌最为常见(37株)。胆汁细菌培养阳性率为86.4％(51/59);64.4％(38/59)的标本为厌氧菌与兼性厌氧菌混合污染。22.0％(13/59)的标本中仅分离到兼性需氧菌。提示,胆汁中污染的类杆菌及大肠杆菌是胆道手术后感染的主要原因菌。