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Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4 , which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4 -related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump ( ABCB11 ) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection. Neonatal cholestasis is a result of elevated bile acid levels, and is associated with mutations in genes regulating bile acid homeostasis. Here the authors identify mutations in the bile acid sensing farnesoid X receptor in four individuals with neonatal cholestasis from two unrelated families.

Cholestatic liver injuries, characterized by regional damage around the bile ductular region, lack curative therapies and cause considerable mortality. Here we generated a high-definition spatiotemporal atlas of gene expression during cholestatic injury and repair in mice by integrating spatial enhanced resolution omics sequencing and single-cell transcriptomics. Spatiotemporal analyses revealed a key role of cholangiocyte-driven signaling correlating with the periportal damage-repair response. Cholangiocytes express genes related to recruitment and differentiation of lipid-associated macrophages, which generate feedback signals enhancing ductular reaction. Moreover, cholangiocytes express high TGFβ in association with the conversion of liver progenitor-like cells into cholangiocytes during injury and the dampened proliferation of periportal hepatocytes during recovery. Notably, Atoh8 restricts hepatocyte proliferation during 3,5-diethoxycarbonyl-1,4-dihydro-collidin damage and is quickly downregulated after injury withdrawal, allowing hepatocytes to respond to growth signals. Our findings lay a keystone for in-depth studies of cellular dynamics and molecular mechanisms of cholestatic injuries, which may further develop into therapies for cholangiopathies. A comprehensive time series characterization of a mouse model of cholestatic liver injury with spatial enhanced resolution omics sequencing and single-cell RNA sequencing identifies zonal responses to insult, such as cholangiocyte signaling recruiting lipid-associated macrophages.

Deficiencies in the development of epithelial structures and delays in cellular maturation can increase the susceptibility of neonates to disease early in life. To investigate human biliary development and its vulnerability to biliary atresia, a severe pediatric cholangiopathy, we engineered multi-compartment biliary organoids (MBOs) from co-cultures of human liver-derived epithelial organoid cells with human endothelial and mesenchymal cells. MBOs derived from normal livers effectively replicated the epithelial structure of the bile duct epithelium and peribiliary glands (PBGs). Conversely, MBOs from diseased livers exhibited defective epithelial layers, a significant epithelial-mesenchymal transition (EMT), and an activation of the TGF-β/Activin-SMAD2/3 signaling, primarily due to intermediary cell sub-populations. Inhibition of TGF-β signaling suppressed EMT and promoted biliary epithelial development in human MBOs and suppressed the phenotype of experimental biliary atresia in neonatal mice. Thus, the modulation of TGF-β-dependent EMT regulates bile duct epithelial development and influences the susceptibility of neonates to biliary injuries. The study reveals that TGF-β–driven epithelial-mesenchymal transition disrupts bile duct development in biliary atresia. Inhibiting this pathway restores epithelial structure and reduces disease features, highlighting a potential therapeutic target.

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3 −/− ) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro . We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3 −/− mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

Etiology and pathogenesis of biliary atresia (BA) remain elusive. Evidence points to a T cell mediated autoimmune response contributing to the disease by driving the ongoing hepatic inflammation. CD26, also known as dipeptidylpeptidase 4 (DPP4), is an immunoregulatory protein also expressed on T-lymphocytes. We aimed to investigate the potential of pharmacological DPP4 inhibition on cytokine production of T cells and potential therapeutic benefits in experimental BA. We analyzed the expression and regulation of CD26/DPP4 on αβ and γδ T cells in mice suffering from rotavirus-induced BA. Enzymatic DPP4 activity in murine and human serum was examined. In cell cultures, lymphocytes were incubated with the DPP4-inhibitor Sitagliptin to study the effects of DPP4 inhibition on cytokine production. Clinical effects were assessed by intraperitoneal Sitagliptin injection of mice suffering from BA. Analyses included flow cytometry, qPCR, serum analysis and histological examinations. In mice suffering from BA, CD26/DPP4 was strongly expressed and upregulated on αβ and to an even greater extent on γδ T cells compared to healthy controls. DPP4-inhibition led to a dose-dependent suppression of the pro-inflammatory cytokines IL-17 and IFN-γ produced by Th1, Th17 and γδ T cells. Therapeutic administration of Sitagliptin in experimental BA led to reduced serum levels of GOT and Bilirubin as well as decreased hepatic infiltration with F4/80 + macrophages but had no effect on overall survival. In humans, serum DPP4-activity was upregulated in infants suffering from BA compared to healthy children. To our knowledge, this is the first time an upregulation of CD26 expression has been demonstrated for γδ T cells in the setting of an autoimmune inflammatory response. Mechanistically, we could demonstrate that DPP4/CD26 is upregulated on T cells in experimental BA and that pharmacological inhibition decreased their pro-inflammatory potential. However, this translated to only mild clinical benefits in the mouse model. Thus, although the protein appears to play a role in BA, further research is needed to elucidate the potential to serve as a therapeutic target.

Although macrophages are recognized as important players in the pathogenesis of chronic liver diseases, their roles in cholestatic liver fibrosis remain incompletely understood. We previously reported that long noncoding RNA-H19 (lncRNA-H19) contributes to cholangiocyte proliferation and cholestatic liver fibrosis of biliary atresia (BA). We here show that monocyte/macrophage CD11B mRNA levels are increased significantly in livers of BA patients and positively correlated with the progression of liver inflammation and fibrosis. The macrophages increasingly infiltrate and accumulate in the fibrotic niche and peribiliary areas in livers of BA patients. Selective depletion of macrophages using the transgenic CD11b-diphtheria toxin receptor (CD11b-DTR) mice halts bile duct ligation (BDL)-induced progression of liver damage and fibrosis. Meanwhile, macrophage depletion significantly reduces the BDL-induced hepatic lncRNA-H19. Overexpression of H19 in livers using adeno-associated virus serotype 9 (AAV9) counteracts the effects of macrophage depletion on liver fibrosis and cholangiocyte proliferation. Additionally, both H19 knockout (H19 −/− ) and conditional deletion of H19 in macrophage (H19 ΔCD11B ) significantly depress the macrophage polarization and recruitment. lncRNA-H19 overexpressed in THP-1 macrophages enhance expression of Rho-GTPase CDC42 and RhoA. In conclusions, selectively depletion of macrophages suppresses cholestatic liver injuries and fibrosis via the lncRNA-H19 and represents a potential therapeutic strategy for rapid liver fibrosis in BA patients.

To explore an effective noninvasive tool for monitoring liver fibrosis of children with biliary atresia (BA) is important but evidences are limited. This study is to investigate the predictive accuracy of supersonic shearwave elastography (SSWE) in liver fibrosis for postoperative patients with BA and to compare it with aspartate aminotransferase to platelet ratio index (APRI) and fibrosis-4 (FIB-4). 24 patients with BA received SSWE and laboratory tests before scheduled for liver biopsy. Spearman rank coefficient and receiver operating characteristic (ROC) were used to analyze data. Metavir scores were F0 in 3, F1 in 2, F2 in 4, F3 in 7 and F4 in 8 patients. FIB-4 failed to correlate with fibrosis stage. The areas under the ROC curves of SSWE, APRI and their combination were 0.79, 0.65 and 0.78 for significant fibrosis, 0.81, 0.64 and 0.76 for advanced fibrosis, 0.82, 0.56 and 0.84 for cirrhosis. SSWE values at biopsy was correlated with platelet count (r = −0.426, P  = 0.038), serum albumin (r = −0.670, P  < 0.001), total bilirubin (r = 0.419, P  = 0.041) and direct bilirubin levels (r = 0.518, P =  0.010) measured at 6 months after liver biopsy. Our results indicate that SSWE is a more promising tool to assess liver fibrosis than APRI and FIB-4 in children with BA.

Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse neonatal survival and is estimated to impact between 0.4 and 5% of pregnancies worldwide. Here we show that maternal cholestasis (due to Abcb11 deficiency) produces neonatal death among all offspring within 24 h of birth due to atelectasis-producing pulmonary hypoxia, which recapitulates the neonatal respiratory distress of human ICP. Neonates of Abcb11-deficient mothers have elevated pulmonary bile acids and altered pulmonary surfactant structure. Maternal absence of Nr1i2 superimposed on Abcb11 deficiency strongly reduces maternal serum bile acid concentrations and increases neonatal survival. We identify pulmonary bile acids as a key factor in the disruption of the structure of pulmonary surfactant in neonates of ICP. These findings have important implications for neonatal respiratory failure, especially when maternal bile acids are elevated during pregnancy, and highlight potential pathways and targets amenable to therapeutic intervention to ameliorate this condition. The mechanisms underlying perinatal mortality due to intrahepatic cholestasis of pregnancy are not fully understood. Here, the authors show that absence of the nuclear receptor and bile acid regulator Nrli2 and the biliary transporter Abcb11 strongly reduces maternal serum bile acid levels, improving neonatal survival.

Cholestasis is a clinical disorder defined as an impairment of bile flow, and that leads to toxic bile acid (BA) accumulation in hepatocytes. Here, we investigated the hepatoprotective effect of Yinchenhaotang (YCHT), a well-known formulae for the treatment of jaundice and liver disorders, against the cholestasis using the α-naphthylisothiocyanate (ANIT)-induced cholestasis in male Wistar rats. ANIT feeding induced significant cholestasis with substantially increased intrahepatic retention of hydrophobic BAs. The dynamic changes of serum and liver BAs indicated that YCHT was able to attenuate ANIT-induced BA perturbation, which is consistent with the histopathological findings that YCHT significantly decreased the liver damage. YCHT treatment substantially reduced serum alanine aminotransferase (ALT), alkaline phosphatase (AST), total bilirubin (TBIL) and direct bilirubin (DBIL) with minimal bile duct damage in the ANIT treated rats. Elevated mRNA expression of liver IL-6, IL-17A, IL-17F, TGF-β1, α-SMA, TGR5, NTCP, OATP1a1, and ileum ASBT and decreased liver IL-10, FXR, CAR, VDR, BSEP, MRP2, MRP3, MRP4 was also observed in ANIT-induced cholestasis but were attenuated or normalized by YCHT. Our results demonstrated that the BA profiles were significantly altered with ANIT intervention and YCHT possesses the hepatoprotective potential against cholestatic liver injury induced by hepatotoxin such as ANIT.