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Biliary atresia (BA) is a condition unique to infancy. It results from inflammatory destruction of the intrahepatic and extrahepatic bile ducts. It is the most frequent surgically correctable liver disorder in infancy and the most frequent indication for liver transplantation in paediatric age. Clinical presentation is in the first few weeks of life with conjugated hyperbilirubinaemia (dark urine and pale stools); other manifestations of liver disease, such as failure to thrive, splenomegaly and ascites, appear only later, when surgery is unlikely to be successful. Hence, all infants with conjugated hyperbilirubinaemia must be urgently referred to specialised centres for appropriate treatment. Success of surgery depends on the age at which it is performed. With corrective surgery, followed, when necessary, by liver transplantation, the overall survival rate is approximately 90%. The cause of BA is unknown, but there is evidence for the involvement of infectious, genetic and immunologic mechanisms, which will be discussed in this review.

Biliary atresia (BA) is a devastating fibro-inflammatory disease characterized by the obstruction of extrahepatic and intrahepatic bile ducts in infants that can have fatal consequences, when not treated in a timely manner. It is the most common indication of pediatric liver transplantation worldwide and the development of new therapies, to alleviate the need of surgical intervention, has been hindered due to its complexity and lack of understanding of the disease pathogenesis. For that reason, significant efforts have been made toward the development of experimental models and strategies to understand the etiology and disease mechanisms and to identify novel therapeutic targets. The only characterized model of BA, using a Rhesus Rotavirus Type A infection of newborn BALB/c mice, has enabled the identification of key cellular and molecular targets involved in epithelial injury and duct obstruction. However, the establishment of an unleashed chronic inflammation followed by a progressive pathological wound healing process remains poorly understood. Like T cells, macrophages can adopt different functional programs [pro-inflammatory (M1) and resolutive (M2) macrophages] and influence the surrounding cytokine environment and the cell response to injury. In this review, we provide an overview of the immunopathogenesis of BA, discuss the implication of innate immunity in the disease pathogenesis and highlight their suitability as therapeutic targets.

Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.

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.

Macrophages play an important role in disease progression of pediatric cholestatic liver disease, particularly biliary atresia (BA); however, the restorative versus pathogenic role for precise macrophage subsets remains poorly defined. We aimed to distinguish the transcriptional profiles and roles of defined macrophage subset(s) in murine BA. We used multiparameter flow cytometry and RNA-sequencing analysis to profile recruited CD11b CD64+ hepatic macrophages by cell surface expression of MHCII and Ly6c in the Rhesus rotavirus (RRV)-induced murine model of BA versus saline controls. Modulation of macrophage numbers via intra-peritoneal injections of clodronate-loaded liposomes was performed to determine the association between macrophage numbers and histologic injury (Ishak score). Ly6c+ macrophages demonstrated the greatest increase in numbers and percent of total macrophages in murine BA versus saline controls whereas MHCII+ macrophages decreased. Transcriptional changes in murine BA MHCII+ macrophages included reduced expression of the Kupffer cell gene signature, lower expression of genes involved in homeostatic processes, and increased expression of genes involved in inflammatory processes. Ly6c+ macrophages in murine BA showed increased expression for and other genes involved in the cellular response to hypoxia. Among all subsets, the number of Ly6c+ macrophages exhibited the strongest correlation with severity of histologic liver injury by Ishak score. Our data identify specific pathways upregulated in Ly6c vs MHCII+ macrophage subsets in murine BA. Transcriptional similarities between murine BA and human cholestatic macrophages may enable translation of future mechanistic studies to new macrophage subset-specific therapies.

IL-17A+ γδT cells are involved in biliary atresia (BA) inflammatory injury; however, the mechanism underlying their activation remains unclear. The present study aimed to elucidate the mechanism by which γδT cells are activated to secrete IL-17A in BA. Clinical samples from 27 patients with BA and 23 control individuals were collected and analyzed using reverse transcription-quantitative PCR (RT-qPCR), western blotting, ELISA, flow cytometry, Opal multiplex immunohistochemistry and immunohistochemistry. In addition, wild-type (WT) and Tcrδ−/− BA murine models were developed through infection with rhesus rotavirus (RRV) and assessed by RT-qPCR, western blotting, ELISA, flow cytometry, immunohistochemistry and hematoxylin and eosin staining. The release of high-mobility group box-1 (HMGB1) from RRV-infected biliary epithelial cells (BECs) and macrophages was investigated by ELISA and immunofluorescence confocal microscopy. In addition, co-culture systems of γδT cells with BECs or macrophages were established. The results revealed that the levels of hepatic IL-17A were higher in patients with BA, with γδT cells serving an important role in producing IL-17A. In the BA murine model, the IL-17A levels increased from day 3 to 14, and IL-17A+ γδT cells peaked on day 7. The Tcrδ−/− BA murine model exhibited lower IL-17A levels, improved liver and bile duct morphology, and lower BA incidence compared with in the WT BA murine model, which was reversed through adoptive transfer of murine IL-17A+ γδT cells. HMGB1, and Toll-like receptor (TLR)2 and 4 were upregulated in liver tissues from both patients with BA and the BA murine model. By contrast, hepatic IL-17A levels were decreased in the BA murine model treated with the HMGB1 inhibitor. In addition, HMGB1 was released from RRV-infected BECs or macrophages, and exogenous HMGB1 stimulation enhanced IL-17A production in γδT cells. The Tlr2−/− and Tlr4−/− γδT cell co-culture system inhibited IL-17A production and NF-κB activation, whereas NF-κB inhibition abolished HMGB1-induced IL-17A production in WT γδT cells. In conclusion, HMGB1 released from injured BECs or macrophages may activate IL-17A+ γδT cells to mediate BA inflammatory injury via the HMGB1-TLR2/4-NF-κB pathways. The present study was registered with the Chinese Clinical Trial Registry (registration ID: ChiCTR2000039619; registered November 3, 2020).

Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn’s susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells. The pathogenesis of biliary atresia remains poorly understood. Here, the authors report that maternal butyrate treatment alters the gut microbiome and glutamine/hypoxanthine metabolites similar to human subjects, and suppresses biliary atresia in newborn mice.

The pathogenesis of biliary atresia (BA) is unclear to date and no therapies targeting immune regulatory pathways exist. Here we characterized potent effector liver tissue resident memory CD8 T cells (Trm) and monocytic cells in children with advanced BA and an age-matched control group to gain insight into BA pathogenesis and immunologic regulation. Liver explants from 18 children with biliary atresia and 10 with metabolic disease and normal histology were analyzed by multicolor flow-cytometry and immunohistochemistry. Cytokines and cytotoxic mediators were quantified by intracellular staining and bead-based arrays in culture supernatant. The frequency of CD103 CD69 CD8 Trm cells and CD14 CD16 monocytes was significantly higher in BA than in the control group. In BA, T cells showed elevated expression of CD103, CD69, CD39 and production of TNF-α and Granzyme-B , which could be reproduced by allowing cell-contact with monocytes. Cytotoxic CD8 Trm cells and intrahepatic monocytes might contribute to tissue destruction in BA. Therapies targeting Trm cells or the TNF-α signaling pathway could be explored to delay progression to cirrhosis in BA.

Background Biliary atresia (BA) is a rare pediatric cholestatic disorder characterized by progressive bile duct inflammation and fibrosis. The underlying molecular mechanisms of BA remain poorly defined. This study aimed to identify susceptibility genes causally associated with BA by integrating genome-wide association study (GWAS) and transcriptomic data, and to explore their potential immunopathological roles. Methods Two independent BA transcriptomic datasets from the Gene Expression Omnibus (GEO) were analyzed, and Mendelian randomization (MR) was applied to assess causal associations between differentially expressed genes (DEGs) and BA. Co-expressed genes were further evaluated for biological pathway enrichment and immune cell infiltration patterns. Expression levels of candidate genes were validated using quantitative real-time PCR (qRT-PCR) in liver tissues from 20 BA patients and 10 normal controls. Representative liver histopathology was also examined. Results We identified 816 DEGs, including 458 upregulated and 358 downregulated genes. MR analysis highlighted seven co-expressed genes with potential causal relevance to BA, including C12orf75, PSD3, CRIM1, CHIT1 (upregulated), and SEC14L4, MAPRE3, TCEA3 (downregulated). qRT-PCR validation confirmed significantly elevated expression of C12orf75, PSD3, and CHIT1, and reduced expression of TCEA3 in BA liver tissues compared to controls ( P  < 0.05), consistent with MR predictions. Histopathological analysis revealed severe portal fibrosis, bile duct proliferation, and pseudolobule formation in BA samples, whereas normal controls exhibited preserved hepatic architecture with minimal fibrotic changes. Conclusion This study identifies a panel of immune- and transcription-related genes with potential causal roles in BA and validates their expression in human liver tissue. These findings offer new insights into the genetic and molecular basis of BA, supporting future efforts in subtype classification and immunomodulatory therapeutic development.

Regulatory T cells (Tregs) and CD4 T helper (Th) cells have important roles in bile duct injury of biliary atresia (BA). However, their impacts on liver fibrosis are undefined. Between 2013 and 2016, 146 patients with various stages of BA were enrolled in this study. Peripheral blood, liver biopsy and lymph node samples were collected. Flow cytometry, magnetic cell sorting and immunostaining were used to characterize lymphocytes from BA patients. Deficiency of Tregs was observed along with increased Th1, Th2 and Th17 frequencies in the peripheral blood and livers of BA patients. The levels of peripheral and intrahepatic Th1 cells positively correlated with the stage of liver fibrosis. Furthermore, Th1 cells were located in close proximity to activated hepatic stellate cells (HSCs) and areas of fibrosis in BA livers. In culture, Th1 cells accelerated the proliferation and secretion of profibrogenic markers of HSCs through the IFN-γ/STAT1 pathway. Of note, Tregs blocked the Th1-stimulated effects on HSCs by inhibiting Th1-induced activation of STAT1. Consistent with the results of in vitro study, intrahepatic IFN-γ/STAT1 levels increased in relation to the severity of liver fibrosis in BA patients, and the altered balance between MMP2 and TIMP1 expressions in livers may contribute to increased deposition of extracellular matrix and fibrosis. Finally, to identify the effects of Th1 cells on Tregs, we demonstrated that Th1 cells upregulated the proportion of aTreg cells by secreting IFN-γ cytokine. Thus, aberrant Th1 immune responses in BA promote the proliferation and secretion of HSCs through the IFN-γ/STAT1 pathway. The regulation of HSCs by the interactions between Tregs and Th1 cells might be part of the mechanism underlying progressive liver fibrosis and may be a suitable target for therapy.