Pranavkumar Shivakumar

Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFN-γ in experimental biliary atresia

The etiology and pathogenesis of bile duct obstruction in children with biliary atresia are largely unknown. We have previously reported that, despite phenotypic heterogeneity, genomic signatures of livers from patients display a proinflammatory phenotype. Here, we address the hypothesis that production of IFN-gamma is a key pathogenic mechanism of disease using a mouse model of rotavirus-induced biliary atresia. We found that rotavirus infection of neonatal mice has a unique tropism to bile duct cells, and it triggers a hepatobiliary inflammation by IFN-gamma-producing CD4(+) and CD8(+) lymphocytes. The inflammation is tissue specific, resulting in progressive jaundice, growth failure, and greater than 90% mortality due to obstruction of extrahepatic bile ducts. In this model, the genetic loss of IFN-gamma did not alter the onset of jaundice, but it remarkably suppressed the tissue-specific targeting of T lymphocytes and completely prevented the inflammatory and fibrosing obstruction of extrahepatic bile ducts. As a consequence, jaundice resolved, and long-term survival improved to greater than 80%. Notably, administration of recombinant IFN-gamma led to recurrence of bile duct obstruction following rotavirus infection of IFN-gamma-deficient mice. Thus, IFN-gamma-driven obstruction of bile ducts is a key pathogenic mechanism of disease and may constitute a therapeutic target to block disease progression in patients with biliary atresia.

Coordinate expression of regulatory genes differentiates embryonic and perinatal forms of biliary atresia

The molecular basis for the embryonic and perinatal clinical forms of biliary atresia is largely undefined. In this study, we aimed to: 1) determine if the clinical forms can be differentiated at the transcriptional level, and 2) search for molecular mechanisms underlying phenotypic differences. To this end, we generated biotinylated cRNA probes from livers of age‐matched infants with the embryonic (n = 5) and perinatal (n = 6) forms of biliary atresia at the time of diagnosis and hybridized them against the Affymetrix human HG‐U133 A and B microarrays containing 44,760 gene products. Data filtering and two‐way cluster analysis of the gene expression platform identified 230 genes with an expression profile that is highly distinctive of the clinical phenotypes. Functionally, the profile did not reveal a higher‐order function for a specific cell type; instead, it uncovered a coordinated expression of regulatory genes. These regulatory genes were predominantly represented in the embryonic form (45% of genes), with a unique pattern of expression of genes involved in chromatin integrity/function (Smarca‐1, Rybp, and Hdac3) and the uniform overexpression of five imprinted genes (Igf2, Peg3, Peg10, Meg3, and IPW), implying a failure to downregulate embryonic gene programs. In conclusion, embryonic and perinatal forms of biliary atresia are distinguished by gene expression profiling. The coordinate expression of regulators of chromatin structure/function and of imprinted genes provides evidence for a transcriptional basis for the pathogenesis of the embryonic form of biliary atresia. Further studies exploring these biological processes are required to determine the significance of these findings. Supplementary material for this article can be found at http://genet.cchmc.org. (HEPATOLOGY 2004;39:954–962.)

Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia

BACKGROUND & AIMS Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. METHODS We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. RESULTS While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFalpha and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. CONCLUSIONS (1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.

Temporal-spatial activation of apoptosis and epithelial injury in murine experimental biliary atresia†

Biliary atresia is a fibro‐inflammatory cholangiopathy that obstructs the extrahepatic bile ducts in young infants. Although the pathogenesis of the disease is undefined, studies in livers from affected children and neonatal mice with experimental biliary atresia have shown increased expression of proapoptosis molecules. Therefore, we hypothesized that apoptosis is a significant mechanism of injury to duct epithelium. To test this hypothesis, we quantified apoptosis using terminal transferase dUTP nick end labeling and active caspase‐3 staining in livers and extrahepatic bile ducts from Balb/c mice infected with Rhesus rotavirus (RRV) within 24 hours of birth. RRV induced a significant increase in labeled cells in the portal tracts and in epithelial and subepithelial compartments of extrahepatic bile ducts, with onset within 3 days and peaks at 5–10 days. Exploring mechanisms of injury, we found increased biliary expression of caspases 1 and 4 and of interferon‐gamma (IFNγ)–related and tumor necrosis factor‐alpha (TNFα)–related genes. Using a cholangiocyte cell line, we found that neither IFNγ nor TNFα alone affected cell viability; however, simultaneous exposure to IFNγ and TNFα activated caspase‐3 and decreased cell viability. Inhibition of caspase activity blocked apoptosis and restored viability to cultured cholangiocytes. In vivo, administration of the caspase inhibitor IDN‐8050 decreased apoptosis in the duct epithelium and the extent of epithelial injury after RRV challenge. Conclusion: The biliary epithelium undergoes early activation of apoptosis in a mouse model of biliary atresia. The synergistic role of IFNγ and TNFα in activating caspase‐3 in cholangiocytes and the decreased apoptosis following pharmacologic inhibition of caspases support a prominent role for apoptosis in the pathogenesis of experimental biliary atresia. (HEPATOLOGY 2008.)

Th2 signals induce epithelial injury in mice and are compatible with the biliary atresia phenotype

Biliary atresia (BA) is a destructive cholangiopathy of childhood in which Th1 immunity has been mechanistically linked to the bile duct inflammation and obstruction that culminate in liver injury. Based on reports of decreased Th1 cytokines in some patients and the development of BA in mice lacking CD4+ T cells, we hypothesized that Th1-independent mechanisms can also activate effector cells and induce BA. Here, we tested this hypothesis using Stat1-/- mice, which lack the ability to mount Th1 immune responses. Infection of Stat1-/- mice with rhesus rotavirus type A (RRV) on postnatal day 1 induced a prominent Th2 response, duct epithelial injury and obstruction within 7 days, and atresia shortly thereafter. A high degree of phosphorylation of the Th2 transcription factor Stat6 was observed; however, concurrent inactivation of Stat1 and Stat6 in mice did not prevent BA after RRV infection. In contrast, depletion of macrophages or combined loss of Il13 and Stat1 reduced tissue infiltration by lymphocytes and myeloid cells, maintained epithelial integrity, and prevented duct obstruction. In concordance with our mouse model, humans at the time of BA diagnosis exhibited differential hepatic expression of Th2 genes and serum Th2 cytokines. These findings demonstrate compatibility between Th2 commitment and the pathogenesis of BA, and suggest that patient subgrouping in future clinical trials should account for differences in Th2 status.

Staging of biliary atresia at diagnosis by molecular profiling of the liver.

BackgroundYoung age at portoenterostomy has been linked to improved outcome in biliary atresia, but pre-existing biological factors may influence the rate of disease progression. In this study, we aimed to determine whether molecular profiling of the liver identifies stages of disease at diagnosis.MethodsWe examined liver biopsies from 47 infants with biliary atresia enrolled in a prospective observational study. Biopsies were scored for inflammation and fibrosis, used for gene expression profiles, and tested for association with indicators of disease severity, response to surgery, and survival at 2 years.ResultsFourteen of 47 livers displayed predominant histological features of inflammation (N = 9) or fibrosis (N = 5), with the remainder showing similar levels of both simultaneously. By differential profiling of gene expression, the 14 livers had a unique molecular signature containing 150 gene probes. Applying prediction analysis models, the probes classified 29 of the remaining 33 livers into inflammation or fibrosis. Molecular classification into the two groups was validated by the findings of increased hepatic population of lymphocyte subsets or tissue accumulation of matrix substrates. The groups had no association with traditional markers of liver injury or function, response to surgery, or complications of cirrhosis. However, infants with an inflammation signature were younger, while those with a fibrosis signature had decreased transplant-free survival.ConclusionsMolecular profiling at diagnosis of biliary atresia uncovers a signature of inflammation or fibrosis in most livers. This signature may relate to staging of disease at diagnosis and has implications to clinical outcomes.

Gene expression signature for biliary atresia and a role for interleukin-8 in pathogenesis of experimental disease.

Biliary atresia (BA) is a progressive fibroinflammatory obstruction of extrahepatic bile ducts that presents as neonatal cholestasis. Due to the overlap in clinical, biochemical, and histological features with other causes of cholestasis, the diagnosis requires an intraoperative cholangiogram. Thus, we determined whether diseased livers express a gene expression signature unique to BA. Applying stringent statistical analysis to a genome‐wide liver expression platform of 64 infants with BA at the time of diagnosis, 14 age‐appropriate subjects with intrahepatic cholestasis as diseased controls and seven normal controls, we identified 15 genes uniquely expressed in BA with an accuracy of 92.3%. Among these genes, IL8 and LAMC2 were sufficient to classify subjects with BA distinctly from diseased controls with an area under the curve of 0.934 (95% confidence interval [CI]: 0.84‐1.03), sensitivity of 96.9%, and specificity of 85.7% using their combined first principal component. Direct measurement of interleukin (IL)8 protein in the serum, however, was not different between the two groups. To investigate whether the liver‐restricted increase in IL8 was relevant to disease pathogenesis, we inactivated the signaling of IL8 homologs by genetic targeting of the Cxcr2 receptor in a murine model of experimental BA. Disruption of Cxcr2 shortened the duration of cholestasis, decreased the incidence of bile duct obstruction, and improved survival above wild‐type neonatal mice. Conclusion: The hepatic expression of IL8 and LAMC2 has high sensitivity for BA at diagnosis and may serve as a biomarker of disease, with an important role for the IL8 signaling in experimental BA. (Hepatology 2014;60:211–223)

Dendritic Cells Regulate Natural Killer Cell Activation and Epithelial Injury in Experimental Biliary Atresia

Plasmacytoid and conventional dendritic cells both contribute to natural killer cell activation and epithelial tissue injury in a mouse model of biliary atresia. Unblocking the Road to Treating Biliary Atresia Biliary atresia is the most common cause of liver cirrhosis in young children. With onset of symptoms in the first few weeks of life, the disease is triggered by activation of the neonatal immune system, which targets the epithelial cells that line the bile ducts. Immune system activation is followed rapidly by fibrosis and obstruction of the bile duct lumen. Cessation of bile flow from the liver to the intestines produces jaundice and clay-colored stools within a few weeks of birth. The most effective treatment to restore bile flow is surgical intervention to remove the damaged bile duct and to create a new conduit using a segment of the small intestine. Despite surgery and improved bile flow, liver disease progresses to end-stage cirrhosis in most children. The development of new therapies to block progression to cirrhosis has been hampered by our limited knowledge of the earliest events underpinning biliary atresia pathogenesis. To identify the early triggers of this disease, Saxena et al. used a neonatal mouse model in which biliary atresia is induced by infection with rotavirus. Because dendritic cells (DCs) are known to be important for priming innate and adaptive immunity, Saxena and colleagues first examined liver DCs in early mouse postnatal life and after rotavirus infection. They found spontaneous expression of the activation marker CD80 in a subtype of DCs called conventional DCs (cDCs) and discovered that the rotavirus preferentially infected plasmacytoid DCs (pDCs) in early postnatal life. A potential role for DCs in the pathogenesis of the human disease was suggested by gene expression and immunostaining of liver biopsies from infants diagnosed with biliary atresia. These analyses showed an increased number of activated pDCs in the portal tracts, which contain small bile duct branches that collect bile for drainage into the intestine. To directly explore whether pDCs are involved in bile duct injury, the investigators used cocultures of DCs, T lymphocytes, and natural killer (NK) cells to demonstrate that pDCs produce the cytokine interleukin-15 (IL-15) and that both DC subtypes are required for the proliferation of T lymphocytes and the activation of NK cells. Notably, disruption of this cellular network by depletion of pDCs or blockade of IL-15 signaling in neonatal mice infected with rotavirus prevented injury to bile duct epithelial cells, maintained anatomic continuity of bile ducts, and promoted long-term survival of the animals. These findings identify DCs as cellular triggers of biliary injury. The presence of activated DCs in the livers of human infants at the time of diagnosis with biliary atresia points to DCs and IL-15 as potential therapeutic targets that could be blocked to halt the progression of liver disease in these infants. Biliary atresia is the most common cholangiopathy of childhood. During infancy, an idiopathic activation of the neonatal immune system targets the biliary epithelium, obstructs bile ducts, and disrupts the anatomic continuity between the liver and the intestine. Here, we use a model of virus-induced biliary atresia in newborn mice to trace the initiating pathogenic disease mechanisms to resident plasmacytoid (pDCs) and conventional (cDCs) dendritic cells. We found pDCs to be the most abundant DC population in the livers of newborn mice, and we observed pDCs in the livers of infants at the time of diagnosis. In the livers of newborn mice, cDCs spontaneously overexpressed the costimulatory molecule CD80 soon after birth, and pDCs produced the cytokine interleukin-15 (IL-15) in response to a virus insult. Both subtypes of primed DCs were required for the proliferation of T lymphocytes and the activation of natural killer cells. Disruption of this cellular network by depletion of pDCs or blockade of IL-15 signaling in mice in vivo prevented epithelial injury, maintained anatomic continuity of the bile duct, and promoted long-term survival. These findings identify cellular triggers of biliary injury and have implications for future therapies to block the progression of biliary atresia and liver disease.

Identification of intramural epithelial networks linked to peribiliary glands that express progenitor cell markers and proliferate after injury in mice

Peribiliary glands (PBGs) are clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts (EHBDs). Though their function is largely undefined, they may represent a stem cell niche. Here, we hypothesized that PBGs are populated by mature and undifferentiated cells capable of proliferation in pathological states. To address this hypothesis, we developed a novel whole‐mount immunostaining assay that preserves the anatomical integrity of EHBDs coupled with confocal microscopy and found that PBGs populate the entire length of the extrahepatic biliary tract, except the gallbladder. Notably, in addition to the typical position of PBGs adjacent to the duct mucosa, PBGs elongate and form intricate intramural epithelial networks that communicate between different segments of the bile duct mucosa. Network formation begins where the cystic duct combines with hepatic ducts to form the common bile duct (CBD) and continues along the CBD. Cells of PBGs and the peribiliary network stain positively for α‐tubulin, mucins, and chromogranin A, as well as for endoderm transcription factors SRY (sex determining region Y)‐box 17 and pancreatic and duodenal homeobox 1, and proliferate robustly subsequent to duct injury induced by virus infection and bile duct ligation. Conclusion: PBGs form elaborate epithelial networks within the walls of EHBDs, contain cells of mature and immature phenotypes, and proliferate in response to bile duct injury. The anatomical organization of the epithelial network in tubules and the link with PBGs support an expanded cellular reservoir with the potential to restore the integrity and function of the bile duct mucosa in diseased states. (Hepatology 2013;58:1486–1496)

Expansion of prominin‐1‐expressing cells in association with fibrosis of biliary atresia

Biliary atresia (BA), the most common cause of end‐stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long‐term transplant‐free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus‐induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ‐1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen‐1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E‐CADHERIN, biliary marker CYTOKERATIN‐19, and mesenchymal markers VIMENTIN and alpha‐SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor‐beta (TGFβ) signaling. In vitro cotreatment of PROM1‐expressing Mat1a−/− hepatic progenitor cells with recombinant human FGF10 and TGFβ1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen‐1α1, Fibronectin, and α‐Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. Conclusion: Expansion of collagen‐producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFβ pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA. (Hepatology 2014;60:941–953)