Rebecca M. Tucker

Biliary Atresia Is Associated with CD4 + Th1 Cell–Mediated Portal Tract Inflammation

A proposed mechanism in the pathogenesis of biliary atresia involves an initial virus-induced, progressive T cell–mediated inflammatory obliteration of bile ducts. The aim of this study was to characterize the inflammatory environment present within the liver of infants with biliary atresia to gain insight into the role of a primary immune-mediated process versus a nonspecific secondary response to biliary obstruction. Frozen liver tissue obtained from patients with biliary atresia, neonatal giant cell hepatitis, total parenteral nutrition (TPN)–related cholestasis, choledochal cysts, and normal control subjects was used for fluorescent immunohistochemistry studies of cellular infiltrates, cytokine mRNA expression, and in situ hybridization for localization of cytokine-producing cells. Immunohistochemistry revealed increases in CD8+ and CD4+ T cells and Kupffer cells (CD68+) in the portal tracts of biliary atresia. Reverse transcription–PCR analysis of biliary atresia tissue showed a Th1-type cytokine profile with expression of IL-2, interferon-γ, tumor necrosis factor-α, and IL-12. This profile was not seen in normal, neonatal hepatitis or choledochal cyst livers but was present in TPN-related cholestasis. In situ hybridization revealed that the Th1 cytokine–producing cells were located in the portal tracts in biliary atresia and in the parenchyma of TPN-related cholestasis. A distinctive portal tract inflammatory environment is present in biliary atresia, involving CD4+ Th1 cell–mediated immunity. The absence of similar inflammation in other pediatric cholestatic conditions suggests that the portal tract inflammation in biliary atresia is not a secondary response to cholestasis but rather indicates a specific immune response involved in the pathogenesis of biliary atresia.

Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia

Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus‐induced, subsequent autoimmune‐mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)‐ induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN‐γ–producing T cells from RRV‐diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV‐diseased mice into naïve syngeneic SCID recipients resulted in bile duct–specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV‐diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia–specific T cell–mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research. (HEPATOLOGY 2006;44:1231–1239.)

Cytomegalovirus-specific T cell reactivity in biliary atresia at the time of diagnosis is associated with deficits in regulatory T cells

Biliary atresia (BA) is a progressive, inflammatory cholangiopathy that culminates in fibrosis of extrahepatic and intrahepatic bile ducts. A leading theory on the pathogenesis of BA is that the bile duct damage is initiated by a virus infection, followed by a bile duct‐targeted autoimmune response. One mechanism of autoimmunity entails a diminished number or function of regulatory T cells (Tregs). The aim of this study was to identify potential virus‐specific liver T cells from infants with BA at the time of diagnosis, implicating the virus involved in early bile duct damage. A subaim was to determine if the presence of virus infection was associated with quantitative changes in Tregs. Liver T cells from BA and control patients were cultured with antigen‐presenting cells in the presence of a variety of viral or control proteins. 56% of BA patients had significant increases in interferon‐gamma‐producing liver T cells in response to cytomegalovirus (CMV), compared with minimal BA responses to other viruses or the control group CMV response. In addition, a positive correlation between BA plasma CMV immunoglobulin M (IgM) and liver T‐cell CMV reactivity was identified. Investigation of peripheral blood Tregs revealed significant deficits in Treg frequencies in BA compared with controls, with marked deficits in those BA patients who were positive for CMV. Conclusion: Liver T‐cell responses to CMV were identified in the majority of BA patients at diagnosis, suggesting perinatal CMV infection as a plausible initiator of bile duct damage. Deficiency of Tregs in BA implies decreased inhibition of inflammation and autoreactivity, potentially allowing for exaggerated bile duct injury. (HEPATOLOGY 2012)

α-Enolase Autoantibodies Cross-Reactive to Viral Proteins in a Mouse Model of Biliary Atresia

BACKGROUND & AIMS Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology. The bile duct injury that occurs in patients with BA might result from a hepatobiliary viral infection followed by an autoimmune response against the bile duct epithelia. We aimed to identify autoantigens recognized by serum antibodies in the Rhesus rotavirus (RRV)-induced mouse model of BA; findings were correlated with BA in humans. METHODS Bile duct epithelial proteins were screened for their reactivity with serum antibodies from the mouse model of BA using immunoblot assays. Unique proteins that reacted with sera antibodies were identified by mass spectrometry and verified using enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Candidate autoantibodies in BA patient sera were analyzed by ELISA. RESULTS A bile duct epithelial antigen that reacted strongly with serum immunoglobulin (Ig) G from the mouse model of BA was identified as α-enolase. α-Enolase autoantibody specificity was confirmed by ELISA and immunoblot analyses. Anti-RRV and anti-enolase antibodies cross-reacted with enolase and RRV proteins; we identified regions of sequence homology between RRV and enolase. Serum samples from patients with BA had increased levels of anti-enolase IgM and IgG. CONCLUSIONS We have identified autoantibodies against α-enolase in a mouse model of BA (infected with RRV) and in serum samples from patients, indicating a role of humoral autoimmunity in disease pathogenesis. The cross-reactivity between an anti-enolase antibody and RRV proteins indicates that molecular mimicry might activate humoral autoimmunity in BA patients; further studies are required.

Genetic Control of Glycoprotein 70 Autoantigen Production and Its Influence on Immune Complex Levels and Nephritis in Murine Lupus

The F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune disease that serves as a model for human systemic lupus erythematosus. Autoimmunity in (NZB × NZW)F1 mice includes the production of autoantibodies to the endogenous retroviral envelope glycoprotein, gp70, and gp70-anti-gp70 immune complexes (gp70 IC) have been implicated in the development of lupus nephritis in these animals. We used backcross and intercross combinations of C57BL/6 (B6; low gp70 levels) and NZB mice (high gp70 levels) to examine the contribution of serum gp70 Ag levels to the development of gp70 IC and nephritis. Analysis of (B6.H2z × NZB)F1 × NZB backcross mice and (NZB × B6)F2 mice showed a much stronger association of gp70 IC with kidney disease compared with IgG anti-chromatin autoantibodies in both populations of mice. Serum levels of gp70 correlated with production of gp70 IC in mice producing autoantibodies, although the overall effect on nephritis appeared to be small. Genetic mapping revealed three NZB-derived regions on chromosomes 2, 4, and 13 that were strongly linked with increased gp70 levels, and together, accounted for over 80% of the variance for this trait. However, additional linkage analyses of these crosses showed that loci controlling autoantibody production rather than gp70 levels were most important in the development of nephritogenic immune complexes. Together, these studies characterize a set of lupus-susceptibility loci distinct from those that control autoantibody production and provide new insight into the components involved in the strong association of gp70 IC with murine lupus nephritis.

Cholangiocytes as immune modulators in rotavirus-induced murine biliary atresia.

Background/Aims: Biliary atresia (BA) is a progressive disease characterized by bile duct inflammation and fibrosis. The aetiology is unknown and may be due to a virus‐induced, autoimmune‐mediated injury of cholangiocytes. Cholangiocytes are not only targets of injury but may also modulate hepatic inflammation. The aim of this study was to determine the immune profile of murine cholangiocytes and the ability to function as antigen‐presenting cells (APCs) in culture with Rhesus rotavirus (RRV), poly I:C (viral mimic) or interferon‐γ/tumour necrosis factor‐α.

Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresia

BACKGROUND & AIMS Biliary atresia (BA) is a pediatric inflammatory disease of the biliary system which leads to cirrhosis and the need for liver transplantation. One theory regarding etiology is that bile duct injury is due to virus-induced autoreactive T cell-mediated inflammation. Regulatory T cell (Treg) abnormalities in BA could result in unchecked bystander inflammation and autoimmunity targeting bile ducts. The aim of this study was to determine if Tregs are dysfunctional in the rotavirus-induced mouse model of BA (murine BA). METHODS Murine BA resulted from infection of BALB/c neonates with Rhesus rotavirus (RRV). RESULTS Liver Tregs from BA mice were decreased in number, activation marker expression, and suppressive function. Adoptive transfer studies revealed that RRV-infected mice that received Tregs had significantly increased survival (84%) compared to controls (12.5%). In addition, ablation of Tregs in older mice, followed by RRV infection, resulted in increased bile duct injury. CONCLUSIONS These studies demonstrate that dysregulation of Tregs is present in murine BA and that diminished Treg function may be implicated in the pathogenesis of human BA.

B Cell Deficient Mice Are Protected from Biliary Obstruction in the Rotavirus-Induced Mouse Model of Biliary Atresia

A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α-/-) mice received RRV shortly after birth. Ig-α-/- RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α-/- mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α-/- mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α-/- mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α-/- mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α-/- mice were not the sole reason for protection from disease. Conclusion: B cell deficient Ig-α-/- mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.

Dissection of Genetic Mechanisms Governing the Expression of Serum Retroviral gp70 Implicated in Murine Lupus Nephritis

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes as an acute phase protein, and it has been thought to be a product of an endogenous xenotropic virus, NZB-X1. However, since endogenous polytropic (PT) and modified polytropic (mPT) viruses encode gp70s that are closely related to xenotropic gp70, these viruses can be additional sources of serum gp70. To better understand the genetic basis of the expression of serum gp70, we analyzed the abundance of xenotropic, PT, or mPT gp70 RNAs in livers and the genomic composition of corresponding proviruses in various strains of mice, including two different Sgp (serum gp70 production) congenic mice. Our results demonstrated that the expression of different viral gp70 RNAs was remarkably heterogeneous among various mouse strains and that the level of serum gp70 production was regulated by multiple structural and regulatory genes. Additionally, a significant contribution of PT and mPT gp70s to serum gp70 was revealed by the detection of PT and mPT, but not xenotropic transcripts in 129 mice, and by a closer correlation of serum levels of gp70 with the abundance of PT and mPT gp70 RNAs than with that of xenotropic gp70 RNA in Sgp3 congenic mice. Furthermore, the injection of lipopolysaccharides selectively up-regulated the expression of xenotropic and mPT gp70 RNAs, but not PT gp70 RNA. Our data indicate that the genetic origin of serum gp70 is more heterogeneous than previously thought, and that distinct retroviral gp70s are differentially regulated in physiological vs inflammatory conditions.

High-dose IgG therapy mitigates bile duct-targeted inflammation and obstruction in a mouse model of biliary atresia

Background:A proposed etiology of biliary atresia (BA) entails a virus-induced, progressive immune-mediated injury of the biliary system. Intravenous Ig (IVIg) has demonstrated clinical benefit in several inflammatory diseases. The aim of this study was to determine the therapeutic effects of high-dose IgG treatment in the rhesus rotavirus (RRV)–induced mouse model of BA.Methods:Newborn mice were infected with RRV, and jaundiced mice were given high-dose IgG or albumin control. Survival, histology, direct bilirubin, liver immune cell subsets, and cytokine production were analyzed.Results:There was no difference in overall survival between RRV-infected groups, however high-dose IgG resulted in decreased bilirubin, bile duct inflammation, and increased extrahepatic bile duct patency. High-dose IgG decreased vascular cell adhesion molecule-1, resulting in limited migration of immune cells to portal tracts. High-dose IgG significantly decreased CD4+ T cell production of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α and CD8+ T cell production of IFN-γ, as well as increased levels of regulatory T cells.Conclusion:High-dose IgG therapy in murine BA dramatically decreased Th1 cell-mediated inflammation and biliary obstruction. This study lends support for consideration of IVIg clinical trials in infants with BA, to diminish the progressive intrahepatic bile duct injury.