Biliary atresia (BA) is a progressive fibroinflammatory obstruction of the extrahepatic biliary tree and the most common indication for pediatric liver transplantation worldwide. It is uniquely restricted to the early neonatal period, and rotavirus (RRV) infection only causes BA in mice when injected during the first 3 days of life. We have reported that regulatory T cells (Tregs) are absent in the liver during this time period, but emerge rapidly following RRV challenge in older mice protected from RRV induced BA. In preliminary studies, we have shown that adoptive transfer of Treg-containing CD4+ cells prior to RRV inoculation constrained hepatic expansion of NK and CD8 lymphocytes and attenuated the BA phenotype, as evidenced by lower serum bilirubin levels and reduced inflammatory bile duct obstruction. Mechanistically, these changes were linked to down-regulation of B7 costimulatory molecules on hepatic myeloid dendritic cells. Adoptive transfer experiments with CD4 cells lacking the chemokine receptor CXCR3 indicated a critical role for local positioning of Tregs in control of hepatic immune responses. In samples of infants with BA, we found trends towards increased production of pro-inflammatory cytokines in circulating T-lymphocytes, up-regulation of B7 molecules on hepatic myeloid dendritic cells, and increased frequency of circulating Tregs lacking expression of CXCR3 compared with age- matched controls without liver injury. Based on these data we propose the overarching hypothesis that Tregs protect from neonatal bile duct injury through local control of effector lymphocyte activation. This hypothesis will be tested in three overlapping specific aims: 1) to determine the mechanisms by which Tregs constrain bile duct injury, 2) to identify pathways of immune regulation by Tregs in BA, and 3) to elucidate molecular determinants for Treg trafficking to the site of inflammation in BA.
For aim 1, the effects of adoptive transfer of Treg/CD4+ cells on cytotoxic activity of NK cells and differentiation of T-lymphocytes will be examined in murine BA, and Treg suppression of cytokine production in circulating T-lymphocytes will be studied in infants with BA and in healthy controls.
For aim 2, the roles of dendritic cells, TGF? and IL10 as cellular and cytokine mediators of Treg suppression will be investigated using knockout and reporter transgenic mice, cell transplantation experiments, cytokine neutralizing antibodies, and co-culture assays measuring the stimulatory capacity of dendritic cells. Preliminary results of up-regulation of B7-molecules on hepatic dendritic cells in infants with BA will be validated in a larger cohort.
For aim 3, the influence of CXCR3 on Treg-migration to the liver will be elucidated in adoptive transfer experiments and by determining the functional profile of circulating and hepatic Tregs in BA subjects and controls. Collectively, the complementary studies in mice and tissue of infants with BA will identify key regulatory pathways by which Tregs control pathogenesis of disease, guide future therapies to block the progression of biliary injury, and improve long term outcomes in children.

Public Health Relevance

Biliary atresia is the number one indication for liver transplantation in children and its cause is unknown. This grant proposes to investigate whether a low number of master immune cells (named regulatory T cells) during the neonatal period predisposes to uncontrolled activation of lymphocytes injuring neonatal bile ducts. The complementary studies in the mouse model and in human samples will advance our knowledge of the disease and facilitate the development of novel non-transplant treatment options for biliary atresia.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01DK095001-03
Application #
8702161
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Doo, Edward
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Miethke, Alexander G; Huppert, Stacey S (2013) Fishing for biliary atresia susceptibility genes. Gastroenterology 144:878-81