The Whitington laboratory is involved in translational research - applying molecular approaches to the study of human specimens and animal and cell-based models in order to understand the mechanisms involved in the pathogenesis of several pediatric liver diseases.
Current Research Programs
Gestational Alloimmune Liver Disease (AKA Neonatal Hemochromatosis)
Neonatal hemochromatosis (NH) is defined phenotypically as severe liver disease in association with iron accumulation (siderosis) in the liver and extrahepatic sites in a distribution similar to that seen in hereditary hemochromatosis. It has been considered to be a very rare condition that is usually lethal to the fetus or neonate. Fetal liver injury is considered to be the precursor of NH, and thus our research has focused on the mechanism(s) of fetal liver injury. Based on clinical insights we hypothesized that NH is a gestational alloimmune disorder, wherein the fetal liver is the target of a maternal humoral immune response. Subsequently, we determined that the livers of infants with NH showed evidence of extensive complement-mediated hepatocyte injury. Since the only reasonably possible mechanism for inciting such complement-mediated injury, which involves activation of the terminal complement cascade and formation of membrane attack complex, is via the classical pathway, maternal IgG must be involved. We named the condition Gestational Alloimmune Liver Disease (GALD). We have shown that with few exceptions (rare cases of bile acid synthetic defects, mitochondrial DNA depletion due to DGUOK mutations and Down syndrome) the NH phenotype is the result of GALD. We have further shown that GALD may produce acute liver injury and liver failure in the fetus, causing intrauterine fetal demise. Many of these cases do not show the NH phenotype due to the acuteness of the injury. Thus, we have shown nearly all NH is due to GALD, yet GALD is the cause of severe liver injury in the absence of the NH phenotype as well.
We have sought to answer the question of what cell type is actually injured in GALD. The acute form seemingly occurs only in fetuses in mid-gestation, and typical NH seems to produce hepatic insufficiency beginning in mid-gestation. To rectify all of these findings, we developed a unifying hypothesis to explain the mechanism by which GALD produces the stereotypic liver pathology seen in typical NH, as well as the pathology of acute GALD. The central figure in this mechanistic paradigm is the “nascent hepatocyte”. We had focused on the complement accumulated in giant cells in NH liver, but now refocused on whether we could identify nascent hepatocyte injury in these same end-stage livers. By changing our approach we can show membrane attack complex (MAC) concentrated in a membranous pattern on cells with the morphology of early hepatocytes. Double staining for MAC and DLK1, a marker for hepatoblasts, shows the antigens to not be expressed in the same cells. DLK+ hepatoblasts are few in number and never show MAC staining. These findings lead us to postulate that the emergence of cells in the hepatocyte lineage from hepatoblast status is associated with the expression of a protein that is the GALD-antigen. We have found that mouse embryonic liver at a specific stage expresses a protein that is bound by GALD-IgG and is likely the orthologue of the human GALD-antigen. We are now identifying the gene involved. With this information, it will be possible to express the protein in a mammalian cell system and thus to prepare it in quantity. If successful, cloning the fetal liver protein target of GALD will permit us to develop an assay for detecting and quantifying antibody in the serum of women at risk and in infants with liver disease.
Liver Fibrosis in Pediatric Liver Disease and Liver Allografts
The Whitington laboratory has produced results that show the importance of the Hedgehog signaling pathway in the development of liver fibrosis in biliary atresia and other pediatric cholestatic diseases and in GALD. This work challenges the precept that inflammation is a necessary precursor to scarring in liver disease. The group in collaboration with Anna Mae Diehl’s laboratory at Duke University has shown that in biliary atresia, the most important liver disease of infancy, the pathway can be turned on by bile duct injury (as might occur in a viral infection) and without inducing inflammation leads to the accumulation of scar-producing cells. This is an innovative finding that is expected to change thinking and lead to new research in this important disease. The collaborative group also has shown that Hedgehog activation is an important contributor to fibrosis associated with fatty liver disease. Recently we have shown that Hedgehog activation in extensive progenitors in GALD leads to fibrosis by way of elaborating the profibrogenic cytokine SPP1. A future aim of this work is to understand why children with liver transplants develop fibrosis (scarring) of the liver graft over time. This basic research project is expected to provide answers for the questions of why and how liver graft fibrosis occurs and provide targets for intervening. This will result in improved health and longer lives for pediatric liver transplant recipients.
Further studies of experimental models in mice and cells in culture will help us determine what molecular pathways need to be examined in pediatric liver transplant recipients to understand the mechanism of late allograft scarring. The team uses various advanced techniques to examine the induction of cell signals at the gene level and at the protein level. These techniques enable the study of the pathways involved in fibrosis in a small portion of a routine liver biopsy. The team expects to find that pathways similar, if not identical to those seen in experimental liver disease, are important in the scarring often seen in long-term liver transplant recipients. If this hypothesis proves true, Whitington and his colleagues will further understand how to prevent the activation of scarring and potentially discovering drug interventions to prevent late allograft loss.
See the full spectrum of studies.
Fatty Liver Disease
Whitington is a site PI for the NIH-NIDDK consortium Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) and is actively involved in various clinical and translational research studies taking place under the auspices of this consortium.