We have produced several Nonobese diabetic (NOD) congenic strains (generically designated """"""""NOD.ABD"""""""" mice) that develop spontaneous, autoimmune biliary disease (ABD) and anti-pyruvate dehydrogenase complex (PDC) autoantibodies, and serve as a model of human primary biliary cirrhosis (PBC). They provide a much needed model system for determining early immunogenetic mechanisms of PBC, which is critically important because the human disease has a long """"""""silent"""""""" phase. We have now produced a novel congenic mouse, termed NOD.Abd3, with a 1.0 megabase chromosome one B6 interval on the NOD background;this strain develops severe, spontaneous ABD with 100% penetrance in male and female mice. This grant will study immunological mechanisms of disease in NOD.Abd3 mice. Our preliminary data show that ABD can be transferred by NOD.ABD, but not NOD, CD8+ cells, implicating NOD.Abd3 CD8 cells in pathogenesis. Our preliminary data also show that the adaptive immune system is central to the ABD disease process. Finally, co- transfer of ABD T regulatory cells prevented CD8 cell mediated ABD in scid recipients. This grant will dissect the immunological basis for this enhanced pathogenicity of NOD.ABD CD8 cells, and protective capacity of NOD.Abd3 Tregs, through the following specific aims:
Aim #1. Mechanism of CD8+ T cell mediated autoimmune biliary disease. We have demonstrated that NOD.ABD, but not NOD, CD8+ T cells are sufficient to transfer biliary pathology to ABD-scid recipients.
This aim dissects major mechanisms of CD8+ T cell mediated disease, using 1) transfer and bone marrow chimeric studies;2) an exhaustive analysis of CD8+ T cell marker expression in NOD.ABD mice;and 3) a thorough analysis of CD8+ T cell cytokine expression in vitro, and in vivo following adoptive transfer.
Aim #2. T helper dependence and antigen specificity of intrahepatic NOD.ABD CD8 T cells.
This aim will address two major issues: 1) Are the NOD CD8 T cell responses CD4+ T cell dependent or independent? 2) Are intrahepatic CD8 T cells antigen specific? We will test these issues by studying ABD with and without either CD4 or CD8 cells and by restricting the CD8+ T cell repertoire and assessing effect on disease.
Aim #3 : Role of T regulatory cells in ABD. We have shown that co-transfer of NOD.Abd3 splenic Tregs can prevent disease in our adoptive transfer model. Why don't NOD.Abd3 Tregs prevent spontaneous disease in vivo? We will exhaustively study the phenotype of hepatic vs. peripheral Tregs and demonstrate the functional role of Tregs in ABD by employing transfer and depletion studies. These studies are important and significant because they will allow us to study mechanisms of immune mediated biliary damage that previously could not be studied due to a lack of a spontaneous animal model of disease. Understanding early mechanisms of altered biliary immunity will lead to new hypotheses regarding the etiology and treatment of human autoimmune biliary disease.
The studies proposed here are of broad relevance to the VA mission and have an impact on understanding many inflammatory liver diseases and conditions. First, these studies will yield novel insights into the early pathogenic cellular mechanisms of autoimmune biliary disease, and identify the critical cell subsets that interact to produce an autoimmune biliary immune microenvironment. The discovery of critical immune cell subsets, and whether or to what extent they are regulated by innate versus adaptive immune events, will raise new therapeutic possibilities for autoimmune biliary disease. Second, the relevance of this work includes the central role hepatic immunity plays in many diseases that involve inflammation of the liver, such as alcoholic hepatitis, over-zealous response to hepatic infectious pathogens, and drug related liver injury, all of which are mediated by the hepatic immune system. We believe our studies will further the understanding of hepatic cellular immune injury and tolerance, and thus have broad relevance to medical science.