Recently, we identified a child born with a genetic deficiency of IL-2 receptor ␣ (IL-2R␣, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2R␣/CD25 deficient (IL-2R␣ ؊/؊ ) mice and wildtype littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2R␣ ؊/؊ , but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4 ؉ and CD8 ؉ T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-␥, TNF-␣, IL-2 and IL-12p40. Of importance is the finding that the IL-2R␣ ؊/؊ mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2R␣ ؊/؊ mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage. (HEPATOLOGY 2006;44:1240-1249.) P rimary biliary cirrhosis (PBC) is a chronic autoimmune liver disease primarily affecting smaller intrahepatic bile ducts and characterized by autoantibodies to the mitochondrially located E2 subunits of pyruvate dehydrogenase and related enzymes (PDC-E2), traditionally known as antimitochondrial antibodies (AMA). 1 A better understanding of the initial pathologic events will lead to better understanding for this eventually morbid and often fatal human disease. In particular, the early immunologic events in PBC have remained obscure, partly because of the long asymptomatic period. Clearly animal models of human PBC would greatly facilitate studies of the etiology of this disease and specially facilitate the identification of the early events that lead to this disease. Two murine models have recently been described that provide data which suggest that indeed this goal may be achieved. The first is the presence of AMA and severe biliary disease in a congenic strain of NOD mice, coined NOD.c3c4. 2,3 The second model is the appearance of autoimmune cholangitis and AMAs in TGF-␤ receptor II dominant-negative mice. 4 We have also seen a PBC-like disease in a child with homozygous IL-2R␣ (CD25) deficiency. 5 This latter finding is of interest since our laboratory has previously documente...

There have been important advances in defining effector mechanisms for several human autoimmune diseases. However, for most human autoimmune diseases, the induction stage is less well defined and there are very few clues on etiology. Our laboratory has focused on defining the molecular basis of autoantibody recognition and epitope modification in primary biliary cirrhosis (PBC). Our work has demonstrated that antibodies to mitochondria (AMA), the hallmark of disease, are directed against a very conserved site of pyruvate dehydrogenase (PDCE2). We have also demonstrated that several chemical xenobiotics, chosen based on quantitative structural activity relationship analysis and rigorous epitope analysis, when coupled to the lysine residue that normally binds the lipoic acid co-factor of PDC-E2, reacts as well or better to PBC sera than native autoantigen. In the present studies, we immunized C57BL/6 mice with one such xenobiotic, 2-octynoic acid (2OA), coupled to bovine serum albumin (BSA) and followed mice for 24 weeks. Animals were studied for appearance of histologic lesions as well as appearance of antibodies to PDC-E2, serum levels of TNF-α and IFN-γ and splenic and liver lymphoid phenotyping by flow cytometry. Mice immunized with 2OA manifest autoimmune cholangitis, typical mitochondrial autoantibodies, increased liver lymphoid cell numbers, an increase in CD8+ liver infiltrating cells, particularly CD8+ T cells that co-express CD44, and finally an elevation of serum TNF-α and IFN-γ. In conclusion, these data provide a persuasive argument in favor of an environmental origin for human PBC.

The interleukin (IL)-12/IL-23 mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, IFN-γ, IL-12RB2, IL-23p40, IL-23p19, IL-17 and IL-23R using liver from PBC (n=51) and non-PBC (n=80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining were detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most importantly, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN-γ expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance pro-fibrotic Th17 signaling and pro-inflammatory IFN-γ production that contribute to PBC pathology. In conclusion, our data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.

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Our laboratory has reported that mice that express a dominant negative form of transforming growth factor ␤ receptor restricted to T cells (dnTGF␤RII) develop an inflammatory biliary ductular disease with elevated serum levels of interleukin (IL)-12p40 and other proinflammatory cytokines and antimitochondrial autoantibodies (AMAs) closely resembling human primary biliary cirrhosis (PBC). We have used this mouse model to address the potential mechanisms of immunomodulation of liver disease by creating two unique genetic strains: IL-12p40 knockout (KO)-dnTGF␤RII mice and IFN-␥ KO-dnTGF␤RII mice. The two colonies of genetically modified mice-and, for purposes of controls, the dnTGF␤RII mice-were monitored for liver immunopathology, AMAs, and intrahepatic cytokine production. Disease expression in the IFN-␥ KO-dnTGF␤RII mice, including liver immunopathology, were similar to those of dnTGF␤RII mice, whereas the IL-12p40 KOdnTGF␤RII mice had a dramatic reduction in histological autoimmune cholangitis and significant decreases in levels of intrahepatic proinflammatory cytokines, but similar levels of AMAs compared with dnTGF␤RII controls. Conclusion: These data indicate that in this mouse model of PBC, signaling by way of IL-12p40 is an essential requirement for the development of autoimmune cholangitis. The results of these studies will play an important role in identifying pathways and reagents that will selectively inhibit IL-12 signaling for the outlining of future therapeutic strategies for human PBC.