William M. Ridgway

Anti-Mitochondrial Antibodies and Primary Biliary Cirrhosis in TGF-β Receptor II Dominant-Negative Mice

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-β receptor type II (dnTGFβRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-βRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

IL‐2 receptor α−/− mice and the development of primary biliary cirrhosis

Recently, we identified a child born with a genetic deficiency of IL‐2 receptor α (IL‐2Rα, 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‐2Rα/CD25 deficient (IL‐2Rα−/−) mice and wild‐type 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‐2Rα−/−, 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‐γ, TNF‐α, IL‐2 and IL‐12p40. Of importance is the finding that the IL‐2Rα−/− 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‐2Rα−/− 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.)

Liver Autoimmunity Triggered by Microbial Activation of Natural Killer T Cells

Summary Humans with primary biliary cirrhosis (PBC), a disease characterized by the destruction of small bile ducts, exhibit signature autoantibodies against mitochondrial Pyruvate Dehydrogenase Complex E2 (PDC-E2) that crossreact onto the homologous enzyme of Novosphingobium aromaticivorans, an ubiquitous alphaproteobacterium. Here, we show that infection of mice with N. aromaticivorans induced signature antibodies against microbial PDC-E2 and its mitochondrial counterpart but also triggered chronic T cell-mediated autoimmunity against small bile ducts. Disease induction required NKT cells, which specifically respond to N. aromaticivorans cell wall α-glycuronosylceramides presented by CD1d molecules. Combined with the natural liver tropism of NKT cells, the accumulation of N. aromaticivorans in the liver likely explains the liver specificity of destructive responses. Once established, liver disease could be adoptively transferred by T cells independently of NKT cells and microbes, illustrating the importance of early microbial activation of NKT cells in the initiation of autonomous, organ-specific autoimmunity.

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti–PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9–10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease.

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, the hallmark of disease, are directed against a very conserved site of pyruvate dehydrogenase, the E2 subunit of pyruvate dehydrogenase (PDC‐E2). 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 cofactor 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, coupled to bovine serum albumin and we followed the mice for 24 weeks. Animals were studied for appearance of histologic lesions as well as appearance of antibodies to PDC‐E2, serum levels of tumor necrosis factor–α and interferon‐γ, and splenic and liver lymphoid phenotyping by flow cytometry. Mice immunized with 2‐octynoic acid manifest autoimmune cholangitis, typical mitochondrial autoantibodies, increased liver lymphoid cell numbers, an increase in CD8+ liver infiltrating cells, particularly CD8+ T cells that coexpress CD44, and finally an elevation of serum tumor necrosis factor–α and interferon‐γ. Conclusion: these data provide a persuasive argument in favor of an environmental origin for human PBC. (HEPATOLOGY 2008.)

Natural killer T cells exacerbate liver injury in a transforming growth factor β receptor II dominant‐negative mouse model of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an organ‐specific autoimmune liver disease characterized by the presence of antimitochondrial antibodies and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of CD1d‐restricted natural killer T (NKT) cells were increased in the livers of patients with PBC. To define a specific role of CD1d‐restricted NKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic CD1d‐restricted NKT cells in our transforming growth factor β (TGF‐β) receptor II dominant‐negative (dnTGFβRII) mouse model of PBC. We generated CD1d−/− and CD1d+/− dnTGFβRII mice and performed a comparative study of liver immunopathology. We report herein that these dnTGFβRII mice demonstrate a massive increase of hyperactive CD1d‐restricted NKT cells within the hepatic tissues. CD1d−/−dnTGFβRII mice, which lack CD1d‐restricted CD1d‐restricted NKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with CD1d+/−dnTGFβRII mice. Interestingly, there was a significant increase in the production of interferon‐γ in hepatic CD1d‐restricted NKT cells activated by α‐galactosylceramide in young but not older dnTGFβRII mice, suggesting an age‐dependent role of CD1d‐restricted NKT cells. Conclusion: These data demonstrate that CD1d‐restricted NKT cells in dnTGFβRII mice are a critical factor in liver injury. (HEPATOLOGY 2008.)

Adoptive transfer of CD8+ T cells from transforming growth factor beta receptor type II (dominant negative form) induces autoimmune cholangitis in mice

We recently reported that mice with a T cell–restricted expression of a dominant negative form of transforming growth factor β receptor type II (dnTGFβRII) spontaneously develop autoimmune cholangitis that resembles human primary biliary cirrhosis (PBC), including antimitochondrial antibodies (AMAs) and extensive portal CD4+ and CD8+ lymphocytic infiltrates. On the basis of these data, we performed a series of experiments to determine whether the pathology was secondary to direct dnTGFβRII disruption of the liver and/or alternatively the appearance of autoreactive T cells. First, using dnTGFβRIIRag1−/− mice, we noted a normal hepatic and biliary structure. Hence, we performed a rigorous series of adoptive transfer studies, transferring Ly5.1+ unfractionated spleen cell CD4+ or CD8+ T cells from dnTGFβRII mice into B6/Rag−/− (Ly 5.2) recipients. In unmanipulated dnTGFβRII mice, there was a marked increase in CD4+ and CD8+ T cell biliary infiltrates with AMA. Indeed, B6/Rag−/− recipients of dnTGFβRII unfractionated cells develop features of liver disease similar to PBC, suggesting that splenic loss of self‐tolerance alone is sufficient to cause disease in this model and therefore that there is no specific abnormality in the biliary targets required for appearance of disease. More importantly, adoptive transfer of CD8+ but not CD4+ T cells into B6/Rag−/− mice led to liver histopathology remarkably similar to PBC, emphasizing a prominent role for CD8 T cell–mediated pathogenesis. In contrast, B6/Rag−/− recipients of CD4+ T cells from dnTGFβRII mice predominantly developed inflammatory bowel disease associated with higher levels of serum interferon γ and tumor necrosis factor α. Conclusion: These data suggest that in this model of PBC, autoreactive CD8+ cells destroy bile ducts. (HEPATOLOGY 2008.)

Genetic Control of Autoimmunity: Protection from Diabetes, but Spontaneous Autoimmune Biliary Disease in a Nonobese Diabetic Congenic Strain

At least 20 insulin-dependent diabetes (Idd) loci modify the progression of autoimmune diabetes in the NOD mouse, an animal model of human type 1 diabetes. The NOD.c3c4 congenic mouse, which has multiple B6- and B10-derived Idd-resistant alleles on chromosomes 3 and 4, respectively, is completely protected from autoimmune diabetes. We demonstrate in this study, however, that NOD.c3c4 mice develop a novel spontaneous and fatal autoimmune polycystic biliary tract disease, with lymphocytic peribiliary infiltrates and autoantibodies. Strains having a subset of the Idd-resistant alleles present in the NOD.c3c4 strain show component phenotypes of the liver disease: NOD mice with B6 resistance alleles only on chromosome 3 have lymphocytic liver infiltration without autoantibody formation, while NOD mice with B10 resistance alleles only on chromosome 4 show autoantibody formation without liver infiltration. The liver disease is transferable to naive NOD.c3c4 recipients using splenocytes from affected NOD.c3c4 mice, demonstrating an autoimmune etiology. Thus, substitution of non-NOD genetic intervals into the NOD strain can prevent diabetes, but in turn cause an entirely different autoimmune syndrome, a finding consistent with a generalized failure of self-tolerance in the NOD genetic background. The complex clinical phenotypes in human autoimmune conditions may be similarly resolved into largely overlapping biochemical pathways that are then modified, potentially by alleles at a few key chromosomal regions, to produce specific autoimmune syndromes.

B Cells Suppress the Inflammatory Response in a Mouse Model of Primary Biliary Cirrhosis

BACKGROUND & AIMS Mice that express a dominant-negative form of transforming growth factor-beta receptor restricted to T cells (dnTGF-betaRII) develop antimitochondrial antibodies and liver inflammation similar to human primary biliary cirrhosis. METHODS To address the role of B cells in this model of primary biliary cirrhosis, we bred B cell-deficient mice (Igmu(-/-)) with dnTGF-betaRII mice, creating Igmu(-/-)dnTGF-betaRII mice, and compared the resulting disease phenotype with that of dnTGF-betaRII mice (controls). We also performed adoptive transfer of dnTGF-betaRII CD8(+) splenocytes, with or without B cells, to 8-week-old female Rag-1(-/-) mice to assess the role of B cells in the inflammatory response. RESULTS The B cell-deficient Igmu(-/-)dnTGF-betaRII mice unexpectedly developed a more severe form of cholangitis than controls (dnTGF-betaRII mice) and had a significantly greater frequency of activated CD4(+) and CD8(+) T cells in the liver. They also had reduced frequency of Foxp3(+) regulatory T cells in the hepatic CD4(+) T-cell population and natural killer (NK) T cells (NK1.1(+) CD3(+)) in hepatic inflammatory cell infiltrates. The Igmu(-/-)dnTGF-betaRII mice had increased levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and developed a more severe form of colitis than controls. Adoptive transfer of CD8(+) splenocytes from dnTGF-betaRII mice and peritoneal cavity-derived, but not spleen-derived, CD19(+) B cells into Rag-1(-/-) mice resulted in decreased amounts of liver inflammation and bile duct damage, compared with Rag-1(-/-) mice in which only CD8(+) splenocytes were transferred. CONCLUSION B cells have a suppressive effect on the inflammatory response in the dnTGF-betaRII model of primary biliary cirrhosis.

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC‐E2) leads to an anti‐mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non‐obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2‐octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti‐mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA‐BSA, but not with BSA alone, develop high titre anti‐mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8+ cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.