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

Abstract

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-alpha and interferon-gamma, 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-alpha and interferon-gamma.

Conclusion: these data provide a persuasive argument in favor of an environmental origin for human PBC.

Figure 1. Structures of lipoic acid and 2-octynoic acid-BSA

2-octynoic acid was conjugated with BSA and used for immunization. “Z” represents any lysine residue of BSA.

Figure 2. Antibody to PDC-E2

A. Immunoblot at 8 weeks after immunization with 2OA-BSA, and BSA. Recombinant PDC-E2 protein was loaded on SDS-PAGE and transferred to a nitrocellulose membrane. All 13 sera from mice immunized with 2OA-BSA versus none of 12 sera from BSA-immunized mice showed detectable reactivity with a component of ~70kDa. Sera from two PBC patients and a mouse anti-PDC-E2 monoclonal antibody (mAb), 2H4, were included as positive controls. B. Quantification of anti-PDC-E2 in the sera by ELISA at 2 weekly intervals after immunization shows significant increases in O.D. values after immunization between responder and control mice. *P<0.05, **P<0.01, ***P<0.001.

Figure 3. Levels of TNF-α and IFN-γ in sera of mice after immunization

The levels of both Th1 cytokines in sera from mice immunized with 2OA-BSA versus BSA-immunized mice were markedly increased. **P<0.01, ***P<0.001.

Figure 4. Microscopic examination of liver tissue from immunized and control mice

A–D. Light microscopy of stained sections of liver at 12 weeks after immunization of mice with BSA (A) shows no abnormalities, and after immunization with 2OA-BSA shows mild infiltration of lymphocytes in portal tracts (B, C, D) particularly surrounding damaged intralobular bile ducts (B, C, green *). Bile duct loss and epithelioid granulomas (yellow arrow) were observed in some portal tracts (D), and granulomas were found also in hepatic parenchyma (C). P, portal vein. (H & E ×400: scale bar, 100μm.) E, F. Immunohistochemical analysis of liver sections from 2OA-BSA-immunized mice at 12 weeks after immunization using mAbs to CD4 (E) and CD8 (F). Both CD4⁺ and CD8⁺ T lymphocytes were distributed in portal tracts with CD8⁺ T cells predominating over CD4⁺ T cells. (×400: scale bar, 100μm.) G. Portal infiltrates and bile duct damages were scored at 12 weeks after immunization; 0 for none, 1 for mild, 2 for moderate. Both portal infiltrates and bile duct damages were observed in all the tested mice for 2OA-BSA (n=6) while none of them were found in all the mice for BSA (n=4).

Figure 5. Lymphoid cell population in liver and spleen from immunized mice at 12 weeks

Cells were stained with mAbs to TCRβ, CD4 and CD8. Analysis was performed with gating of TCRβ⁺ cells. The CD4/CD8 ratio was significantly decreased in liver but not in spleen from mice immunized with 2OA-BSA versus control BSA-immunized mice. **P<0.01.

Figure 6. Numbers of lymphoid cells in liver and spleen from immunized mice

A. Total lymphoid cells were significantly increased in the liver but not in the spleen of -BSA-immunized mice versus control BSA-immunized mice. B–D. Cells were stained with anti-TCRβ, CD4, CD8, CD19 and enumerated by flow cytometry. Graphs show data for CD4⁺ T cells (B), CD8⁺ T cells (C) and CD19⁺ B cells (D). CD8⁺ T cells and CD19⁺ B cells were significantly increased in the liver but not in the spleen of 2OA-BSA-immunized mice versus control BSA-immunized mice. **P<0.01.

Figure 7. Flow cytometry analysis of CD8 and CD44 in liver and spleen

Analysis was performed with gating of TCRβ⁺ and CD8⁺ cells. Frequency of CD44^high memory T cells in CD8⁺ T cells were increased in 2OA-BSA-immunized mice liver but not in the spleen compared with BSA-immunized mice. *P<0.05