Volume 134, Issue 5, Pages 1482-1493 (May 2008) Ae2a,b-Deficient Mice Develop Antimitochondrial Antibodies and Other Features Resembling Primary Biliary Cirrhosis  January T. Salas, Jesús M. Banales, Sarai Sarvide, Sergio Recalde, Alex Ferrer, Iker Uriarte, Ronald P.J. Oude Elferink, Jesús Prieto, Juan F. Medina  Gastroenterology  Volume 134, Issue 5, Pages 1482-1493 (May 2008) DOI: 10.1053/j.gastro.2008.02.020 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Splenomegaly and splenocyte alterations in Ae2a,b−/− mice. (A) Typical spleens from an Ae2a,b−/− mouse and its wild-type littermate. Bar = 0.5 cm. (B) Higher spleen/body weight ratio (spleen index) in Ae2a,b−/− mice than in control littermates. (C) Intracellular alkalinization of splenocytes from Ae2a,b−/− mice compared with control littermates. (D) Cytokine arrays indicate that splenocytes from Ae2a,b−/− mice produce more interferon gamma (IFNγ) and IL-12p70 than those from wild-type littermates. In B and C, each dot represents the value for an individual mouse, and horizontal bars represent mean values. In B, spleen index was calculated for each animal as the ratio of spleen weight ×1000 to body weight. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Expansion of CD8+ population and reduced frequency of Tregs in Ae2a,b−/− spleens. (A) FACScan analysis and subsequent calculation of the absolute number of CD3+CD8+ splenocytes in Ae2a,b−/− mice and control littermates. (B) Typical FACS dot-plot graphs of CD3+CD4+ and CD3+CD8+ populations in an Ae2a,b−/− mouse and control littermates. The reduced proportion of CD3+CD4+ splenocytes in the Ae2a,b−/− mouse results from CD3+CD8+ expansion, because the calculated total number of CD3+CD4+ cells is similar among genotypes. (C) Inversion of CD4+/CD8+ ratio in Ae2a,b−/− mice versus control littermates. (D) FACS dot-plot graphs of CD4+FoxP3+ splenocytes in an Ae2a,b−/− mouse and control littermates indicating a lower proportion of this population in an Ae2a,b−/− mouse. (E) Immunohistochemical analysis for FoxP3 in paraffin-embedded spleen sections of an Ae2a,b−/− mouse and control littermates showing a reduced number of FoxP3+ splenocytes (half of them indicated by arrows) in the Ae2a,b−/− mouse. Bar = 50 μm. (F) Image analysis of the histologic sections and calculation of the proportion of Tregs indicated reduced FoxP3+ cells in Ae2a,b−/− mice compared with wild-type littermates. In A, C, and F each dot represents the value for an individual mouse, and horizontal bars represent mean values. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 AMA developed in Ae2a,b−/− mice recognize the mouse PDC-E2 component. (A) Immunoblot analysis showing that the serum from a 15-month-old Ae2a,b−/− mouse has AMA that recognizes a 65-kilodalton band of PDC partially purified from mouse hearts (right band). The 65-kilodalton band was also recognized by the serum from a wild-type mouse immunized with bovine PDC extract as described17 (left band). (B) Q-TOF/MS analysis of the 65-kilodalton band showed 25% sequence coverage (green) for the E2 component of Mus musculus PDC. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 AMA in Ae2a,b-deficient mice are directed against the PBC-specific PDC-E2 epitope. (A) Chemiluminescence immunoblots indicate that most 15-month-old Ae2a,b−/− mice and some heterozygous littermates developed serum AMA; they recognize the recombinant mouse PDC-E2 peptide 170–313, which encompasses PBC epitopes (see Materials and Methods). The upper diagram represents the chemiluminescence values in mouse litters L1–L11 (filled boxes for Ae2a,b−/− mice and dotted boxes for heterozygous littermates); sera were always diluted 1:3000. Chemiluminescence values are relative to the highest value (100%) obtained in the Ae2a,b−/− mouse from litter L3. In litters L8, L9, and L11, 2 heterozygous littermates were available as controls, while in litter L7 there were no available (n.a.) heterozygous controls. (B and C) Analyses of serum immunoreactivity intensity in Ae2a,b−/− and Ae2a,b+/− mice from litters L8 and L11 at 3, 6, 12, and 15 months of age indicate that the levels of AMA (always IgG autoantibodies) increased over time. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Progressive cholestasis and portal infiltration in Ae2a,b−/− mice. (A) Steady increase in the serum levels of the hepatic isoform of alkaline phosphatase (hALP) in 6-, 12- and 15-month-old Ae2a,b−/− mice versus heterozygous and wild-type littermates; horizontal bars represent mean values. (B) H&E staining of Ae2a,b−/− liver section showing intense mononuclear cell infiltration in portal tracts surrounding damaged bile ducts (see the thick black arrow in the inset magnified 4 times at the right). (C) Ae2a,b−/− liver section stained as in B, in which eosinophils (thin black arrows) are also observed in a portal infiltrate. (D) Portal fibrosis (white arrow) revealed by Masson's trichrome staining in an Ae2a,b−/− liver. Bars = 100 μm. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Immunohistopathology and T-cell populations in Ae2a,b−/− livers. (A) OCT frozen liver sections, either from Ae2a,b−/− or wild-type mice, stained for CD8 and CD4. The intense brown stains in portal infiltrates indicate that a high proportion of infiltrating mononuclear cells are CD8+ T lymphocytes; black arrows in Ae2a,b−/− serial sections indicate the same damaged bile duct. Bar = 200 μm. (B) Typical FACS dot-plot graphs of liver CD3+CD4+ and CD3+CD8+ populations showing an increased proportion of the latter in an Ae2a,b−/− mouse compared with control littermates. (C) Inversion of CD4+/CD8+ ratio in Ae2a,b−/− livers vs control littermates. (D) Typical FACS dot-plot graphs of liver CD4+FoxP3+ lymphocytes showing a lower proportion of this population in an Ae2a,b−/− mouse compared with control littermates. (E) FoxP3+ cells are significantly reduced in the livers from Ae2a,b−/− mice compared with those in wild-type littermates. In C and E, each dot represents the value for an individual mouse, and horizontal bars represent mean values. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Real-time PCR showing differential messenger RNA expression of genes involved in oxidative stress and antigen presentation pathways between cholangiocytes from Ae2a,b−/− mouse and wild-type mouse (see Table 1 for complete gene names). Transcript levels in Ae2a,b−/− mouse cholangiocytes are represented as n-fold changes relative to the levels in wild-type mouse cholangiocytes (mean ± SD; n = 5 each). *P < .05; **P < .01; ***P < .001. Gastroenterology 2008 134, 1482-1493DOI: (10.1053/j.gastro.2008.02.020) Copyright © 2008 AGA Institute Terms and Conditions