Decreased cell infiltrates and IFN-γ production of liver mononuclear cells in 2-OA-BSA immunized CD1d^-/- mice.

<p>(A-C) CD1d^-/- and wild type mice were immunized with 2-OA-BSA at 0, 2, 4, 6 and 8 and sacrificed at week 12. (A) Liver total mononuclear cells (MNCs) were measured. (B) The numbers of T (CD3⁺ NK1.1^-), NKT (CD3⁺NK1.1⁺), NK (CD3^-NK1.1⁺) and B (CD19⁺) cells were measured. (C) The numbers of CD4⁺ and CD8⁺ T cells were detected. n = 16–20 per group. *, p<0.05. (D) CD1d^-/- or wild type mice were immunized with 2-OA-BSA at weeks 0, 2, and 4 and sacrificed 3 days after last immunization. IFN-γ production of liver mononuclear cells stimulated with anti-CD3 and anti-CD28 Abs for 2 days was measured by ELISA. n = 8 mice for PBS treated group, n = 13–17 mice for 2-OA-BSA immunized group. *, p<0.05; **, p<0.01.</p

Lower serum levels of IFN-γ after OCH injection.

<p>C57BL/6 mice were intravenously injected with α-GalCer, OCH, or PBS. Serum samples were collected at 2 and 18 hours after α-GalCer, OCH, or PBS injection. IFN-<i>γ</i> (A) and IL-4 (B) were measured by ELISA. n = 10 mice per group. ***, p<0.001.</p

Decreased AMAs in 2-OA-BSA immunized CD1d^-/- mice.

<p>(A) iNKT cell deficiency in CD1d^-/- mice was confirmed by staining with CD3 and CD1d tetramer. (B) CD1d^-/- and wild type mice were immunized with 2-OA-BSA at 0, 2, 4, 6 and 8 and sacrificed at week 12. Serum levels of IgM and IgG to mPDC-E2 were measured by ELISA. n = 10 mice for each group. *, p<0.05.</p

OCH administration increased cell infiltrates and activation of T cells in mice.

<p>(A) C57BL/6 mice were intravenously injected with α-GalCer, OCH, or PBS. Liver total mononuclear cells (MNC) were counted 3 days after α-GalCer, OCH, or PBS injection. n = 10–13 mice per group. ***, p<0.001. (B-E) Wild type mice were immunized with 2-OA-BSA and α-GalCer (group name: 2-OA/a-GC), OCH (group name:2-OA/OCH) or PBS (group name: 2-OA/PBS) at weeks 0, 2, 4, 6 and 8 and sacrificed at week 12. (B) Liver total mononuclear cells (MNC) were measured. (C) The numbers of T (CD3⁺ NK1.1^-), NKT (CD3⁺NK1.1⁺), NK (CD3^-NK1.1⁺) and B (CD19⁺) cells were measured. (D) The numbers of CD4⁺ and CD8⁺ T cells were detected. (E) The expression of CD69 and CD44 in CD4⁺ and CD8⁺ T cells was measured by flowcytometry. n = 9–10 mice per group. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Increased serum AMAs in mice injected with 2-OA-BSA/OCH.

<p>Wild type mice were immunized with 2-OA-BSA and α-GalCer (group name: 2-OA/a-GC), OCH (group name: 2-OA/OCH) or PBS (group name: 2-OA/PBS) at weeks 0, 2, 4, 6 and 8. At week 12, serum levels of autoantibodies to mPDC-E2 were measured by ELISA. n = 9–10 mice per group. *, p < 0.05 in 2-OA/a-GC to 2-OA/PBS; #, p < 0.05 in 2-OA/OCH to 2-OA/PBS.</p

The increase of portal inflammation and fibrosis in mice injected with 2-OA-BSA/OCH.

<p>Mice were immunized with 2-OA-BSA and α-GalCer (group name: 2-OA/a-GC), OCH (group name: 2-OA/OCH) or PBS (group name: 2-OA/PBS) at weeks 0, 2, 4, 6 and 8 and sacrificed at week 12. (A) Representative stained liver sections of haematoxylin and eosin (H&E) and Masson’s trichrome stain. (B) Histopathological scores of individual livers on portal inflammation and fibrosis. 0 = no significant change, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe pathology. Individual symbols each represent a single mouse.</p

Pathological changes in the liver of IFN-γ^−/− mice immunized with 2OA-BSA.

<p>(A) Representative H&amp;E stained of liver sections from IFN-γ^−/− mice compared with WT mice 8 weeks after 2OA-BSA immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed in WT mice; normal bile ducts in IFN-γ^−/− mice (blue arrows). (B) Scoring of portal inflammation and bile duct damage in sections of liver from WT and IFN-γ^−/− mice (each group, n = 16).</p

IL-12p40 is required for 2OA-BSA-induced cholangitis.

<p>(A) H&amp;E staining of representative liver from WT, IL-12p40^−/− (p40^−/−), IL-23^−/− (p19^−/−) and IL-12p35^−/− (p35^−/−) mice 8 weeks after 2OA-BSA immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed in the liver of WT, p19^−/− and p35^−/− mice but not p40^−/− mice; normal bile ducts in p40^−/− mice (blue arrow). (B) Portal inflammation and bile duct damage were examined in individual animals. The pathological score of portal inflammation and biliary cell damage were evaluated in WT (n = 17), p19^−/− (n = 14), p35^−/− (n = 3) and p40^−/− (n = 5) mice. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001. (C) Immunohistochemical analysis of the liver of WT mice and IL-23p19^−/− mice at 8 weeks after 2OA-BSA immunization using mAbs to CD4 and CD8.</p

Pathological changes in liver of mice immunized with 2OA-BSA.

<p>(A) Representative H&amp;E staining profiles of liver from IL-17A^−/−, IL-17F^−/− and IL-22^−/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F^−/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A^−/− and IL-22^−/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A^−/− (n = 19), IL-17F^−/− (n = 13) and IL-22^−/− (n = 14) mice.</p

Inflammatory cytokine production in mice immunized with 2OA-BSA.

<p>(A) Serum levels of IFN-γ and IL-17A in B6 and IL-23p19^−/− mice 2 and 4 weeks after initial 2OA-BSA immunization. (B) Production of IFN-γ and IL-17A in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs for 3 days (C) inflammatory cytokines in extracted liver protein from WT mice (n = 8) and IL-23p19^−/− (n = 8) mice. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001.</p