Deletion of interleukin-12p40 suppresses autoimmune cholangitis in dominant negative transforming growth factor beta receptor type II mice

Abstract

Our laboratory has reported that mice that express a dominant negative form of transforming growth factor beta receptor restricted to T cells (dnTGFbetaRII) 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)-dnTGFbetaRII mice and IFN-gamma KO-dnTGFbetaRII mice. The two colonies of genetically modified mice-and, for purposes of controls, the dnTGFbetaRII mice-were monitored for liver immunopathology, AMAs, and intrahepatic cytokine production. Disease expression in the IFN-gamma KO-dnTGFbetaRII mice, including liver immunopathology, were similar to those of dnTGFbetaRII mice, whereas the IL-12p40 KO-dnTGFbetaRII 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 dnTGFbetaRII 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.

Figure 1

Histological evidence of cholangitis in the liver of IFN-γKO-dnTGFβRII mice. A. HE-stained liver sections of IFN-γKO-dnTGFβRII mice demonstrate lymphoid cell infiltration in portal tracts around bile ducts (arrow, left panel) and a damaged bile duct within the cell-infiltrated portal area (double arrow, right panel). B. Scoring of liver portal inflammation and bile duct damage in IFN-γKO-dnTGFβRII mice (left) compared to wild-type dnTGFβRII mice (right) was coded as follows: 0, no inflammation (or bile duct damage); 1, mild inflammation (or bile duct damage); 2, moderate inflammation (or bile duct damage); 3, severe inflammation (or bile duct damage). The scores were deemed to be equivalent.

Figure 2

Protection from cholangitis in liver sections from IL-12p40KO-dnTGFβRII mice. A. HE-stained tissue sections of liver from dnTGFβRII mouse (left panel) demonstrate lymphoid infiltration in portal tract. In contrast, liver sections from IL-12p40KO-dnTGFβRII mice (right panel) demonstrate absence of lymphoid infiltration. B. Scoring of portal inflammation and bile duct damage in the liver of dnTGFβRII mice, IL-12p40KO-dnTGFβRII mice and normal B6 mice were coded as noted in Fig. 1B. * P <0.05, ** P <0.01, *** P <0.001 using Kruskal-Wallis test followed by Dunn's multiple comparisons test.

Figure 3

Flow cytometric analysis of the number of total mononuclear cells (MNC), CD19⁺ B cells, CD4⁺ T cells and CD8⁺ T cells among liver and spleen cells from dnTGFβRII mice (n=7), IL-12p40KO-dnTGFβRII mice (n=7) and B6 mice (n=6). Data are shown as mean +/- S.D. *P<0.05, ** P <0.01, *** P <0.001 determined using the one-way ANOVA test followed by Bonferroni multiple comparisons test.

Figure 4

Levels of immunoglobulins G and A, and anti-PDC-E2 antibody, in serum of dnTGFβRII mice, IL-12p40KO-dnTGFβRII mice, and normal B6 mice. Horizontal bars represent median values. An OD values of anti-PDC-E2 antibody > 3 standard deviations above the means of that for normal B6 mice was considered positive. ** P <0.01, ***P<0.001 determined by one-way ANOVA test followed by Bonferroni multiple comparisons test.

Figure 5

Hepatic profiles of inflammatory cytokines and chemokines in whole protein extracts prepared from the liver of dnTGFβRII (n=7), IL-12p40KO-dnTGFβRII (n=7), and B6 (n=6) mice. Assays for levels of (A) IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10, and IL-6, and (B) IP-10 (CXCL10) and MIG (CXCL9). Results are shown as mean levels +/- S.D. *P<0.05, ** P <0.01, *** P <0.001 determined using the one-way ANOVA test followed by Bonferroni multiple comparisons test.