Interferon-γ-dependent immune responses contribute to the pathogenesis of primary sclerosing cholangitis in mice

Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of interferon-γ (IFNγ) positive lymphocytes and IFNγ-induced chemokines have been detected in liver tissue and plasma of PSC patients. Aim of this study is to analyze the role of IFNγ as well as IFNγ-producing cell populations in the immune pathogenesis of PSC. We used multidrug resistance protein 2 knockout (Mdr2-/-) mice, an established mouse model resembling PSC.

The contribution of T cells to liver pathology was assessed in Mdr2-/-Rag1-/- mice and following T cell depletion using an anti-CD90.2 antibody. The role of IFNγ for liver pathology was determined in Mdr2-/-IFNγ-/- mice and following anti-IFNγ antibody treatment. The severity of liver injury was assessed by plasma levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Histological analysis of H&E stained liver tissue sections allowed the examination of inflammation and fibrosis. The cytotoxic potential of hepatic non-parenchymal cells (NPCs) was analyzed ex vivo via flow cytometry after restimulation with PMA and Ionomycin. Real time RT-PCR was applied to examine the gene expression of targets involved in the IFNγ signaling pathway. In addition a bead-based multiplex assay was used to investigate cytokine production.

Our studies showed that hepatic CD8+ T cells and NK cells were the primary source of IFNγ in Mdr2-/- mice. Depletion of CD90.2+ cells, which are mainly T cells, significantly reduced the production of inflammatory cytokines including IFNγ, NK cell cytotoxicity and overall liver injury. Similar results were obtained in Mdr2-/-Rag1-/- mice, which lack T and B lymphocytes. Neutralisation of IFNγ in Mdr2-/- mice attenuated fibrotic remodeling indicated by reduced hydroxyproline level and reduced Sirius Red positive areas. Additionally the neutralization resulted in reduced frequencies of IFNγ producing NK cells and CD8+ T cells with reduced cytotoxic potential. Complete absence of IFNγ in the Mdr2-/-IFNγ-/- mice resulted in reduced hepatic mRNA expression of Cxcl9 and Cxcl10, both of which are highly induced in Mdr2-/- mice. The frequencies of NK cells and CD8 T cells expressing granzyme B and TRAIL were significantly reduced in the Mdr2-/-IFNγ-/- mice in comparison to Mdr2-/-. Mdr2-/-IFNγ-/- mice also showed reduced fibrosis displayed by significantly reduced col3a1 gene expression, hydroxyproline level and Sirius Red positive areas.

Overall the data suggests, that CD8+ T cells and NK cells are one of the major producers of IFNγ in Mdr2-/-mice. Moreover IFNγ appears to have profibrotic properties in the Mdr2-/-mice, since absence of IFNγ reduces the fibrotic response. Further experiments will be performed to elucidate the role of IFNγ in PSC progression with special focus on downstream targets of the IFNγ signaling pathways and induction of cytotoxicity in liver lymphocytes.