Hintergrund: Die autoimmune Hepatitis (AIH) ist eine seltene autoimmune Lebererkrankung mit einer erheblichen phänotypischen Heterogenität. Trotz großen Fortschritten in der Diagnose und Behandlung der AIH sind die zugrunde liegenden molekularen Mechanismen weitestgehend unbekannt. In der vorliegenden Studie sollte mittels gesamtgenomischen Analysen ein Einblick in die molekularen Pathomechanismen der AIH gewonnen werden, um klinisch relevante Subgruppen für neue therapeutische Ansätze zu identifizieren.

The pathogenesis of primary sclerosing cholangitis (PSC) remains poorly understood. Since PSC predominantly occurs in patients with inflammatory bowel disease, autoimmunity triggered by activated T cells migrating from the gut to the liver is a possible mechanism. We hypothesized that T cells primed in the gut-associated lymphoid tissue (GALT) by a specific antigen migrate to the liver and cause cholangitis when they recognize the same antigen on cholangiocytes. We induced ovalbumin-dependent colitis in mice that express ovalbumin in biliary epithelia (ASBT-OVA mice) and crossed ASBT-OVA mice with mice that express ovalbumin in enterocytes (iFABP-OVA mice). We analyzed T-cell activation in the GALT and crossreactivity to the same antigen in the liver as well as the effects of colitis per se on antigen-presentation and T-cell activation in the liver. Intrarectal application of ovalbumin followed by transfer of CD8 OT-I T cells led to antigen-dependent colitis. CD8 T cells primed in the GALT acquired effector function and the capability to migrate to the liver, where they caused cholangitis in a strictly antigen-dependent manner. Likewise, cholangitis developed in mice expressing ovalbumin simultaneously in biliary epithelia and enterocytes after transfer of OT-I T cells. Dextran sodium sulfate colitis led to increased levels of inflammatory cytokines in the portal venous blood, induced activation of resident liver dendritic cells, and promoted the induction of T-cell-dependent cholangitis. Conclusion: Our data strengthen the notion that immune-mediated cholangitis is caused by T cells primed in the GALT and provide the first link between colitis and cholangitis in an antigen-dependent mouse model. (Hepatology 2014;59:601–611)

Contents List of Figures v List of Tables vii List of Abbreviations viii Zusammenfassung xi Summary xiii 1 Introduction 1 1.1 The liver, its anatomy and function .

BACKGROUND & AIMS:The enterohepatic circuit of T cells may be responsible for the development of autoimmune liver disease. We employed transgenic mice to characterize phenotype and migration patterns of CD8 T cells activated in liver and gut. METHODS:We studied the migration of antigen-specific CD8 T cells primed in liver or gut after transfer in wild-type mice or mice that express ovalbumin in liver or gut. We performed transcriptome analysis of these two distinct T cell populations and confirmed our findings by flow cytometry. RESULTS:Specific migration patterns were induced by activation of CD8 T cells in gut or liver. Gut-activated CD8 T cells expressed α4β7 and CCR9 and migrated to the gut and to the liver. Liver-activated T cells expressed integrins α4, α6, β1, α4β7 as well as CD62L, Ly6C, and neuropilin-1 and retained the capability to re-circulate through lymph nodes. Presence of the antigen increased retention of both types of activated T cells in the liver, but migration of liver-activated T cells to the gut was prohibited. CONCLUSIONS:CD8 T cells primed in the liver in vivo are not capable of migrating to the gut, implying that the enterohepatic circuit of CD8 T cells is in fact a one-way road from the gut to the liver. Priming of CD8 T cells in the liver results in a distinct phenotype with attributes of central memory cells and induces a unique homing pattern. Gut-primed T cells preferentially home to the liver, in principle enabling them to induce autoimmune liver disease.

POSTERSand biochemical features (presentation, prognosis, response to treatment) also evolved over the last 20 years.Figure 1.New cases of autoimmune hepatitis during 2001-2012. Conclusion:Incidence of autoimmune hepatitis is increasing, as other autoimmune diseases in developed countries.The main recognised hypothesis is the 'hygienist theory' (improvement of hygien leading to a decrease in infections) with few putative non exclusive mechanisms involving antigenic competition, extension of immune regulation induced by exogenous bacterial antigen or Toll Like Receptors.

Autoimmune hepatitis (AIH) is a major chronic liver disease characterized by diverse and pleiotropic spectrum of different phenotypes. Despite recent progress in clinical management of AIH patients, the exact molecular pathogenesis remains largely unknown. Here, we used a comparative genomic approach to define molecular characteristics and test the predictive value of gene expression profiles in this important liver disease. Gene expression profiles of 124 patients with different chronic liver diseases (AIH, HBV, HCV) were generated from FFPE samples using Illumina DASL assay and computationally integrated with expression data from a T-cell induced mouse model of autoimmune liver disease.

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.

Background and Aims:The contribution of different types of antigen-presenting cells in the pathogenesis of autoimmune hepatitis is not known.TF-OVA mice express ovalbumine in hepatocytes and develop immune-mediated liver injury after transfer of naïve T-cell receptor transgenic CD8 T-cells (OT-I cells).Full effector function of OT-I cells is only achieved in the presence of bone-marrow derived APCs.We tested whether dendritic cells (DCs) or Kupffer cells (KCs) are required for the activation of OT-I cells in the liver.Methods: The contribution of KCs was investigated in TF-OVA mice treated with clodronate liposomes (every other day starting at day -1) to deplete KCs.The contribution of DCs was investigated in TF-OVA mice crossed to CD11cDTR mice (Jung S et al., Immunity 2002:17, 211-20), in which a DTR-GFP fusion protein marks the cross-presenting CD11c high subset of DCs with GFP and renders them susceptible to depletion with diphteria toxin (DT).Mice were treated with DT at days 0 and 2. The degree of T-cell activation was judged from the severity of hepatitis as indicated by ALTlevels at the peak of inflammation, by histology, and by release of interferon-gamma after restimulation of liver-derived CD8 T-cells.Results: Depletion of KCs increased the severity of hepatitis compared with untreated mice as indicated by ALT levels (1017±241 vs. 510±39U/l, p = 0.02), suggesting that KCs are dispensable for the activation of CD8 T-cells in the liver.Rather, they may play a role in the control of the immune response.In contrast, depletion of DCs with DT in TF-OVAxCD11cDTR mice reduced ALT-levels when compared to untreated controls (210±25 vs. 314±43, p = 0.05), while DT in the absence of OT-I cells caused only mildly elevated ALT-levels (113±25 U/l).Restimulation of CD8 T-cells derived from livers of DT-treated mice led to significantly less production of interferon-gamma compared with OT-I cells retrieved from untreated mice.Conclusion: Our date indicate that CD11c high DCs in the liver are required for the cross presentation of an endogenous antigen and for the activation of self-reactive CD8 T-cells while KCs are dispensable and may rather play a role in suppressing T-cell responses.

T-Zellen spielen bei der Pathogenese der Autoimmunhepatitis eine entscheidende Rolle. Während CD8 T-Zellen als Effektorzellen zu Inflammation führen, können CD4 T-Zellen als Effektoren die Inflammation sowohl verstärken als auch als Regulatoren unterdrücken. Die Bedeutung regulatorischer T-Zellen bei der Autoimmunhepatitis ist bislang weitgehend unbekannt.

Background and Aims: Recently we have reported a high prevalence of IgG and/or IgM anticardiolipin antibodies (aCL) in patients with autoimmune liver diseases, namely autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), which were independent of the respective isotypes of antibodies against b2glycoprotein I (anti-b2GPI).IgA aCL and IgA anti-b2GPI are the least studied of the three specific isotypes either in antiphospholipid syndrome (APS) or in other conditions.Therefore, we investigated the prevalence and clinical significance of IgA anti-b2GPI and IgA aCl in a consecutive group of patients with autoimmune liver diseases.Methods: 192 patients namely, 59 with AIH, 96 with PBC and 39 with PSC were investigated using commercial ELISAs (INOVA Diagnostics, San Diego, CA).The disease controls consisted of 50 with HCV, 50 with HBV, 30 with alcoholic liver disease (ALD) and 30 with non-alcoholic steatohepatitis (NASH) while 45 blood donors served as healthy controls. Results:The prevalence of IgA anti-b2GPI was higher in AIH (45.8%) compared to PBC (p = 0.027), PSC (p = 0.002), NASH (p = 0.001), ALD (p = 0.032) and HCV (p = 0.004).The titers also were significantly higher in AIH compared to any other group of the study (PBC, p < 0.001; PSC, p = 0.003; NASH, p < 0.001; ALD, p = 0.001; HCV, p < 0.001 and HBV, p < 0.05).The prevalence of IgA aCl was significantly higher in AIH (20.3%) compared to PSC (p = 0.014), NASH (p = 0.007), ALD (p = 0.007) and HCV (p = 0.003).There was no evidence for any association of IgA anti-b2GPI or IgA aCl and clinical manifestations of APS (thrombosis, pregnancy morbidity, thrombocytopenia) in patients with liver autoimmunity. of note, IgA anti-b2GPI and IgA aCL were associated with clinical and biochemical markers of disease severity in AIH and PBC patients.Conclusions: We demonstrated for the first time a high prevalence of IgA anti-b2GPI in patients with AIH compared to any other liver disease of the study.IgA anti-b2GPI and IgA aCL were associated with the severity and biochemical activity of AIH and PBC but prospective studies with long-term follow-up of IgA anti-b2GPI and/or IgA aCL positive AIH and PBC patients are needed in order to address whether this new finding is of clinical importance.