The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver

Blockade but not overexpression of the junctional adhesion molecule C influences virus-induced type 1 diabetes in mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans

Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection

Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6–infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, “fused” liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6–infected mice generated type 1 liver kidney microsomal–like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6–infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases

Autoantibodies in Autoimmune Hepatitis: Can Epitopes Tell Us about the Etiology of the Disease?

Autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) are serious autoimmune liver diseases that are characterized by a progressive destruction of the liver parenchyma and/or the hepatic bile ducts and the development of chronic fibrosis. Left untreated autoimmune liver diseases are often life-threatening, and patients require a liver transplantation to survive. Thus, an early and reliable diagnosis is paramount for the initiation of a proper therapy with immunosuppressive and/or anticholelithic drugs. Besides the analysis of liver biopsies and serum markers indicating liver damage, the screening for specific autoantibodies is an indispensable tool for the diagnosis of autoimmune liver diseases. Such liver autoantigen-specific antibodies might be involved in the disease pathogenesis, and their epitope specificity may give some insight into the etiology of the disease. Here, we will mainly focus on the generation and specificity of autoantibodies in AIH patients. In addition, we will review data from animal models that aim toward a better understanding of the origins and pathogenicity of such autoantibodies

The many roles of cell adhesion molecules in hepatic fibrosis

Fibrogenesis is a progressive scarring event resulting from disrupted regular wound healing due to repeated tissue injury and can end in organ failure, like in liver cirrhosis. The protagonists in this process, either liver-resident cells or patrolling leukocytes attracted to the site of tissue damage, interact with each other by soluble factors but also by direct cell-cell contact mediated by cell adhesion molecules. Since cell adhesion molecules also support binding to the extracellular matrix, they represent excellent biosensors, which allow cells to modulate their behavior based on changes in the surrounding microenvironment. In this review, we focus on selectins, cadherins, integrins and members of the immunoglobulin superfamily of adhesion molecules as well as some non-classical cell adhesion molecules in the context of hepatic fibrosis. We describe their liver-specific contributions to leukocyte recruitment, cell differentiation and survival, matrix remodeling or angiogenesis and touch on their suitability as targets in antifibrotic therapies

Cytochrome P450 2D6 as a Model Antigen

Cytochrome P450 2D6 (CYP2D6) has been identified as the major autoantigen in type 2 autoimmune hepatitis (AIH). However, because of a lack of appropriate animal models, the etiology of AIH is still poorly understood. We generated a mouse model for AIH using the human CYP2D6 as a triggering molecule for autoimmunity. We infected wild-type FVB mice with an adenovirus expressing human CYP2D6 (Ad-2D6) to break self-tolerance to the mouse CYP2D6 homologues. Ad-2D6-infected mice showed persistent features of liver damage including hepatic fibrosis, cellular infiltrations, focal-to-confluent necrosis and generation of anti-CYP2D6 antibodies, which predominantly recognized the identical immunodominant epitope recognized by LKM-1 antibodies from AIH patients. Interestingly, Ad-2D6 infection of transgenic mice expressing the human CYP2D6 (CYP2D6 mice) resulted in delayed kinetics and reduced severity of liver damage. However, the quantity and quality of anti-CYP2D6 antibodies was only moderately reduced in CYP2D6 mice. In contrast, the frequency of CYP2D6-specific CD4 and CD8 T cells was dramatically decreased in CYP2D6 mice, indicating the presence of a strong T cell tolerance to human CYP2D6 established in CYP2D6 mice, but not in wild-type mice. CYP2D6-specific T cells reacted to human CYP2D6 peptides with intermediate homology to the mouse homologues, but not to those with high homology, indicating that molecular mimicry rather than molecular identity breaks tolerance and subsequently causes severe persistent autoimmune liver damage. The CYP2D6 model provides a platform to investigate mechanisms involved in the immunopathogenesis of autoimmune-mediated chronic hepatic injury and evaluate possible ways of therapeutic interference

Cross-talk of Integrin α3β1 and Tissue Factor in Cell Migration

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is β1 integrin–dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing αvβ3 or certain β1 integrin heterodimers (α3β1, α4β1, α5β1, α6β1, α9β1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates α3β1-dependent migration on laminin 5. Expression of TF suppresses α3β1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2–dependent phosphorylation of TF. In both cases, release of α3β1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility