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

Abstract

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology 1,2 . In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease [3][4][5] . However, in various models the induction of disease was rather complex and often hepatitis was only transient [3][4][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 trigger 6 . Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-2 7,8 . 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 field 9 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.

Animal models of chronic liver diseases

“…In line, injecting wild-type FVB/N mice with an adenovirus expressing human cytochrome P-450 2D6 (Ad-CYP2D6) leads to a chronic form of severe autoimmune liver damage with subcapsular fibrosis (63,64). Interestingly, transgenic FVB/N mice expressing the identical human CYP2D6 in their liver failed to develop persistent hepatitis upon injection with Ad-CYP2D6, indicating the presence of a stronger immunological tolerance.…”

Pathogen Infection as a Possible Cause for Autoimmune Hepatitis

“…This approach indeed succeeded in generating a robust immune response against CYP2D6 that resulted in the breakdown of tolerance to the mouse Cyp homologues. Subsequently, the mice developed persistent AIH characterized by cellular infiltrations in the peri-portal area, fibrosis, and the generation of LKM-1-like autoantibodies [59,102,105]. Further, CYP2D6-specific CD4 and CD8 T cells were generated that accumulated in the liver [106].…”

“…In contrast, CYP2D6, which constitutes one of the best-characterized autoantigens in AIH had not been used in a virus-infection setting. Thus, we generated the CYP2D6 mouse model in which wild-type FVB or C57BL/6 mice are infected with an Adenovirus expressing the human CYP2D6 (Ad-2D6) [59,102]. The model is based on the concept of molecular mimicry [103,104], since Ad-2D6-infected wildtype mice are only expressing the mouse Cyp homologues of human CYP2D6 (i.e.…”

Immunopathogenic Mechanisms of Autoimmune Hepatitis: How Much Do We Know from Animal Models?

“…In contrast, the CYP2D6 mouse model utilizes the immunodominant antigen (human CYP2D6) in human AIH type 2 [28,29], as a triggering antigen. The targeted delivery of CYP2D6 to the liver with an adenovirus (Ad-2D6) ensures at first an acute hepatic inflammation that subsequently develops into a chronic AIH-like disease [117,118]. Infection of mice with Ad-2D6 results in interface hepatitis with cellular infiltrates that predominantly exhibit B cells and CD4 T cells.…”