Li, Wen

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper‐gammaglobulinemia. There are two types of AIH, type 1 (AIH‐1) and type 2 (AIH‐2), characterized by distinct autoimmune serology. Patients with AIH‐1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH‐2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles ‐DR3, ‐DR4, and ‐DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA‐DR3 transgenic mouse on the nonobese‐diabetic background by immunization of HLA‐DR3– and HLA‐DR3+ nonobese‐diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA‐DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA‐DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

Abstract The genetic factors that contribute to the etiology of type 1 diabetes are still largely uncharacterized. However, the genes of the MHC (HLA in humans) have been consistently associated with susceptibility to disease. We have used several transgenic mice generated in our laboratory, bearing susceptible or resistant HLA alleles, in the absence of endogenous MHC class II (Aβo), to study immune responses to the autoantigen glutamic acid decarboxylase (GAD) 65 and its relevance in determining the association between autoreactivity and disease pathogenesis. Mice bearing diabetes-susceptible haplotypes, HLA DR3 (DRB1*0301) or DQ8 (DQB1*0302), singly or in combination showed spontaneous T cell reactivity to rat GAD 65, which is highly homologous to the self Ag, mouse GAD 65. The presence of diabetes-resistant or neutral alleles, such as HLA DQ6 (DQB1*0602) and DR2 (DRB1*1502) prevented the generation of any self-reactive responses to rat GAD. In addition, unmanipulated Aβo/DR3, Aβo/DQ8, and Aβo/DR3/DQ8 mice recognized specific peptides, mainly from the N-terminal region of the GAD 65 molecule. Most of these regions are conserved between human, mouse, and rat GAD 65. Further analysis revealed that the reactivity was mediated primarily by CD4+ T cells. Stimulation of these T cells by rat GAD 65 resulted in the generation of a mixed Th1/Th2 cytokine profile in the Aβo/DR3/DQ8, Aβo/DR3, and Aβo/DQ8 mice. Thus, the presence of diabetes-associated genes determines whether immune tolerance is maintained to islet autoantigens, but autoreactivity in itself is not sufficient to induce diabetes.

Although DQA1*0301/DQB1*0302 is the human histocompatibility leukocyte antigen (HLA) class II gene most commonly associated with human type 1 diabetes, direct in vivo experimental evidence for its diabetogenic role is lacking. Therefore, we generated C57BL/6 transgenic mice that bear this molecule and do not express mouse major histocompatibility complex (MHC) class II molecules (DQ8+/mII−). They did not develop insulitis or spontaneous diabetes. However, when DQ8+/mII− mice were bred with C57BL/6 mice expressing costimulatory molecule B7-1 on β cells (which normally do not develop diabetes), 81% of the DQ8+/mII−/B7-1+ mice developed spontaneous diabetes. The diabetes was accompanied by severe insulitis composed of both T cells (CD4+ and CD8+) and B cells. T cells from the diabetic mice secreted large amounts of interferon γ, but not interleukin 4, in response to DQ8+ islets and the putative islet autoantigens, insulin and glutamic acid decarboxylase (GAD). Diabetes could also be adoptively transferred to irradiated nondiabetic DQ8+/mII−/B7-1+ mice. In striking contrast, none of the transgenic mice in which the diabetes protective allele (DQA1*0103/DQB1*0601, DQ6 for short) was substituted for mouse MHC class II molecules but remained for the expression of B7-1 on pancreatic β cells (DQ6+/mII−/B7-1+) developed diabetes. Only 7% of DQ−/mII−/B7-1+ mice developed diabetes at an older age, and none of the DQ−/mII+/B7-1+ mice or DQ8+/mII+/B7-1+ mice developed diabetes. In conclusion, substitution of HLA-DQA1*0301/DQB1*0302, but not HLA-DQA1*0103/DQB1*0601, for murine MHC class II provokes autoimmune diabetes in non–diabetes-prone rat insulin promoter (RIP).B7-1 C57BL/6 mice. Our data provide direct in vivo evidence for the diabetogenic effect of this human MHC class II molecule and a unique “humanized” animal model of spontaneous diabetes.