Profiles of Immune Cells and Reproduction of Immune-mediated Drug-induced Liver Injury

Abstract

Introduction: Immune-mediated drug-induced liver injury (DILI) is induced by various drugs that can evoke immune responses. However, the mechanisms of immune-mediated DILI have not been clarified yet. The aim of the study is to determine the immune profiles in the peripheral blood of DILI subjects and the patterns of liver injury, and to investigate whether CYP3A4-mediated active metabolites of drugs can participate in inducing the immune responses. Methods: 1. We analyzed the proportions of immune cells in peripheral blood mononuclear cells (PBMCs) of DILI using BD FACS canto II, comparing 18 healthy controls with 6 acute hepatitis subjects (type A 2cases, type B 4cases). PBMCs were stained with the following 4 sets of antibodies. 1. DC:CD1c, CD14, CD19, CD86, CD141, CD303, CD304, PD-L1; 2.Tcell: CD3, CD4, CD8a, CD25, CD127, PD-1; 3.NK/NKT:CD3, CD16, CD56, NKG2D, 4. Myeloid-derived suppressor cells (MDSC); CD11b, CD14, CD16, CD33, PD-L1, HLA-DR. 2. PBMCs from immune-mediated DILI patients or healthy subjects were cultured with CYP3A4 expressing HepG2 cells and amiodarone (AMD) using a transwell system. We collected PBMCs in the upper chamber and analyzed the proportions of the various kinds of immune cells at 48 hours after co-incubation by BD FACS canto II. To assess the effects of the metabolites of AMD, we measured the concentrations of AMD and N-desethyamiodarone, a major metabolite of AMD, using LS/MS. Moreover, PBMCs from DILI subjects treated with asunaprevir were analyzed the same way since asunaprevirmediated DILI could be induced by immune-dysfunction. Results: 1. There was significantly elevated proportion of MDSCs in the PBMCs of DILI and acute hepatitis(type A, type B), compared with those of healthy controls(P<0.0001). The frequency of regulatory T cells (Tregs) in the PBMCs of DILI was higher than in Tregs of acute hepatitis (type A, type B) and healthy controls (P=0.0862). Comparisons of immune profiles between the dominant injury of hepatocytes and the dominant injury of bile ducts did not reveal any significant differences. 2. The in vitro co-culture system could partially reproduce the immune profiles in some DILI patients. Conclusion: These results demonstrated that immune-mediated DILI was associated with specific immune subsets. The profiles of immune cells in peripheral blood appear to be prognostic. We might reproduce immune-mediated drug-induced liver injury with the in vitro co-culture system.Figure 1Figure 2