The Mechanism Of Drug-induced Liver Injury And Characterization Of B Cells In Humanized Mice

Acetaminophen is usually used as over-the-counter analgesic and antipyretic drug. However, acetaminophen overdose cause acute liver injury. And intentional or accidental overdose has become frequent cause of death. In the United States, APAP overdose has become a major burden to the public health system. In China, there are much more patients with liver diseases because of HBV infection. The acetaminophen toxicity may be more severe for those people.Acetaminophen is metabolized by hepatic cytochrome p450CYP2E1into toxic intermediate, N-acetyl-p-benzoquinone-imine (NAPQI), detoxified by hepatic glutathione. However, excessive NAPQI consumes hepatic glutathione and covalently binds to cellular proteins, nextly caused oxidative stress reaction, mitochondrium dysfunction and DNA damage, finally resulting in hepatocytes nercosis. Drug-induced hepatocytes necrosis only contributes to a part of the whole liver injury.Since innate immune response following hepatocytes necrosis caused a second wave of liver destruction, which is more severe than initial destruction. Many innate immune cell populations are involved in the acetaminophen induced-liver injury, such as natural killer cells, macrophages, dendrtic cells and neutrophils. Many studies focus on the role of neutrophils in that a large number of neutrophils infiltrate into the liver after acetaminophen challenge.In this study, mice were treated with overdose acetaminophen and got acute liver injury. Acute liver injury was evaluated by serum levels of alanine aminotransferase (ALT) and histochemical staining. The populations and percentages were determined by the flow cytometric analysis and cell counting. The concentrations of cytokines were measured by enzyme-linked immunosorbent assay (ELISA).The function of innate immune cells was analyzed by depletion or in vitro stimulation. The roles of cytokines were determined by neutralizing antibodies in vivo.Through the methods as above mentioned, we get the major results as following:1. Overdose acetaminophen induces liver injury and recruitment of neutrophils into liver We found that serum ALT started to elevate early and picked at24hours after acetaminophen challenge. Histochemical staining demonstrated the presence of many necrotic areas around the central port veins in the liver. The amount of total leukocytes in the liver increased, and neutrophils but not lymphocytes were the major cells in the increased leukocytes.2. Recruitment of neutrophils into liver depends on production of IL-17AWe found that the concentration of IL-17A in serum gradually increased and picked at24hours after acetaminophen challenge.After inhibiting the function of IL-17A with a neutralizing antibody, the number of neutrophils in murine liver decreased. Meanwhile, the sera ALT level in anti-IL-17A-treated mice was less than that of control group. Accordingly, the survival rate of mice pretreated with the neutralizing antibody was better than that of control mice.3. γδ T cells are main producer of IL-17A in APAP-induced liver injuryWe found that only IL-17A+CD3+CD4-NK1.1-γδ TCR+cells were significantly increased after acetaminophen challenge. After depletion of γδ T cells but neither CD4+T cells nor NKT with mAb, the concentration of IL-17A in serum was significantly reduced. Meanwhile, we found that liver injury was attenuated and the survival rate was markedly improved after depletion of γδ T cells.Total number of neutrophils in the liver significantly reduced in absence of γδ T cells.The reduction of neutrophils in the livers also was observed in TCRδ-/-mice compared to that in the same-aged C57BL/6control mice after acetaminophen challenge.4. IL-23is critical in generation of IL-17A-producing γδ T cellsWhat could induce the IL-17A production from γδ T cells? We found that the IL-23level in the serum and the liver tissue significantly increased after acetaminophen challenge. Serum IL-17A significantly reduced after neutralizing IL-23or using p40-deficient mice。 Moreover, infiltration of neutrophils into the liver was significantly ameliorated in absence of IL-23. According to experiments in vitro we found that stimulation of the hepatic γδT cells with alone IL-23could induce γδ T cells to produce IL-17A.5. HMGB-1-TLR4pathway mediates production of IL-23by macrophages Where did the IL-23come from? We found that the concentrations of serum IL-23and IL-17A were reduced after inhibition of macrophages. Whereas serum high-mobility group boxl (HMGB1) increased after treatment with acetaminophen. HMGB1inhibitor glycyrrhizin markedly reduced the production of IL-23, IL-17A and hepatic neutrophils recruitment. Soluble HMGB1stimulated the production of IL-23by TLR4+/+macrophages but not by TLR4-/-macrophages. The concentrations of serum IL-23and IL-17A significantly reduced in TLR4-/-mice compared to C57BL/6mice.Conclusion:In the present study, we demonstrated that crosstalk between macrophages and y8T cells play an important role in acetaminophen-caused tissue damage-induced acute liver inflammation. Serum HMGB1, a damage-associated molecule released from necrotic hepatocytes after acetaminophen challenge, stimulate the production of IL-23by hepatic macrophages in TLR-4-dependent manner, and IL-23helps the generation of IL-17A-producing γδ T cells in the liver. IL-17A secreted by γδ T cells then recruits hepatic neutrophils. Thus, HMGBl-TLR4-IL-23-IL17A axis between macrophages and γδ T cells contributes to tissue damage-induced liver inflammation and accumulation of neutrophils.