Role of Innate and Adaptive Immune Systems in Drug-Induced Liver Disease

先天性和适应性免疫系统在药物性肝病中的作用

• 批准号: 8939856
• 负责人: Lance R Pohl
• 金额: $ 100.38万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939856
• 关键词: Acids; Animal Model; Animals; Antibodies; Anticonvulsants; Area; CD4 Positive T Lymphocytes; CD8B1 gene; Categories; Cells; Characteristics; Clinical; Cytokine Signaling; Data; Diffuse; Disease; Disulfiram; Drug toxicity; Eosinophil Granule Proteins; Eosinophilia; Epithelial; Erythromycin; Ethanol; Etiology; Female; Generations; Growth Factor; Halothane; Hepatic; Hepatocyte; Hepatotoxicity; Hour; Human; ITGAM gene; IgE; IgG1; Immune; Immune response; Immune system; Immunity; Incidence; Infiltration; Injury; Interleukin-13; Interleukin-4; Interleukin-5; Investigation; Knockout Mice; Liver; Mediating; Metabolism; Modeling; Mus; Myelogenous; Nature; Necrosis; Pathologic; Patients; Pharmaceutical Preparations; Play; Process; Proteins; Reaction; Regulation; Reporting; Research Personnel; Role; Serum; Severities; Signal Pathway; Staining method; Stains; Sulfonamides; Suppressor-Effector T-Lymphocytes; T-Lymphocyte; Testing; Time; Toxic effect; Wild Type Mouse; adduct; chemokine; cytokine; cytotoxic; drug induced liver disease; eosinophil; hepatic necrosis; immunopathology; liver injury; neutrophil; paracrine; prevent; receptor; response; success; troglitazone

Although clinical evidence suggests that many cases of DILD are mediated by hepatic protein adducts of drugs and the adaptive and innate immune systems, detailed experimental proof for this mechanism of toxicity has remained elusive due to the lack of animal models. We have hypothesized that DILD is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver, which consists of multiple negative regulators of the adaptive immune system. This idea has now been tested in an established murine model of halothane-induced liver injury where the toxicity is initiated by the metabolism of halothane to form trifluoroacetylated liver proteins and enhanced by the innate immune system. Twenty-four hours after female Balb/cJ mice were treated with halothane, analysis of the liver revealed perivenous necrosis and an infiltration of CD11b+ Gr-1High neutrophils, as reported by other researchers. Further study revealed that the neutrophils contained a subpopulation of myeloid-derived suppressor cells (MDSC) that inhibited the proliferation of both CD4+ and CD8+ T cells isolated from naive mice. When MDSC were depleted from the liver with Gr-1 antibodies prior to two treatments with halothane, enhanced liver injury was observed nine days after the second exposure of halothane as compared to mice that were pretreated with isotype control antibodies before halothane treatments. Moreover, the liver injury was associated with elevated levels of hepatic eosinophils and hepatic T cells and serum antibodies that both reacted with trifluoroacetylated liver proteins isolated from halothane treated mice. The resulting liver injury appears to be mediated at least in part by CD4+ T cells, as indicated by significant protection against liver injury when CD4+ T cells are depleted prior to the onset of hepatotoxicity. Further investigation of TFA-adduct specific serum antibodies reveals a significant increase in IgG1 and IgE subclasses indicative of a Type II adaptive immune response. Liver eosinophilia has been associated with incidences of drug-induced liver disease (DILD) for more than 50 years, though its role in the etiology of this disease has remained unclear. We reported for the first time a pathogenic role of eosinophils in DILD using a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected in the liver within 12 hours and increased thereafter proportionally to liver damage. Eosinophil associated chemokines, eotaxins, and the activator/growth factor interleukin-5 (IL-5) increased in response to halothane-treatment. Immunohistochemical staining for major basic protein (MBP), a cytotoxic eosinophil granule protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis and appeared to show signs of degranulation as MBP staining was more diffuse than in the livers from vehicle controls. The severity of HILI was decreased significantly when the study was repeated in wild-type mice partially depleted of eosinophils and in the eosinophil knockout mice. Conversely, animals with selective depletion of neutrophils, which have been previously reported to play a pathogenic role in this model, failed to reduce the extent of HILI when levels of eosinophils remained unchanged. These findings indicate that eosinophils, not neutrophils, have a pathologic role in HILI in mice. More recently, we have shown that the epithelial derived cytokine, thymic stromal lymphopoetin (TSLP), and its corresponding receptor TSLPR also play a pathogenic role in HILI in mice that is mediated by type 2 immunity. In this regard,the severity of HILI was reduced in both TSLPR and IL-4 knockout mice and was accompanied by decreases in serum levels of IL-5 and eotaxins and hepatic eosinophilia. In addition, we found that murine and human hepatocytes treated with IL-4 secreted TSLP and eotaxins. Conclusion: Collectively, these data provide a rational approach for developing animal models of DILD that are mediated by the adaptive and innate immune system and suggest that deficiencies in liver tolerance may predispose patients to DILD. These findings also establish a pathologic role for type 2 immunity in HILI in mice and suggest that similar signaling pathways may be involved in DILD caused by a variety of drugs in humans.

虽然临床证据表明，许多DILD病例是由药物的肝脏蛋白加合物和适应性免疫系统和先天免疫系统介导的，但由于缺乏动物模型，这种毒性机制的详细实验证据仍然难以获得。我们假设，DILD在动物中和在人类中一样罕见，至少部分原因是肝脏的耐受性，它由适应性免疫系统的多个负面调节因素组成。这一想法现在已经在氟烷诱导的肝损伤的小鼠模型中进行了测试，在这种模型中，毒性是由氟烷的代谢形成三氟乙酰化的肝脏蛋白并由天然免疫系统增强的。在雌性Balb/CJ小鼠接受氟烷治疗24小时后，肝脏分析显示静脉周围坏死和CD11b+Gr-1高中性粒细胞的渗透，这是其他研究人员所报道的。进一步的研究表明，中性粒细胞含有髓系抑制细胞(MDSC)亚群，该亚群抑制了从幼小鼠分离的CD4+和CD8+T细胞的增殖。当在两次氟烷治疗前用Gr-1抗体从肝脏中清除MDSC时，与在氟烷治疗之前用同型对照抗体预处理的小鼠相比，第二次氟烷治疗9天后观察到肝损伤加重。此外，肝脏损伤与肝脏嗜酸性粒细胞、肝脏T细胞和血清抗体水平升高有关，这些抗体都与从氟烷处理的小鼠中分离的三氟乙酰化肝脏蛋白发生反应。由此产生的肝损伤似乎至少部分是由CD4+T细胞介导的，当CD4+T细胞在肝毒性发生之前被耗尽时，对肝损伤具有显著的保护作用。对TFA加合物特异性血清抗体的进一步研究显示，指示II型适应性免疫反应的IgG1和IgE亚类显著增加。 肝脏嗜酸性粒细胞增多症与药物性肝病(DILD)的发病率相关已有50多年，尽管其在该疾病的病因中所起的作用尚不清楚。我们首次使用氟烷诱导的小鼠肝损伤(HILI)模型报道了嗜酸性粒细胞在DILD中的致病作用。给雌性Balb/CJ小鼠注射氟烷后，12小时内可在肝脏中检测到嗜酸性粒细胞，此后随肝损伤而成比例增加。嗜酸性粒细胞相关趋化因子、嗜酸性粒细胞趋化因子和激活剂/生长因子白介素5(IL-5)在氟烷治疗后增加。主要碱性蛋白(MBP)是一种细胞毒性嗜酸性粒细胞颗粒蛋白，免疫组织化学染色显示，嗜酸性粒细胞仅聚集在肝细胞坏死区周围，并显示脱颗粒的迹象，因为MBP染色比赋形剂对照组的肝脏更弥漫。当在部分去除嗜酸性粒细胞的野生型小鼠和嗜酸性粒细胞敲除小鼠中重复这项研究时，HALI的严重程度显著降低。相反，在嗜酸性粒细胞水平保持不变的情况下，中性粒细胞选择性耗竭的动物未能减少HALI的程度，此前已有报道称中性粒细胞在这一模型中发挥了致病作用。这些发现表明，嗜酸性粒细胞，而不是中性粒细胞，在小鼠的HALI中起着病理作用。最近，我们发现上皮源性细胞因子胸腺基质淋巴生成素(TSLP)及其相应的受体TSLPR也在通过2型免疫介导的小鼠HALI中发挥致病作用。在这方面，在TSLPR和IL-4基因敲除的小鼠中，HALI的严重程度都有所减轻，并伴随着血清IL-5和嗜酸性粒细胞趋化因子水平的下降以及肝脏嗜酸性粒细胞增多。此外，我们还发现，经IL-4处理的小鼠和人肝细胞可以分泌TSLP和嗜酸性粒细胞趋化因子。 结论：总的来说，这些数据为建立由适应性免疫系统和先天免疫系统介导的DILD动物模型提供了一种合理的方法，并提示肝脏耐受性缺陷可能使患者更容易发生DILD。这些发现还建立了小鼠HILI中2型免疫的病理作用，并表明类似的信号通路可能参与了由多种药物引起的人类DILD。