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

尽管临床证据表明许多DILD病例是由药物的肝蛋白加合物以及适应性和先天性免疫系统介导的，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捉摸。 我们假设DILD在动物中和在人类中一样罕见，至少部分是由于肝脏的致耐受性，肝脏由适应性免疫系统的多种负调节因子组成。这一想法现已在氟烷诱导肝损伤的既定小鼠模型中进行了测试，其中毒性由氟烷代谢形成三氟乙酰化肝蛋白引发，并由先天免疫系统增强。在用氟烷处理雌性Balb/cJ小鼠24小时后，肝脏分析显示静脉周围坏死和CD 11b + Gr-1High中性粒细胞浸润，如其他研究人员所报告。进一步的研究表明，中性粒细胞含有髓源性抑制细胞（MDSC）的亚群，抑制从幼稚小鼠分离的CD 4+和CD 8 + T细胞的增殖。 当在用氟烷进行两次处理之前用Gr-1抗体从肝脏中耗尽MDSC时，与在氟烷处理之前用同种型对照抗体预处理的小鼠相比，在第二次暴露氟烷后9天观察到增强的肝损伤。 此外，肝损伤与肝嗜酸性粒细胞和肝T细胞以及血清抗体水平升高相关，这些抗体均与从氟烷处理的小鼠中分离的三氟乙酰化肝蛋白反应。 由此产生的肝损伤似乎至少部分由CD 4 + T细胞介导，如在肝毒性发作前CD 4 + T细胞耗尽时对肝损伤的显著保护所示。 TFA-加合物特异性血清抗体的进一步研究显示IgG 1和IgE亚类显著增加，表明II型适应性免疫应答。 肝嗜酸性粒细胞增多症与药物性肝病（DILD）的发生有关已有50多年的历史，但其在该病病因学中的作用仍不清楚。 我们首次报道了嗜酸性粒细胞在使用氟烷诱导的肝损伤（HILI）小鼠模型的DILD中的致病作用。 当雌性Balb/cJ小鼠给予氟烷时，在12小时内在肝脏中检测到嗜酸性粒细胞，此后与肝损伤成比例增加。 嗜酸性粒细胞相关的趋化因子，嗜酸性粒细胞趋化因子，和激活剂/生长因子白细胞介素-5（IL-5）的增加，响应氟烷治疗。主要碱性蛋白（MBP）（一种细胞毒性嗜酸性粒细胞颗粒蛋白）的免疫组织化学染色显示，嗜酸性粒细胞仅在肝细胞坏死区域周围聚集，并且似乎显示脱粒迹象，因为MBP染色比溶剂对照组的肝脏中更弥散。 当在嗜酸性粒细胞部分耗竭的野生型小鼠和嗜酸性粒细胞敲除小鼠中重复该研究时，HILI的严重程度显著降低。 相反，动物与嗜中性粒细胞的选择性耗竭，这已经被报道在这个模型中发挥致病作用，未能减少HILI的程度时，嗜酸性粒细胞的水平保持不变。 这些发现表明，嗜酸性粒细胞，而不是中性粒细胞，在小鼠HILI的病理作用。最近，我们已经表明，上皮细胞衍生的细胞因子，胸腺基质淋巴细胞（TSLP），及其相应的受体TSLPR也发挥致病作用，在HILI小鼠2型免疫介导的。在这方面，HILI的严重程度在TSLPR和IL-4基因敲除小鼠中均降低，并伴有IL-5和嗜酸性粒细胞趋化因子的血清水平和肝嗜酸性粒细胞增多症的降低。 此外，我们发现用IL-4处理的鼠和人肝细胞分泌TSLP和嗜酸性粒细胞趋化因子。 结论：总的来说，这些数据提供了一个合理的方法来开发DILD的动物模型，介导的适应性和先天性免疫系统，并表明，肝脏耐受性的缺陷可能使患者容易患DILD。这些发现也确立了2型免疫在小鼠HILI中的病理作用，并表明类似的信号传导途径可能参与了多种药物引起的人类DILD。

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.