INFLUENCE OF IRON METABOLISM ON THE REGULATION OF INTRA-HEPATIC INFLAMMATORY RESPONSES

终身免疫抑制通常被认为是固体器官接受者的义务，以避免因排斥反应而导致的移植物丢失的风险。然而，慢性免疫抑制治疗与副作用(感染、肾毒性、糖尿病、高血压和癌症)有关，这些副作用对患者死亡率有负面影响。因此，人们对成功地最小化和/或取消免疫抑制，同时保持正常的同种异体移植功能和组织学的策略有着浓厚的兴趣。不是所有的受者都需要永久的免疫抑制来维持他们的移植物，这一证明主要来自于一小部分患者，他们通过不依从性、出于医疗需要或在药物停药试验中停止了传统的免疫抑制，但仍维持着正常的移植物功能。这些患者被认为是“手术”耐受的。最近的研究表明，在肝移植中，这种现象的发生率比以前估计的要高：15%到40%或更多，这取决于受者的年龄和离移植的距离有多远。这使得耐受性被认为是肝移植的一个切实的临床机会。我们的小组最近描述了耐受者的肝组织样本的特征是参与铁代谢调节的基因的差异表达。这些标记物与铁蛋白和海普西丁等铁状态相关参数的血清水平相关，在预测免疫抑制停药方案的结果方面非常准确。这些观察首次表明，铁稳态的变化，即使在正常的参考范围内，也可能在调节同种异体免疫反应和肝内炎症方面发挥作用。为了研究铁稳态是否真的与肝移植耐受有关，我们最近完成了一系列床边到工作台的初步动物实验，在这些实验中，喂养铁缺乏饮食的小鼠被一种诱导淋巴细胞介导性肝炎的化合物挑战。低铁饮食可引起大鼠肝内基因表达的显著变化。此外，它显著改善了肝炎的严重程度，这与T淋巴细胞的激活不佳有关。考虑到肝内同种异体反应性T淋巴细胞的激活是建立肝移植耐受所必需的，这些初步数据为我们临床试验中观察到的结果提供了一个可信的解释。此外，这些结果表明，铁代谢可能是肝脏中调节耐受和免疫平衡的一个基本的和先前未知的机制。本研究的目的是详细探讨低铁和/或低海普西丁调节肝脏炎症和阻止肝移植耐受的机制。这将通过以下特定目标实现：1)确定铁和/或海普西丁影响急、慢性肝损伤后肝脏炎症微环境的机制；2)确定铁和/或海普西丁如何影响淋巴细胞迁移和激活以及调节性T细胞的生成；3)探讨低铁和/或海普西丁阻碍大鼠肝移植耐受发展的机制。4)确定铁稳态是否直接影响肝再生。总之，该项目将有助于阐明肝移植后移植耐受形成的机制，并将为未来进行旨在调节铁代谢作为调节肝脏炎症的手段的停药试验和研究提供理论基础。这项研究可能具有更广泛的意义，因为细胞内铁的调节可能是调节T细胞功能的基本机制。

Life-long immunosuppression is commonly regarded as obligatory for solid-organ recipients to avoid the risk of graft loss due to rejection. Chronic immunosuppressive therapy however is associated with side effects (infections, nephrotoxicity, diabetes, hypertension and cancer) that have a negative impact on patient mortality. As a result, there is intense interest in strategies to successfully minimise and/or withdraw immunosuppression while maintaining normal allograft function and histology. The demonstration that not all recipients require perpetual immunosuppression to maintain their graft mainly derives from the small subset of patients who discontinue conventional immunosuppression through non-compliance, out of medical necessity, or within drug withdrawal trials, and yet sustain normal graft function. These patients are considered as "operationally" tolerant. Recent studies indicate that in liver transplantation the prevalence of this phenomenon is greater than previously estimated: 15 to 40% or more depending on recipient age and how remote from transplant. This has resulted in tolerance being recognized as a tangible clinical opportunity in liver transplantation. Our group recently described that liver tissue samples from tolerant recipients are characterized by the differential expression of genes involved in the regulation of iron metabolism. These markers correlated with serum levels of iron status-related parameters such as ferritin and hepcidin, and were very accurate at predicting the outcome of an immunosuppressive withdrawal protocol. These observations suggested for the first time that changes in iron homeostasis, even within normal reference ranges, could be playing a role in the regulation of alloimmune responses and intra-hepatic inflammation. To investigate if iron homeostasis is truly causatively linked to liver allograft tolerance, we recently completed a series of bedside-to-bench preliminary animal experiments in which mice fed an iron-deficient diet were challenged with a compound that induces lymphocyte-mediated hepatitis. The low iron diet induced marked changes in intra-hepatic gene expression. Furthermore, it substantially improved the severity of hepatitis, and this was associated with suboptimal activation of T lymphocytes. Considering that intra-hepatic activation of alloreactive T lymphocytes is required for the establishment of liver allograft tolerance, these preliminary data provide a plausible explanation to account for the results observed in our clinical trial. Furthermore, these results suggest that iron metabolism could be a fundamental and previously unrecognized mechanism through which the balance between tolerance and immunity is regulated in the liver.The goal of the current proposal is to explore in detail the mechanisms through which low iron and/or low hepcidin regulate liver inflammation and preclude liver transplantation tolerance. This will be accomplished through the following specific aims: 1) to determine the mechanisms through which iron and/or hepcidin influence the liver inflammatory microenvironment following acute and chronic liver injuries ; 2) to define how iron and/or hepcidin influence lymphocyte migration and activation and the generation of regulatory T cells; 3) to interrogate the mechanisms by which low iron and/or hepcidin hamper the development of liver allograft tolerance in rats.4) to determine if iron homeostasis directly influences liver regeneration. Altogether, the project will contribute to elucidate the mechanisms responsible for the development of transplantation tolerance following liver transplantation, and will provide the rationale to conduct future drug withdrawal trials and studies aiming at modulating iron metabolism as a means to regulate liver inflammation. This research could have wider implications, as modulation of intracellular iron could be a fundamental mechanism through which T cell function is regulated.