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

对于实体器官受者来说，终身免疫抑制通常被认为是必须的，以避免由于排斥而导致移植物损失的风险。然而，慢性免疫抑制疗法与副作用（感染、肾毒性、糖尿病、高血压和癌症）相关，对患者死亡率有负面影响。因此，在维持同种异体移植物正常功能和组织学的同时，成功地减少和/或撤销免疫抑制的策略引起了人们的强烈兴趣。并非所有受者都需要持续的免疫抑制来维持移植物，这一证明主要来自一小部分患者，他们由于不遵守规定，出于医疗需要，或在停药试验中停止了传统的免疫抑制，但仍维持了正常的移植物功能。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先前估计的要高：15%至40%或更多，具体取决于受体的年龄和离移植的距离。这导致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受体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。这些标志物与血清铁状态相关参数（如铁蛋白和hepcidin）水平相关，并且在预测免疫抑制停药方案的结果方面非常准确。这些观察结果首次表明，即使在正常参考范围内，铁稳态的变化也可能在同种免疫反应和肝内炎症的调节中发挥作用。为了研究铁稳态是否真的与同种异体肝移植耐受有因果关系，我们最近完成了一系列从床边到实验台的初步动物实验，在实验中，给缺乏铁的小鼠喂食一种诱导淋巴细胞介导肝炎的化合物。低铁饮食引起肝内基因表达的显著变化。此外，它大大改善了肝炎的严重程度，这与T淋巴细胞的次优激活有关。考虑到肝内激活同种异体T淋巴细胞是建立肝脏移植耐受所必需的，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合理的解释。此外，这些结果表明，铁代谢可能是肝脏调节耐受性和免疫平衡的一个基本的、以前未被认识到的机制。本研究的目的是详细探讨低铁和/或低hepcidin调节肝脏炎症和阻止肝移植耐受的机制。这将通过以下具体目标来实现：1)确定铁和/或hepcidin影响急性和慢性肝损伤后肝脏炎症微环境的机制；2)确定铁和/或hepcidin如何影响淋巴细胞迁移和激活以及调节性T细胞的产生；3)探讨低铁和/或hepcidin阻碍大鼠同种异体肝移植耐受发展的机制。4)确定铁稳态是否直接影响肝脏再生。总之，该项目将有助于阐明肝移植后移植耐受发展的机制，并将为开展未来的药物停药试验和研究提供理论基础，旨在调节铁代谢作为调节肝脏炎症的手段。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.