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

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.

A sizeable proportion of liver transplant recipients spontaneously develop a state of immune tolerance that allows them to discontinue all immunosuppression and yet sustain normal graft function. In an attempt to characterize this group of patients we recently completed a large multi-centre drug withdrawal trial. Tolerance was found to be associated with iron metabolism parameters (i.e recipients requiring lifelong immunosuppression displayed lower hepcidin and serum iron levels than tolerant patients). To investigate the potential causal link between iron metabolism, intra-hepatic alloimmune responses, and tolerance, we have performed preliminary experiments in rodent models of immune-mediated liver damage that indicate that decreased iron and/or hepcidin levels markedly interfere with baseline intra-hepatic gene expression, inhibit intra-hepatic T cell function, and could interfere with regulatory T cell generation and/or function. These preliminary results suggest that iron homeostasis plays a previously unrecognised role in regulating the balance between liver inflammation and immune tolerance. The aims of the current proposal are to elucidate the exact cellular and molecular pathways through which iron and/or hepcidin modulate inflammatory liver injury, interfere with lymphocyte function and/or migration, and regulate tolerance. Experiments will be performed in murine models of inflammatory liver damage and in a rat model of orthotopic liver transplantation. The project will contribute to elucidate the mechanisms responsible for the development of transplantation tolerance following liver transplantation and will provide insight into the pathogenesis of liver inflammatory disorders.

Following liver transplantation, the need to administer chronic immunosuppressive therapy results in significant side effects such as infections, nephrotoxicity, diabetes, hypertension and cancer. These co-morbidities have a negative impact on patient mortality and are in part responsible for the stagnation in long-term recipient outcomes observed over the past two decades in liver transplantation. The achievement of liver allograft tolerance (i.e. successful discontinuation of all
immunosuppressive drugs) would therefore have a major impact on the health and well-being of liver transplant recipients. Recent findings by our group have revealed that tolerant liver recipients exhibit specific transcriptional and blood cell phenotypic biomarkers. Furthermore, these results have shown that the regulation of iron homeostasis is closely associated with the development of liver allograft tolerance. Our preliminary experiments in animal models are consistent with the results of our clinical studies and suggest that modulation or iron and/or hepcidin could be a fundamental and previously unrecognized mechanisms through which liver inflammation and tolerance is regulated.

The aims of the current project are to investigate the mechanisms through which iron metabolism influences intra-hepatic inflammation and tolerance in a series of clinically-relevant experimental animal models. Liver transplant recipients will be the main beneficiaries of our research. Elucidation of the mechanisms responsible
for allograft tolerance in clinical liver transplantation will open the door to therapeutic interventions that may intentionally induce tolerance in those recipients who do not spontaneously develop this state. Altogether this would constitute a major paradigm shift in the way liver transplant patients are medically managed and would provide obvious health benefits in terms of decreased morbidities, increased life expectancy and quality of life of liver transplant recipients.In addition to these health benefits associated with the discontinuation of immunosuppressive therapy, the current proposal would also provide an obvious pharmacoeconomic benefit given the high cost of currently administered immunosuppressive medications (more than £4000 yearly per patient). Beyond liver transplantation, the results of the current proposal are likely to be generalizable to non-transplanted patient populations suffering from inflammatory liver conditions (e.g. steatohepatitis, viral hepatitis, autoimmune liver disease), and could have implications for extra-hepatic inflammatory and autoimmune diseases.