|Abstract: ||Liver transplantation is a well-established, life-saving treatment for patients with end-stage liver disease. Liver transplantation is inevitably accompanied by some degree of ischaemia/reperfusion injury. Ischaemia/reperfusion injury (IRI) can have important implications for both short and long-term transplantation outcomes. Increasing demand for donor livers has necessitated the use of “extended criteria donor” organs. Frequently, these livers are affected by steatosis. Significant steatosis of the donor liver is associated with increased susceptibility to injury during donor brain death and warm ischaemia, and increased rates of primary graft non-function. Consequently, steatosis is the most common reason for procured liver grafts to be deemed unsuitable for transplantation. Better understanding of the mechanisms involved in these processes could help enlarge the pool of available donor livers and allow safer utilization of steatotic grafts. Approximately 20% of liver transplant recipients in the clinical setting can be safely weaned off immunosuppressive medications without the expected graft rejection. As an initial trigger of immune responses, IRI is an important factor which can impede the development of operational tolerance. Spontaneous liver transplantation tolerance has been consistently associated with the deletion or clonal exhaustion of alloreactive T cells following their initial activation, thus preventing their ability to mediate graft destruction.
The first part of this study examined the hypothesis that the higher propensity for liver grafts to be tolerated compared with other organs, is due to a relative insufficiency of T cell survival signals, transmitted through the cytokines, interleukin (IL)-2, 4, 7, 9, 15 and 21 and their receptors, which incorporate the common gamma chain. Transplantation of hearts or kidneys in the PVG to DA rat strain combination led to rejection of the graft. Livers transplanted in the same strain combination are tolerated without the need for immunosuppression. By examining the relative gene expression of the common gamma chain cytokines and their receptors, we found that an association exists between high expression of IL-2, IL-2Rα, IL-4 and IL-15Rα and rejection. In contrast to these, expression of IL-7Rα was significantly higher in tolerated livers than in rejecting hearts and kidneys. IL-7Rα expression is inversely related to the global levels of common gamma chain signalling, further supporting the postulate that graft-infiltrating T cells in tolerated livers have been exposed to lower concentrations of common gamma chain cytokines.
The second part of this study was aimed at establishing a model of partial, warm liver IRI in the steatotic liver and characterizing some of the factors contributing to the increased susceptibility of fatty livers to IRI. This was achieved through feeding male C57BL/6J mice a 45% fat diet for 12 weeks. Lean mice and those with steatotic livers were then subjected to a moderate ischaemic insult, followed by reperfusion. HMGB1 is a critical mediator of inflammatory signalling during liver IRI. We found that HMGB1 was localised to the nucleus of hepatocytes in lean livers, whereas in steatotic livers a striking redistribution of HMGB1 from the nucleus to the cytoplasm had taken place. As early as 15 minutes post-reperfusion, serum levels of HMGB1 were significantly higher in the HFD-fed than in the lean mice, and the severity of ischaemic injury was considerably greater in the HFD-fed group as determined by increased serum alanine aminotransferase, more extensive liver necrosis and apoptosis, and increased synthesis and/or systemic release of proinflammatory cytokines such as IL-6, TNFα, IL-1β and IL-17. The damaging effects of IRI in steatotic livers were accentuated by a reduced rate of hepatocyte regeneration. Moreover, persistently elevated IL-7 levels in the HFD mice on d1 post-reperfusion may be a means of rescuing activated alloreactive T cells if IRI is occurring in the context of an allogeneic liver transplant. IL-6 and IL-17 are also recognised contributors to the blockade of transplant tolerance induction, implying that the more intense innate immune response in steatotic liver not only engenders more severe tissue damage and poorer immediate graft function, but could oppose the development of operational tolerance in these grafts.
The third part of this study examines the effect of overexpression of endogenous soluble Receptor for Advanced Glycation End-products (esRAGE) on the outcome of partial warm IRI in lean and fatty livers. esRAGE acts as a competitive antagonist to alarmins such as HMGB1 and reduces proinflammatory signal transduction. By administering an adenoassociated viral vector encoding esRAGE, persistent high systemic levels of esRAGE were achieved without the need for repeated intraperitoneal injections. An AAV vector encoding Green Fluorescent Protein (GFP) served as a control. Systemic esRAGE expression resulted in reduced serum HMGB1 and conferred protection from IRI in lean livers, but not in steatotic livers. In the esRAGE-transduced lean livers we found minimal necrosis, fewer apoptotic cells and subdued neutrophil infiltration, compared with GFP-transduced lean livers at day 1 and 3 post-reperfusion. Conversely, esRAGE expression in steatotic hepatocytes resulted in a greater incidence of frank necrosis, and hepatocyte apoptosis, associated with more intense neutrophil infiltration and increased expression of various components of inflammatory signalling pathways.
In conclusion, liver-directed gene transfer of esRAGE to lean livers protects them against warm IRI. In the steatotic hepatocytes, esRAGE transduction increases susceptibility to tissue injury, possibly due to augmentation of pre-existing endoplasmic reticulum stress. An alternative approach to harnessing the potential hepatoprotective effects of AAV vector-mediated esRAGE gene transfer in the setting of hepatic steatosis could involve choosing skeletal muscle rather than liver as the site for transgene expression.|