Impact of Machine Perfusion on the Immune Response After Liver Transplantation - A Primary Treatment or Just a Delivery Tool

Abstract

The frequent use of marginal livers forces transplant centres to explore novel technologies to improve organ quality and outcomes after implantation. Organ perfusion techniques are therefore frequently discussed with an ever-increasing number of experimental and clinical studies. Two main approaches, hypothermic and normothermic perfusion, are the leading strategies to be introduced in clinical practice in many western countries today. Despite this success, the number of studies, which provide robust data on the underlying mechanisms of protection conveyed through this technology remains scarce, particularly in context of different stages of ischemia-reperfusion-injury (IRI). Prior to a successful clinical implementation of machine perfusion, the concept of IRI and potential key molecules, which should be addressed to reduce IRI-associated inflammation, requires a better exploration. During ischemia, Krebs cycle metabolites, including succinate play a crucial role with their direct impact on the production of reactive oxygen species (ROS) at mitochondrial complex I upon reperfusion. Such features are even more pronounced under normothermic conditions and lead to even higher levels of downstream inflammation. The direct consequence appears with an activation of the innate immune system. The number of articles, which focus on the impact of machine perfusion with and without the use of specific perfusate additives to modulate the inflammatory cascade after transplantation is very small. This review describes first, the subcellular processes found in mitochondria, which instigate the IRI cascade together with proinflammatory downstream effects and their link to the innate immune system. Next, the impact of currently established machine perfusion strategies is described with a focus on protective mechanisms known for the different perfusion approaches. Finally, the role of such dynamic preservation techniques to deliver specific agents, which appear currently of interest to modulate this posttransplant inflammation, is discussed together with future aspects in this field.

Keywords: hypothermic oxygenated perfusion; innate immune activation; ischemia reperfusion injury; machine perfusion; marginal livers; mitochondrial injury.

Figure 1

Schematic presentation of ischemia-reperfusion-injury with pro-inflammatory signaling. The rapid succinate metabolism at complex II leads to the initial key event of ROS release when oxygen is reintroduced into ischemic tissues. Based on the level of accumulated succinate during ischemia, a number of cells are severely affected and die with subsequent release of mitochondrial DNA and Damps (A). Specific receptors are expressed on liver and immune cells, which trigger transcription and upregulation of pro-cytokines, creating an inflammatory milieu. Various Damps molecules instigate proinflammatory signals and the assembly of the inflammasome (NLRP-3) plus caspase cleavage, which activates available pro-cytokines (B). Such features lead to the injury of previously less activated and affected cells, which releases additional damps and cytokines. ATP, Adenosine-trisphosphate; Damps, danger associated molecular patterns; Hmgb-1, High mobility group box protein-1; IL, Interleukin; ROS, reactive oxygen species; TLR, toll like receptor.

Figure 2

Cascade of ischemia-reperfusion-injury with specific downstream activation of the innate immune system; Different stages of ischemia reperfusion injury (IRI) with innate immune activation and resolution with or without pharmacological treatment in liver transplantation are presented. According to the accumulated succinate, ROS are released at reperfusion (1) with subsequent release of mitochondrial DNA and Damps molecules, which link IRI to innate immune response. (2) Subsequently, dendritic cells and macrophages become activated and present their well-known surface receptors to circulating T cells, which become in turn activated (3). Recipient neutrophils and other immune cells are also attracted by activated endothelial cells in the organ. With the involvement of other liver and recipient immune cells, including stellate cells and fibroblasts, T and B cells plus killer cells, the inflammatory milieu achieves a chronic stage (4) and is resolved by pharmacological upstream treatment (5). Chronic processes may also become very advanced and severe with subsequent fibrosis of the implanted organ (6) if resolution mechanisms fail.

Figure 3

Timing of treatment modalities to address IRI-associated inflammation. Overview on the current timings when therapeutics are administered from the donor to implantation. Most molecules target genes or downstream receptors beyond the instigating processes, instead of succinate and subsequent ROS release.

Figure 4

Impact of HOPE on innate immune response after allogeneic liver transplantation; To address the accumulated succinate with a slow oxidation at complex II is a key mechanism to avoid the massive ROS release and subsequent IRI cascade with complications after transplantation. Machine perfusion is therefore a well explored new method to improve and assess metabolic processes. Hypothermic oxygenated perfusion (HOPE) before implantation was shown to reduce the accumulated succinate and to improve complex I and II function. Using a model of allogeneic liver transplantation, the protective effect of HOPE on the innate immune system was demonstrated with a lower number of activated of Kupffer cells (through less Damps release) and subsequently a lower number of infiltrating T cells in transplanted livers, compared to untreated controls (without HOPE and without immunosuppression). Of note, HOPE treatment achieved the best protection from Kupffer cell and dendritic cell activation early after implantation, e.g., at 24hrs, also compared to the group with full dosage of immunosuppression, which requires time until the most effective blood levels are seen. The protective effect of HOPE was still present 4 weeks after implantation, although the delayed immune response became visible. HOPE treatment was therefore combined with a low dose of immunosuppression, which led to acute rejection when applied alone. HOPE with reduced immunosuppression protected recipients from innate immune activation and acute rejection, similarly to recipients which received the full dose of tacrolimus (immunosuppression). These images were obtained from samples from the study presented in reference (42) (samples and histological images were not published before in this reference). CD, cluster of differentiation.

Figure 5

Therapeutic strategies to target ischemia-reperfusion injury and improve outcomes after liver transplantation; This figure provides an overview of currently applied pharmacological and non-pharmacological modalities with impact on IRI associated features. From the donor, procurement to preservation and reperfusion (transplantation) modalities, their targets and results as well as challenges are presented. The majority of strategies affects individual genes or receptors, which might lead to an even higher proinflammatory response by other genes not affected.