Refining Ex Situ Normothermic Machine Perfusion: Balancing Inflammation and Repair in Liver Transplantation

Liver transplantation remains the gold standard for treating patients with end-stage liver failure. However, ischemia/reperfusion (IR) injury (IRI) poses a significant challenge, affecting both early and long-term outcomes of transplanted organs. The global shortage of donor organs has deepened this crisis, leading to an increasing number of deaths among patients on waiting lists. To address this shortage, the use of extended criteria donor (ECD) organs, including those from donors after circulatory death (DCD), has become more common. Unfortunately, these organs are more prone to IRI and are associated with higher rates of primary nonfunction, vascular complications, and reduced survival due to cholangiopathy.1 Developing effective strategies to prevent or mitigate this injury is essential to safely expand their use. IRI involves cell death and subsequent inflammatory response, primarily driven by the innate immune system and mitochondrial reactive oxygen species. Nonparenchymal liver cells, including neutrophils, platelets, Kupffer cells, and a large population of monocyte-derived macrophages, are crucial in initiating and resolving inflammation. During ischemia, succinate accumulates, and on reperfusion, it is reoxidized, driving extensive reactive oxygen species production by reverse electron transport at mitochondrial complex I. This process disrupts oxidative phosphorylation, depletes ATP, and triggers the release of mitochondrial DNA. These factors and other damage-associated molecular patterns promote and sustain the inflammatory state. Despite extensive research, primarily in animal models, no specific therapies have yet been developed to effectively prevent or overcome IRI.2 Ex situ normothermic machine perfusion (ESNP) was reintroduced into clinical practice in the early 2010s, primarily to extend preservation time and assess organ viability in ECD allografts.3 The principle of ESNP is to maintain liver grafts ex situ in a functioning state by providing oxygen and nutrients at body temperature.4,5 Additionally, ESNP allows for the study of the phenotype and function of hepatic immune cells during liver perfusion and their release dynamics into the perfusate, offering a deeper understanding of the IR process. ESNP also provides a valuable window of opportunity for potential therapeutic interventions. In this issue of Transplantation, Bahadori et al6 evaluated immune events during ESNP in liver allografts from DCD and donation after brain death (DBD) donors. RNA sequencing identified significant transcriptomic changes by comparing postperfusion to preperfusion tissue samples. Notably, in DCD allografts, they observed a significant upregulation of genes encoding clotting factors, particularly SERPINE1, which encodes plasminogen activator inhibitor-1, the inhibitor of tissue plasminogen activator and urokinase, key mediators of fibrinolysis. In addition, the authors identified cytokines and chemokines crucial for recruiting neutrophils (C-X-C motif chemokine ligand 8) and monocytes (C-C motif chemokine ligand 2), along with other proinflammatory mediators (interleukin [IL]-6, C-X-C motif chemokine ligand 10, IL-1β, and tumor necrosis factor-α). Repeated perfusate samples confirmed the increase in these cytokines. Other findings suggest the activation of repair pathways during ESNP, with several prorepair transcripts, including thymic stromal lymphopoietin and various heat shock proteins such as HSP27, being upregulated, indicating a protective response against stress injury. The study also demonstrated a decreased enrichment of genes associated with the oxidative phosphorylation pathway. A novel approach in this work is the use of a leukocyte filter in the perfusion circuit, which significantly reduced transcriptional changes, including tumor necrosis factor-α and IL-1β levels in the perfusate. Although the removal of leukocytes may seem promising, it is essential to recognize their critical role in controlling inflammation and facilitating organ recovery and repair. Neutrophils and macrophages/monocytes, although potentially harmful during the acute phase of tissue damage, play a pivotal role in orchestrating repair during the resolution phase of inflammation. Consequently, depleting these cells might not always be beneficial, as their absence could delay the resolution of inflammation and disrupt immunoregulation.2 Additionally, research has shown that ESNP can increase the population of CD4+CD25+FOXP3+ regulatory T cells within the liver graft, highlighting the importance of these immune cells in maintaining balance and promoting recovery.4 Thus, targeting proinflammatory leukocytes could be a double-edged sword, as their removal might also eliminate cells essential for healing and immune regulation. The increased expression of genes that negatively regulate fibrinolysis could contribute to the formation of microthrombi within the microcirculation, potentially leading to occlusion of the peribiliary microvasculature and, consequently, cholangiopathy in DCD allografts. Although histological evidence was not provided in this study, the authors’ previous work demonstrated a link between intravascular fibrin deposition, biliary epithelial damage, and stromal necrosis in DCD livers. Notably, that study showed that administering tissue plasminogen activator into the perfusate during ESNP could prevent these adverse effects. These findings are currently being validated in a larger study.7 The authors further demonstrated differences in transcript profiles between DCD and DBD livers before ESNP, with upregulation of genes involved in metabolic pathways in DCD grafts and immune-mediated pathways in DBD grafts. This is significant because, although the perfusion equipment and perfusate are standardized for all allografts (heparinized red cells supplemented with nutrition and antibiotics), the metabolic and immune/inflammatory profiles of the livers are different before ESNP. Therefore, modifications to the perfusate should be based on findings from investigations into liver tissues and perfusate from different types of ECD allografts. In an era where maximizing organ utilization is critical, understanding graft pathophysiology and assessing damage before implantation are paramount. ESNP, despite its limitations, provides valuable insights into events following reperfusion, paving the way for personalized and targeted therapeutic strategies. A notable advancement in this work is the integration of a leukocyte filter, which opens new avenues for refining perfusion techniques and improving transplant outcomes. Over the past decade, the safety and efficacy of normothermic machine perfusion have been established, and further optimization is expected as our understanding of organ pathophysiology during ESNP deepens. The ongoing research by the Cambridge group exemplifies this progress and holds significant promise for advancing the field of transplantation.

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