Hypothermic oxygenated and normothermic machine perfusion mitigate innate systemic and hepatic inflammation after liver transplantation

Background Immune-mediated injury drives ischaemia-reperfusion injury (IRI) following liver transplantation. While hypothermic oxygenated (HOPE) and normothermic machine perfusion (NMP) improve clinical outcomes, their immunological effects in humans remain unclear. Objective To examine hepatic and systemic immune responses across preservation strategies in a randomised controlled clinical setting. Design In this mechanistic substudy of the HOPE-NMP randomised controlled trial ( NCT04644744 ), we analysed immune responses in n=47 recipients of extended criteria donor livers from brain-dead donors randomised to static cold storage (SCS), end-ischaemic HOPE or end-ischaemic NMP. Liver biopsies were obtained at organ arrival and early IRI. Blood samples were collected preoperatively, during reperfusion and on postoperative days 1, 2, 3 and 7. Methods Analyses included spectral flow cytometry, multiplex immunofluorescence with spatial analyses, cytokine profiling, cellular bioenergetics and mass spectrometry. Results In human liver allografts, both HOPE and NMP reduced neutrophil infiltration during early IRI, without altering intrahepatic spatial distribution. Machine perfusion induced phenotypic modulation of graft-infiltrating myeloid cells, characterised by downregulation of pro-inflammatory and adhesion markers. Peripheral lymphocyte numbers, particularly of NK cells and T cells, normalised faster after HOPE, compared with SCS and NMP groups, correlating with reduced liver injury. Absolute and relative systemic neutrophil numbers were lower in HOPE-recipients. Machine perfusion decreased the inflammatory activation of circulating myeloid and innate lymphoid cells, while inhibitory immune checkpoints were upregulated postoperatively. Conclusions In a randomised controlled trial using human samples, HOPE and NMP differentially modulate hepatic and systemic immune responses, attenuating neutrophil-driven inflammation and promoting immune regulation after liver transplantation.