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Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

ABSTRACT: Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1⁺/2⁺ (CXCR1⁺/CXCR2⁺) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

SUBMITTER: Hautz T

PROVIDER: S-EPMC10121614 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

Hautz T T Salcher S S Fodor M M Sturm G G Ebner S S Mair A A Trebo M M Untergasser G G Sopper S S Cardini B B Martowicz A A Hofmann J J Daum S S Kalb M M Resch T T Krendl F F Weissenbacher A A Otarashvili G G Obrist P P Zelger B B Öfner D D Trajanoski Z Z Troppmair J J Oberhuber R R Pircher A A Wolf D D Schneeberger S S

Nature communications 20230421 1

Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine recepto ...[more]

PMID: 37085477

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Project description:BackgroundNormothermic machine perfusion (NMP) allows viability assessment and potential resuscitation of donor livers prior to transplantation. The immunological effect of NMP on liver allografts is undetermined, with potential implications on allograft function, rejection outcomes and overall survival. In this study we define the changes in immune profile of human livers during NMP.MethodsSix human livers were placed on a NMP device. Tissue and perfusate samples were obtained during cold storage prior to perfusion and at 1, 3, and 6 hours of perfusion. Flow cytometry, immunohistochemistry, and bead-based immunoassays were used to measure leukocyte composition and cytokines in the perfusate and within the liver tissue. Mean values between baseline and time points were compared by Student's t-test.ResultsWithin circulating perfusate, significantly increased frequencies of CD4 T cells, B cells and eosinophils were detectable by 1 hour of NMP and continued to increase at 6 hours of perfusion. On the other hand, NK cell frequency significantly decreased by 1 hour of NMP and remained decreased for the duration of perfusion. Within the liver tissue there was significantly increased B cell frequency but decreased neutrophils detectable at 6 hours of NMP. A transient decrease in intermediate monocyte frequency was detectable in liver tissue during the middle of the perfusion run. Overall, no significant differences were detectable in tissue resident T regulatory cells during NMP. Significantly increased levels of pro-inflammatory and anti-inflammatory cytokines were seen following initiation of NMP that continued to rise throughout duration of perfusion.ConclusionsTime-dependent dynamic changes are seen in individual leukocyte cell-types within both perfusate and tissue compartments of donor livers during NMP. This suggests a potential role of NMP in altering the immunogenicity of donor livers prior to transplant. These data also provide insights for future work to recondition the intrinsic immune profile of donor livers during NMP prior to transplantation.

Project description:BackgroundReliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation.MethodsIn a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24.ßh. Biopsy and perfusate samples were collected at the end of cold storage, at 1.ßh, 6.ßh, end of NMP, and 1.ßh after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy.FindingsA considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6.ßh of NMP correlated with L-GrAFT.InterpretationsBioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation.FundingThis study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the Tiroler Wissenschaftsfond granted to TH.

Project description:A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and improvement of marginal allografts. The most exciting development in the past decade has been continuous mechanical perfusion of livers with blood or preservation solution to mitigate ischemia-reperfusion injury in contrast to the current standard of static cold storage. Two variations of machine perfusion have emerged in clinical practice. During hypothermic oxygenated perfusion the liver is perfused using a red blood cell-free perfusate at 2-10°C. In contrast, normothermic machine perfusion mimics physiologic liver perfusion using a red blood cell-based solution at 35.5-037.5°C, offering a multitude of potential advantages. Putative effects of normothermic perfusion include abrogation of hyperfibrinolysis after reperfusion and inflammation, glycogen repletion, and regeneration of adenosine triphosphate. Research in normothermic machine perfusion focuses on development of biomarkers predicting allograft quality and susceptibility to ischemia-reperfusion injury. Moreover, normothermic perfusion of marginal allografts allows for application of a variety of therapeutic interventions potentially enhancing organ quality. Both methods need to be subjected to translational investigation and evaluation in clinical trials. A clear advantage is transformation of an emergency procedure at night into a planned daytime surgery. Current clinical trials suggest that normothermic perfusion not only increases the use of hepatic allografts but is also associated with milder ischemia-reperfusion injury, resulting in a reduced risk of early allograft dysfunction and less biliary complications, including ischemic cholangiopathy, compared to static cold storage. The aim of this review is to give a concise overview of normothermic machine perfusion and its current applications, benefits, and possible advances in the future.

Dataset Information

Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

Publications

Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

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