Inflammatory gene expression in livers undergoing ex situ normothermic perfusion is attenuated by leukocyte removal from the perfusate
ABSTRACT: Background. Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion. Methods. We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion. Results. During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes. Conclusions. Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP. Overall design: experiment1: Comparative gene expression profiling analysis of RNA-seq data of DCD (donation after circulatory death) liver biopsies taken pre and post machine perfusion. experiment2: Comparative gene expression profiling analysis of RNA-seq data of DCD (donation after circulatory death) liver biopsies taken pre and post machine perfusion with the addition of a leukocyte filter. experiment3: Comparative gene expression profiling analysis of RNA-seq data of DCD (donation after circulatory death) liver biopsies of pre-perfusion samples with a raised 2h perfusate alanine transaminase levels experiment4: Comparative gene expression profiling analysis of RNA-seq data of DBD (donation after brainstem death) liver biopsies of pre-perfusion samples with a raised 2h perfusate alanine transaminase levels