OXYGEN CARRIERS ALTER LIVER-RESIDENT IMMUNE CELLS DURING EX VIVO LIVER PERFUSION
Heather Jennings, Kristin Carlson, Joshua Verhagen, Bret Verhoven, Weifeng Zeng, Stacey McMorrow, Peter Chlebeck, David P. Al Adra*
Surgery, University of Wisconsin-Madison, Madison, WI
Normothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows functional assessment of the liver prior to transplantation. This functional assessment of the liver may allow the expansion of the donor pool to include marginal organs that would otherwise be discarded. However, the effect of NEVLP on liver-resident immune cells (key mediators of organ rejection after transplant) is largely unknown, with reports of either proinflammatory or anti-inflammatory consequences. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells or synthetic hemoglobin have unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells.
Adult Lewis rat livers underwent NEVLP using three different oxygen carriers: human red blood cells (RBC), rat RBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). After four hours of perfusion, the perfusate was collected for downstream analysis and livers were digested to isolate immune cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure common immune cytokines in the perfusate and the immune cells underwent phenotypic characterization with flow cytometry.
Liver perfusion quality and liver function were assessed throughout each NEVLP experiment. There were no differences in lactate clearance, bile production, liver damage enzymes, or histology between the three oxygen carrier groups. qRT-PCR revealed the gene expression of NF-?B and the inflammatory cytokines and chemokines, IL-1?, CCL2 and CD14, were significantly upregulated in the human RBC group compared with rat RBC and Oxyglobin groups. Flow cytometry demonstrated the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident immune cells from both the rat and human RBC groups compared to the Oxyglobin group. Other marker of immune activation, such as CD40, CD11b/c, and MHC II expression levels were not significantly different between groups.
Liver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the cytokine production and phenotype of liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of immune activation when compared to human or rat RBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier. Future studies will assess the effects of oxygen carriers in a transplant model.
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