Interleukin‐10 and Transforming Growth Factor‐β Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver

Abstract

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor β (TGFβ) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGFβ (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGFβ treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGFβ treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGFβ.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion

“…This is demonstrated by the lactate clearance and bile production of the livers during perfusion ( Figure 2 ). Although the enzyme markers of liver damage increased throughout perfusion, in accordance with prior studies ( 20 , 21 ), these increases were mostly independent of the oxygen carrier as shown by multiplex ELISA ( Figure 3 ). The notable exception to this trend was AST in the human pRBC group, which increased rapidly and significantly throughout NEVLP when compared to those of both rat pRBC and Oxyglobin.…”

“…Normothermic perfusion can create an inflammatory environment within the organ with subsequent increase in DAMPs and activation of tissue-resident immune cells ( 20 , 21 , 29 , 30 ). These activated immune cells can have negative downstream consequences, and mitigating their inflammatory potential is of major clinical interest.…”

“…Slides cut at 4-μm thickness were stained with hematoxylin and eosin (H&E). An expert liver pathologist (YL) blindly scored the severity of histologic damage to the liver according to the Suzuki criteria ( 21 ). Representative micrographs were collected at appropriate magnification using an Olympus DP73 equipped microscope (Olympus, Tokyo, Japan).…”

“…Liver-resident immune cells were isolated as previously described ( 21 ). Briefly, 100 ml of warmed 1× Hank’s balanced salt solution (HBSS, Worthington, Lakewood, NJ, USA) containing 25 mM N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid (HEPES) (Sigma-Aldrich), 4.2 mM NaHCO 3 (Sigma-Aldrich), and 83 μM Ethylenediaminetetraacetic acid (EDTA) (Sigma- Aldrich) was perfused through the portal vein.…”

“…It has been previously shown that NEVLP increases the expression of damage-associated molecular pattern (DAMP) proteins in the liver ( 20 ). Furthermore, we found that NEVLP-associated inflammation in the liver affects the phenotype of liver-resident dendritic cells (DCs), which may have undesirable downstream immunological consequences ( 21 ). Therefore, reducing the inflammatory environment within the liver during NEVLP for experimental or clinical purposes is of critical importance.…”