Acute phase liver graft injury significantly mobilized circulating endothelial progenitor cells, myeloid-derived suppressor cells and regulatory T cells through TLR4 signaling

Abstract

Introduction and objective: Higher incidence of tumor recurrence is a major obstacle of living donor liver transplanatation (LDLT) for the patients with hepatocellular carcinoma (HCC). The acute phase small-for-size graft injury is an important factor contributing to late phase tumor invasion and recurrence. In the current study, we aim to investigate the impact of acute phase liver graft injury on mobilization of circulating endothelial progenitor cells (EPCs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in a rat liver transplantation model and to explore the underlying mechanism. Materials and Methods: A rat orthotopic liver transplantation model was established using cirrhotic recipient with the application of whole graft (100%) or small-for-size (50%) graft. Circulating EPCs, MDSCs and Tregs were detected by flow cytometry at day 1, 3 and 5 after transplantation. Intragraft mRNA levels of TLR4, Tie2 (angiogenesis) and CXCR4 (acute phase inflammation and stem cell homing) were compared by qRT-PCR. Intragraft infiltration of circulating cells (EPCs) from recipient source was confirmed using the recipient rat with green fluorescence protein (GFP) labeling. Tregs (FOXP3+) accumulation in the grafts was detected by immunostaining. The numbers of MDSCs in circulating blood and bone marrow were further compared in TLR4 mutant mice after partial hepatic ischemia-reperfusion injury (I/R injury) together with major hepatectomy. Results: There were more circulating EPCs (day 3: 1220 vs 550/105PBMC, p=0.032; day 5: 1064 vs 633/105PBMC, p=0.046) and MDSCs (day 3: 875 vs 220/WBC, p=0.038; day 5: 358 vs 106/105WBC, p=0.043) at day 3 and 5, and more Tregs at day 5 (2604 vs 1414/105WBC, p=0.037) after transplantation using small-for-size graft compared to whole graft. The intragraft TLR4 mRNA levels were significantly higher (TLR4: 1.96 folds of whole graft, p=0.012) at day 2 as well as the intragraft CXCR4 and Tie2 mRNA levels were significantly higher (CXCR4: 1.99 folds of whole graft, p=0.0064; Tie2: 2.01 folds of whole graft, p=0.019) at day 3 after transplantation with small-for-size graft. The circulating MDSCs were significantly decreased in TLR4 mutant mice (0.245% vs 0.665% PBMC, p=0.002) at day 1 as well as the MDSCs in the bone marrow (day 1: 0.415% vs 0.75% , p=0.047; day 3: 0.12% vs 0.36%, p=0.0027) 1 day and 3 days after hepatic I/R injury with left lobe hepatectomy in comparison with wild type mice. There were more Tregs and inflammatory cells infiltration, and severer eosinophilic degeneration in the small-for-size graft. Intragraft infiltration of the circulating cells from the recipient source was confirmed by detection of GFP positive signals in the graft. Involvement of angiogensis by recipient source EPCs was indicated by the detection of GFP+CD31+ and GFP+vW factor+ cells in the grafts. Conclusion: Acute phase small-for-size liver graft injury may play an important role in mobilization of circulating EPCs, MDSCs and Tregs through up-regulation of TLR4/CXCR4 signaling. Higher population of circulating EPCs may contribute to intragraft new vessel formation, leading to angiogenesis and tumor recurrence. TLR4 is important for the mobilization of MDSCs, which may cause tumor escape from the immune system.