Hepatic IR injury induced de-differentiation of neutrophils which exacerbated graft inflammation

Abstract

Introduction: Neutrophils, as main amplifier of hepatic ischemia-reperfusion injury (IRI), has been well recognized. Several studies have shown elimination of excessive neutrophils leads to reduction of liver inflammation. However, the characteristics and function of neutrophils in inflammatory microenvironment remains elusive. Our preliminary data demonstrated increase of a novel infiltrated immature neutrophils (CD66b+ CD10-) following hepatic IRI. With recent evidences of neutrophils cross-linking with adaptive immunity, we hypothesize IRI induced intrahepatic phenotypic change of neutrophil that promote post-transplant graft injury. Methods: Expression of CD66b, HLA-DR and CD10 gene expression in 31 human liver graft biopsy samples collected 2 hours after portal vein reperfusion were quantified using quantitative PCR. Changes in Ly6G and HLA-DR markers in blood and liver at various time points were monitored in C57 mice subjected to partial hepatic IR injury. Mouse Ly6Gintimmature neutrophils and Ly6Ghimature neutrophils were isolated followed by gene profiling and functional experiments. Results: qPCR and flow cytometry data showed that CD10 was down-regulated in post-reperfusion liver grafts (Fig 1). Patients with lower CD10 level developed a higher post-operative SGOT (eta2:0.186, p< 0.01), SGPT (eta2:0.119, p< 0.05) and total bilirubin (eta2: 0.155, p< 0.05). Neutrophil marker CD66b and HLA-DR mRNA level were elevated in post-reperfusion samples, and significantly correlated with each other (p< 0.001). Consistent findings were confirmed in IHC and flow cytometry (Fig 2). Mouse models demonstrated intra-hepatic induction of HLA-DR on neutrophils and de-differentiation of immature neutrophils from mature neutrophils following IR injury (Fig 3-4). Gene profiling demonstrated higher expression level of inflammatory-related genes HLA-DR, CD86, IL17A and MAC-1 (CD11a/CD18) in immature neutrophils compared to mature neutrophils. Conclusion: The results collectively indicated the novel immunostimulatory role of immature neutrophils in promoting inflammation and post-transplant graft injury. Targeting de-differentiation pathway and HLA-DR induction pathway may represent potential therapeutic strategy.