*Purpose: Neutrophils are key mediators of ischemia/reperfusion injury (IRI) in orthotopic liver transplantation (OLT). Once activated, they release nuclear DNA web-like structures called neutrophil extracellular traps (NETs), which exacerbate liver damage. We have established that matrix metalloproteinase-9 (MMP-9) facilitates neutrophil recruitment in mouse liver IRI. This translational study assesses MMP-9 and NETs in human OLTs and explores the role of MMP-9 in NET formation during liver IRI.

*Methods: Liver biopsies were collected from 34 human OLT recipients before abdominal closure and fixed in formalin. Mice were subject to 90min partial ischemia followed by 6h of reperfusion. Functionally mature neutrophils were isolated from bone marrow.

*Results: Infiltrating neutrophils were the main source of MMP-9 in post-reperfusion human biopsies. MMP-9 expression correlated significantly with worse recipient graft function and histological IRI score. Moreover, MMP-9 and NET-specific histone H3 hyper-citrullination (Cit-H3) co-localization evidenced the release of intra-hepatic NETs (rich in MMP-9) post-OLT. To evaluate the significance of NET-bound MMP-9, MMP-9KO mice and wild-type (WT) littermates were subject to hepatic IRI. NET formation measured by Ly-6G+/Cit-H3+ cells and Cit-H3 levels, was significantly depressed in MMP-9KO livers and prominently detected in WT livers. Impaired NET formation in MMP-9KOs was associated with improved histological preservation and significantly reduced inflammation and serum AST/ALT levels. A comparable inhibition of NET release was also achieved with administration of rAAV8-TIMP-1 vectors prior to IRI; rAAV8-TIMP-1 vectors produce liver-specific overexpression of TIMP-1, the major MMP-9 endogenous inhibitor. TIMP-1 overexpression impaired neutrophil infiltration, Ly-6G+/Cit-H3+ NET formation, and the biochemical and histological features of liver IRI. To eliminate the cell infiltration factor, isolated MMP-9KO and WT neutrophils were stimulated with PMA prior to NET assessment; PMA-stimulated WT neutrophils were also treated with conditioned media from TIMP-1 (iHCM-TIMP-1) or GFP (iHCM-GFP) overexpressing hepatocyte cultures. Both MMP-9 deficiency and the iHCM-TIMP-1 treatment markedly hampered NET release in-vitro.

*Conclusions: To our knowledge, this is the first study showing the presence of MMP-9 rich NETs in human-OLT recipients, and to establish a correlation between intra-graft MMP-9, neutrophil infiltration and the severity of human liver IRI. Moreover, our data indicate that inhibition of NET-bound MMP-9 activity impairs NET formation and protects livers from IRI.

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