*Purpose: Ischemia-reperfusion injury (IRI) is a major risk factor of acute and chronic graft rejection in orthotopic liver transplantation (OLT) involving a complex interplay between the innate and adaptive immune systems. We recently showed that intraoperative portal blood samples obtained from IRI+ OLT patients were significantly better than IRI- blood at activating the pattern recognition receptors TLR4 and TLR9, which can both mediate a switch from innate to adaptive responses. The danger-associated molecular pattern HMGB1 has multiple high affinity binding partners, including TLR4 and 9, based on its redox state which has been shown to dramatically alter its immune function. Therefore, we investigated the role of HMGB1 in human OLT-IRI.

*Methods: Portal blood samples obtained immediately post-reperfusion from OLT recipients were analyzed for HMGB1 content and redox state by ELISA and Western blot. The effects of HMGB1+ portal plasma on phenotype and function of healthy third-party PBMC-derived monocytes were evaluated by flow cytometry and ELISA. Multicolor confocal immunofluorescence of 100um-thick tissue sections was used to examine HMGB1 localization in biopsies from OLT patients obtained pre- and post-reperfusion.

*Results: HMGB1 was similarly present in its fully reduced, all-thiol form in the portal blood plasma of both IRI- and IRI+ patients, but was higher in IRI+ patients in its partially oxidized, disulfide form, and was capable of inducing TNFa production in monocytes. Monocytes stimulated with IRI+ plasma increased expression of antigen presentation molecules HLA-DR, CD80 and CD86, whereas those stimulated with HMGB1+ IRI- patient blood downregulated HLA-DR and CD86 and retained low expression of CD80. Three-dimensional subcellular reconstruction of multiparameter immunofluorescence confocal images demonstrated IRI+ biopsies contain mostly chemotaxis-inducing all-thiol HMGB1 pre-transplant, and a large inflammatory infiltrate consisting of disulfide-HMGB1-secreting monocytes/macrophages post-transplant, whereas IRI- biopsies show predominantly non-immunogenic sulfonyl-HMGB1 from ischemic parenchymal cells.

*Conclusions: Together these data suggest a role for HMGB1 redox states in IRI and point to its potential value in recipient matching and/or as a therapeutic intervention during transport for improving OLT outcome.

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