Background: Ischemia-reperfusion injury (IRI) is a major risk factor in the development of acute and chronic graft rejection in orthotopic liver transplantation (OLT); however, no clinical therapeutics or patient-specific diagnostics are currently available. Murine studies have shown the damage-associated molecular pattern (DAMP) high-mobility group box 1 (HMGB1) is released during liver IRI and binds to the pattern recognition receptor (PRR) TLR4 on parenchymal and/or infiltrating innate immune cells, inducing pro-inflammatory cytokine production; however, this has not been investigated in human OLT. Aim: We hypothesize TLR4 is activated on innate cells via HMGB1 released during IRI signaling in human OLT patients. Methods/Results: HMGB1 was measured in plasma samples obtained pre-, intra- and post-transplant from OLT recipients (n=51) and was significantly elevated in initial liver flush (LF) obtained after reperfusion in all patients. HMGB1+ LF from patients identified as IRI+ by histopathology (n=31) induced increased TNF-α production in PBMC-derived monocytes and increased TLR4 activation in HEK-Blue^TM hTLR4 transfected cells compared to IRI- (n=35). However, HMGB1 presence alone did not predict IRI. Interestingly, we found that LF from IRI+ patients did not require canonical TLR4 cofactors CD14 or MD2 to activate hTLR4 cells, whereas IRI- LF samples required CD14 and purified HMGB1 required both for cytokine induction that was unaffected by the addition or removal of LPS. Exploring this further, we identified two key explanations for the discrepancy: 1) patient LF samples (n=28) contained varying levels of one or both redox forms of HMGB1, with TNF-α production and TLR4 activation directly correlating with ratio of oxidized (disulfide) to reduced (all-thiol) HMGB1, which was increased >3-fold in more IRI+ patients (50%) than IRI- (36%) and 2) patient LF samples had varying abilities to activate other PRRs tested thus far including TLR3, TLR5 and NOD1 which might recognize additional DAMPs in the LF to promote monocyte activation. Conclusion: Innate immune signaling induced by IRI provides many potential therapeutic targets for improving OLT outcome, and patient-specific screening of DAMPs/PRRs during OLT could be a novel diagnostic strategy for identifying IRI risk.

CITATION INFORMATION: Sosa R, Zarrinpar A, Rossetti M, Lassman C, Naini B, Datta N, Busuttil R, Gjertson D, Kupiec-Weglinski J, Reed E. Ischemia-Reperfusion Injury-Induced Innate Immune Signaling in Human Orthotopic Liver Transplantation. Am J Transplant. 2017;17 (suppl 3).

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