*Purpose: Ischemia-reperfusion injury (IRI) during liver transplantation (LT) is a pro-inflammatory response linked to poorer outcomes, including increased alloimmunity, post-LT. We previously showed that post-reperfusion portal vein blood (liver flush; LF) from IRI+ LT patients activates TLR4 and induces pro-inflammatory macrophages and that disulfide HMGB1 (diS HMGB1), whose receptors include TLR4, is increased in IRI+ patients. This study aimed to determine the contribution of diS HMGB1-TLR4 signaling to pro-inflammatory macrophage polarization and function and to the previously-observed IRI+ polarized macrophage phenotype.

*Methods: Third-party monocytes were exposed to diS HMGB1 or LPS with or without TAK-242, a TLR4 inhibitor, for 5 days. Surface markers (CD66a, CD86, HLA-DR, PD-L1, TIM-3) and ROS production were analyzed by flow cytometry. Secreted cytokines were analyzed via 38-plex Luminex. Surface marker expression was compared to phenotypes induced by LF from LT patients during previous studies.

*Results: diS HMGB1 mirrored the IRI+ LF macrophage phenotype in 4/5 markers (high HLA-DR, CD86; low PD-L1, CD66a). diS HMGB1 trended with LPS for 1/5 markers (CD66a). High TIM-3 induced by diS HMGB1 mirrored TIM-3 expression induced by IRI- LF. TAK-242 had a dose-dependent effect on 4/5 markers (all but CD66a) for the LPS phenotype but only 1/5 markers (HLA-DR) for the diS HMGB1 phenotype. diS HMGB1 induced higher ROS production than LPS; TAK-242 only affected LPS-induced ROS production. TAK-242 also had a dose-dependent inhibition on 35 LPS-induced cytokines, but only inhibited 4 diS HMGB1-induced cytokines (IL-6, FGF2, MIP1a, and MIP1b).

*Conclusions: diS HMGB1 induces a pro-inflammatory macrophage phenotype with increased antigen presentation (high CD86 and HLA-DR), decreased T-cell inhibition (low PD-L1 and CD66a), high ROS production, and pro-inflammatory cytokine secretion capabilities. However, only HLA-DR expression and IL-6, FGF2, MIP1a, and MIP1b secretion were affected by inhibition of TLR4 signaling. This data suggests that 1) diS HMGB1 contributes to the post-LT immune response in IRI+ patients, 2) these macrophages can contribute to inflammation and alloimmunity post-LT, and 3) the observed diS HMGB1 phenotype is not completely dependent on TLR4. This study reveals that diS HMGB1 released during IRI can polarize macrophages to become pro-inflammatory and contribute post-LT inflammation in IRI+ patients.

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