Figure 2. Redox state of HMGB1+ LF is disulfide in IRI+ and all-thiol in IRI- OLT patients.

Patient LF samples obtained just after reperfusion through the donor allograft were assayed by immunoblotting to determine the ratio of disulfide to all-thiol HMGB1 for each patient. (A) Representative western blot showing 28kD bands that correspond to all-thiol HMGB1 and slightly lower bands at ~25kD corresponding to disulfide-HMGB1 for samples from either IRI+ (+) or IRI- (−) patients. (B) Amount of all-thiol and disulfide-HMGB1 was extrapolated by dividing the total HMGB1 content per sample as determined by ELISA in Fig. 1 by the ratio of HMGB1 redox forms determined by Western blotting in 3A. IRI+, n=27; IRI-, n=31 (C) Disulfide-HMGB1 levels by IRI severity. Data shown as mean±SD for each group. 31 IRI-, 27 IRI+; IRI 0 (n=4), 1 (n=27), 2 (n=18), 3 (n=5), 4 (n=4) (D) HMGB1+ LF from 40 OLT patients (20 IRI-, 20 IRI+) was used to stimulate monocytes from healthy third-party donors for 8 hours (as determined in Fig. S5A following pre-treatment for one hour with either media or anti-TLR4 mAb, and supernatant was assayed for secreted TNFα. (E) Comparison of TLR4 activation, disulfide-HMGB1 levels, and TNFα secretion by monocytes as determined by the common cohort of n=21; 9 IRI-, 12 IRI+ from HEK assay, disulfide-HMGB1 levels and TNFα production from macrophages. Shown is a heat map in which the rows represent patients, the columns represent the groups compared, and the colors represent normalized median values per assay (blue = low, red = high). The rows and columns are ordered based on the results of an unsupervised hierarchical clustering, with dendograms on the left side showing groups of IRI- (salmon) or IRI+ (aqua) OLT recipients with or without functional activation of HEK-Blue hTLR4 cells by their LF. *P < 0.05; **P < 0.01; ****P < 0.0001 by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test was used to determine differences amongst HMGB1 redox state and/or IRI status.