Oxidation of HMGB1 causes attenuation of its pro-inflammatory activity and occurs during liver ischemia and reperfusion - PubMed (original) (raw)
Oxidation of HMGB1 causes attenuation of its pro-inflammatory activity and occurs during liver ischemia and reperfusion
Anding Liu et al. PLoS One. 2012.
Abstract
High mobility group box 1 (HMGB1) is a nuclear transcription factor. Once HMGB1 is released by damaged cells or activated immune cells, it acts as danger molecule and triggers the inflammatory signaling cascade. Currently, evidence is accumulating that posttranslational modifications such as oxidation may modulate the pro-inflammatory potential of danger signals. We hypothesized that oxidation of HMGB1 may reduce its pro-inflammatory potential and could take place during prolonged ischemia and upon reperfusion.Liver grafts were cold preserved for 24 h and flushed with saline in hourly intervals to collect the effluent. Liver grafts, cold-preserved for 6 h, were transplanted into syngeneic recipients to obtain serum and liver samples 24 h after initiation of reperfusion. Addition of the effluent to a macrophage culture induced the synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. The stimulatory activity of graft effluent was reduced after depletion of HMGB1 via immunoprecipitation. Oxidation of the effluent HMGB1 using H(2)O(2) attenuated its stimulatory activity as well. Liver transplantation of cold preserved grafts caused HMGB1 translocation and release as determined by immunohistochemistry and ELISA-assay, respectively. Using Western blot with non-reducing conditions revealed the presence of oxidized HMGB1 in liver samples obtained after 12 h and in effluent samples after 16 h of cold preservation as well as in liver and serum samples obtained 24 h after reperfusion.These observations confirm that post-translational oxidation of HMGB1 attenuates its pro-inflammatory activity. Oxidation of HMGB1 as induced during prolonged ischemia and by reoxygenation during reperfusion in vivo might also attenuate its pro-inflammatory activity. Our findings also call for future studies to investigate the mechanism of the inhibitory effect of oxidized HMGB1 on the pro-inflammatory potential.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Graft effluent stimulates the synthesis of inflammatory cytokines in peritoneal macrophages.
(A) Macrophages were stimulated with effluent (100 µl) and the supernatants were examined for levels of inflammatory cytokines (TNF-α and IL-6) by ELISA 6 h after stimulation. (B) Quantitative PCR was performed to determine the mRNA expression levels of TNF-α and IL-6. TNF-α and IL-6 concentration in the supernatants, as well as intracellular TNF-α and IL-6 mRNA levels were significantly increased after stimulation with effluent obtained after 8 h of cold storage. Data are shown as mean ± SD. *p<0.05, **p<0.001. E, effluent; TNF-α, tumor necrosis factor-alpha; IL, interleukin; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.
Figure 2. Pro-inflammatory activity of graft effluent is decreased after HMGB1 depletion.
HMGB1 concentration in effluent was detected by ELISA-assay (A) or western blot (B), respectively. Effluent HMGB1 was significantly increased as early as 8 h and then upregulated in a time-dependent manner up to 24 h (*p<0.001, **p<0.0001 vs 0 h). (C) The concentration of HMGB1 in effluent obtained after 24 h of cold storage was drastically reduced after HMGB1 depletion using immunoprecipitation. Lane 1–2 showed the HMGB1 in effluent. Lane 3–4 displayed the HMGB1 in flow-through after HMGB1 depletion. (D) Rat peritoneal macrophages were cultured in the presence of effluent (100 µl), or HMGB1-depleted effluent (flow-through; 100 µl) for 6 h. The inflammatory activity of effluent was markedly decreased after HMGB1 depletion when compared with complete effluent. The experiment was performed in triplicates with similar results. Data are shown as mean ± SD. *p<0.001, **p<0.0001 vs blank control; # p<0.001 vs HMGB1-depleted effluent. HMGB1, high mobility group box 1; TNF-α, tumor necrosis factor-alpha; IL, interleukin.
Figure 3. HMGB1 inflammatory activity is attenuated after oxidation in vitro.
(A) HMGB1 was firstly extracted from effluent obtained after 24 h of cold storage and then treated with H2O2 (50 µM) for 1 h on ice. Oxidized HMGB1 was separated on a non-reducing SDS-PAGE gel and detected by western blot with a polyclonal anti-HMGB1 antibody. (B) The gray value of bands was calculated by ImageJ. The relative amount of oxidized HMGB1 was expressed by oxidized HMGB1/total HMGB1. Macrophages were stimulated with effluent HMGB1 (0.5 µg/ml) or oxidized HMGB1 (effluent HMGB1 pretreated with H2O2; 0.5 µg/ml) for 6 h. TNF-α and IL-6 concentration in the supernatants (C), as well as intracellular TNF-α and IL-6 mRNA levels (D) were then determined. Effluent HMGB1 pretreated with H2O2 significantly attenuated its inflammatory activity. The experiment was performed in triplicates with similar results. Data are shown as mean ± SD. *p<0.001, **p<0.0001 vs blank control; # p<0.001 vs HMGB1+H2O2 group. HMGB1, high mobility group box 1; TNF-α, tumor necrosis factor-alpha; IL, interleukin; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; Ox, oxidized; Red, reduced.
Figure 4. HMGB1 inflammatory activity is attenuated after oxidation in vivo.
Recombinant HMGB1 (rHMGB1) was pretreated with 50 µM H2O2 for 1 h on ice. TNF-α mRNA and IL-6 mRNA expression levels were measured by quantitative PCR in liver, lung, heart, kidney and PBMC 6 h after injecting either unmodified (rHMGB1; 350 µg/rat) or oxidized HMGB1 (rHMGB1 pretreated with H2O2; 350 µg/rat) to naive rats. mRNA levels for TNF-α (A) and IL-6 (B) were significantly elevated in lung and PBMC after administration of rHMGB1. In contrast, rHMGB1 pretreated with H2O2 did not upregulate the expression of TNF-α and IL-6. Data are shown as mean ± SD. *p<0.001 vs HMGB1+H2O2 group. HMGB1, high mobility group box 1; TNF-α, tumor necrosis factor-alpha; IL, interleukin; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction.
Figure 5. Liver transplantation causes HMGB1 translocation and release.
(A) Immunohistochemical staining of HMGB1 demonstrated that HMGB1 was translocated from nucleus to cytoplasm in hepatocytes after liver transplantation (a–b, original magnification ×100; c–d, original magnification ×200). Representative images from six rats/group were selected. (B) Expression levels of HMGB1 protein in grafts were significantly increased after liver transplantation. The results were obtained using liver homogenates from six individual animals. (C) Serum concentrations of HMGB1, as measured by ELISA were significantly increased 24 h after liver transplantation. (D) Liver transplantation caused a severe hepatocellular injury as indicated by higher release of AST. (E) Serum HMGB1 levels positively correlated with AST levels in the serum. Data are shown as mean ± SD. *p<0.05, **p<0.001 vs normal control rats. HMGB1, high mobility group box 1; AST, aspartate aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LTx, liver transplantation; ELISA, enzyme-linked immunosorbent assay.
Figure 6. Oxidized HMGB1 is produced after prolonged cold ischemia and reperfusion in liver transplantation.
(A–B) Total hepatic proteins (10 µg) were separated on a non-reducing SDS-PAGE gel and HMGB1 protein was detected by western blot. The gray value of bands was calculated by ImageJ. The relative amount of oxidized HMGB1 was expressed by oxidized HMGB1/total HMGB1. Oxidized HMGB1 was produced in liver graft during cold storage (A) and 24 h after transplantation (B) Each lane represented a separate animal. The results were obtained using liver homogenates from six individual animals per each observation time point. Data are shown as mean ± SD. *p<0.001 vs 0 h. Effluent (C) or serum proteins (D) (10 µl) were separated on a non-reducing SDS-PAGE gel. The gray value of bands was calculated by ImageJ. The relative amount of oxidized HMGB1 was expressed by oxidized HMGB1/total HMGB1. Oxidized HMGB1 was found in effluent after 16 h cold ischemic storage of liver and in serum obtained 24 h after transplantation. The results shown are representative of six animals/group. Data are shown as mean ± SD. *p<0.001 vs normal control rats. HMGB1, high mobility group box 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ox, oxidized; Red, reduced; Con, normal control; LTx, liver transplantation.
References
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