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. 2015 Mar 13;20(1):639-48.
doi: 10.2119/molmed.2014.00076.

Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling

Kerry-Ann Mcdonald  1 , Hai Huang  1 , Samer Tohme  1 , Patricia Loughran  2 , Kimberly Ferrero  1 , Timothy Billiar  1 , Allan Tsung  1
Affiliations

Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling

Kerry-Ann Mcdonald et al. Mol Med. .

Abstract

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1's release from hypoxic hepatocytes in vitro and thereby weakened HMGB1's activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.

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Figures

Figure 1
Figure 1
Pharmacological inhibition of TLR4 with eritoran protects against liver I/R injury. (A) Sham or I/R-treated mice were given eritoran (5 mg/kg body weight) or vehicle control intraperitoneally 30 min before ischemia. Data represent the mean ± SEM (n ≥ 6 mice per group). Gray, eritoran; black, control. *P < 0.05, by one-way ANOVA. (B) Quantification of necrotic hepatocytes in H&E-stained liver sections from sham, control and eritoran-treated animals 6 h after reperfusion. The graph is representative of liver sections from three or more mice per group. *P < 0.05, by one-way ANOVA.
Figure 2
Figure 2
Eritoran suppresses TLR4 signaling pathways and mediates inflammatory signaling in liver I/R. (A) Myd88 and the phosphorylation of IRAK-1 were determined by Western blot and quantitative densitometry analysis of the protein expressions in eritoran-treated mice and control mice that underwent ischemia, and 1 h of reperfusion was determined. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. The blots shown are representative of three experiments with similar results. *P < 0.05 versus control, by Student t test. (B) MAPK activation and phosphorylation at serine 536 of the p65 subunit of NF-κB were determined by Western blot and quantitative densitometry analysis of the protein expressions in eritoran-treated mice and control mice that underwent ischemia and 1 h of reperfusion. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. The blots shown are representative of three experiments with similar results. *P < 0.05 versus control, by Student t test.
Figure 3
Figure 3
Eritoran decreases I/R-induced inflammatory cytokine gene expression. Serum levels of TNF-α and IL-6 obtained from control and eritoran-treated mice at 6 h after reperfusion were measured by RT-PCR. Results are expressed as a relative increase of mRNA expression compared with sham-treated animals. Data represent the mean ± SEM (n ≥ 3 mice per group). *P < 0.05.
Figure 4
Figure 4
Treatment with eritoran attenuates HMGB1-mediated liver I/R injury. (A) Sham, control, eritoran-treated, control with recombinant HMGB1, and eritoran with recombinant HMGB1-treated mice underwent 6 h of reperfusion, and serum ALT levels were analyzed. Eritoran (5 mg/kg body weight) administered intraperitoneally 30 min before ischemia and recombinant HMGB1 (25 μL/mouse) administered intraperitoneally immediately before reperfusion. Data represent the mean ± SEM (n ≥ 3 mice per group). *P < 0.05, by one-way ANOVA. (B) Quantification of necrotic hepatocytes in H&E-stained liver tissue. The graph is representative of liver sections from three or more mice per group. *P < 0.05, by one-way ANOVA. (C) Hepatic TNF-α and IL-6 ELISA. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05, by one-way ANOVA.
Figure 5
Figure 5
Eritoran treatment suppresses HMGB1 release in vivo. (A) Nuclear and cytoplasmic HMGB1 levels were determined by Western blot and quantitative densitometry analysis of the protein expressions in eritoran-treated mice and control mice that underwent ischemia and 6 h of reperfusion determined. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. The blots shown are representative of two experiments with similar results. (B) Serum HMGB1 ELISA after 6 h of reperfusion. Data are representative of two experiments with similar results. Gray, eritoran; white, control. *P < 0.05 when compared against control by one-way ANOVA. (C) Immunofluorescent stain of HMGB1 from sections of sham liver and liver 6 h after I/R in placebo control, and eritoran-treated mice (magnification 40×). Images are representative of liver sections from two mice per group. Red, HMGB1; blue, nuclei; green, F-actin. (D) TLR4 wild-type and knockout mice were given eritoran (5 mg/kg body weight) or vehicle control intraperitoneally 30 min before ischemia. HMGB1 levels in serum were collected 6 h after reperfusion and quantified using ELISA. Data represent the mean ± SEM (n ≥ 6 mice per group). Gray, eritoran; white, control. *P < 0.05, by one-way ANOVA.
Figure 6
Figure 6
Eritoran decreases HMGB1 release by hepatocytes and blocks the inflammatory response of RAW 264.7 cells by HMGB1 in vitro. (A) Eritoran-treated (8 ng/mL) or control hepatocytes were cultured under normoxia or 18 h hypoxia. The media were then analyzed using ELISA for HMGB1. White, control; gray, eritoran. *P < 0.05 when compared against control by one-way ANOVA. (B) Immunofluorescent staining depicting the translocation of HMGB1 in cultured hepatocytes from eritoran-treated (8 ng/mL) or control hepatocytes that were stimulated with either 18 h hypoxia or normoxia was visualized and observed under confocal microscope (magnification ×120). Green, actin; blue, nuclei; red, HMGB1. (C) Hepatocytes were stimulated with LPS (1 μg/mL), recombinant HMGB1 (1 μg/mL), or eritoran (8 ng/mL) in the above combinations for a total of 18 h of stimulation. The media were then analyzed using ELISA for TNF-α and IL-6. Data are representative of two experiments with similar results. White, PBS; gray, eritoran. *P < 0.05 versus control, by one-way ANOVA.
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