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 and Allan Tsung1 1Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America; 2Center for Biologic Imaging, Department of Cell Biology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR re- sponsible 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 con- trol 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 at- tenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepato- cytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an im- portant 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. Online address: http://www.molmed.org doi: 10.2119/molmed.2014.00076 INTRODUCTION been extensively studied and is associ- interferon regulatory factor 3 (IRF3), Hepatic ischemia and reperfusion ated with the activation of pattern recog- mitogen-activated protein kinase (I/R) injury has potentially negative clin- nition receptors such as toll-like receptor (MAPK) and inflammatory cytokines ical implications for patients undergoing 4 (TLR4) by endogenous ligands (2–5). (3,5). Ultimately, activation of TLR4 by liver resection, transplantation and TLR4-deficient mice have decreased lev- endogenous and exogenous ligands trauma. It is characterized by direct cel- els of inflammatory cytokines and are leads to hepatocellular apoptosis and lular damage as a result of the ischemic significantly protected after liver I/R in- necrosis, liver failure and organ injury. insult, as well as delayed dysfunction jury (5–7). It is well known that TLR4 Interest in TLR4 antagonism originated and damage that results from the activa- plays a proinflammatory role in liver I/R from sepsis studies, since TLR4 is known tion of inflammatory pathways (1). The through the activation of myeloid differ- to bind to the gram-negative lipopolysac- mechanism of this injury response has entiation primary response 88 (Myd88), charide (LPS) or endotoxin (8). Eritoran tetrasodium (E5564; Eisai, Andover, MA, USA) is a synthetic lipid A analog of Rhodobacter sphaeroides that has been de- Address correspondence to Allan Tsung, 3459 Fifth Avenue UPMC Montefiore, 7 South Pitts- signed to antagonize the effects of LPS burgh, PA 15213-2582. Phone: 412-692-2001; Fax: 412-692-2002; E-mail: [email protected]. through TLR4 (8–11). Eritoran does not Submitted April 17, 2014; Accepted for publication October 20, 2014; Epub directly interact with TLR4, but competes (www.molmed.org) ahead of print October 31, 2014. with LPS for binding to the hydrophobic pocket of the MD2 portion of the TLR4 re- ceptor complex (9). It has been shown that eritoran binding to the TLR4/MD2 com- MOL MED 20:639-648, 2014 | MCDONALD ET AL. | 639 ERITORAN AMELIORATES LIVER ISCHEMIA AND REPERFUSION INJURY plex blocks the activation of nuclear factor mediated inflammatory responses. Over- Isolation, Culture and Treatment of (NF)-κB and production of inflammatory all, our investigations reveal a dominant Hepatocytes and RAW 264.7 Cells mediators such as tumor necrosis factor role for the TLR4 antagonist, eritoran, in In Vitro (TNF)-α and interleukin (IL)-6, both in ameliorating liver I/R injury. Murine macrophages of the RAW 264.7 vivo and in vitro, in response to LPS cell line (donated from Matthew Rosen- (9–15). Eritoran was investigated as a pos- MATERIALS AND METHODS gart’s laboratory, University of Pittsburgh) sible treatment of sepsis. Unfortunately, were cultured in Dulbecco’s modified the large phase III multicenter ACCESS Materials Eagle medium (DMEM) supplemented Randomized Trial failed to show a differ- Eritoran tetrasodium (E5564) was ob- with 7.5 mL 4-(2-hydroxyethyl)-1- ence in the 28-d all-cause mortality in sep- tained from Eisai, Inc., and prepared in piperazineethanesulfonic acid (HEPES), tic patients who received eritoran com- phosphate-buffered saline (PBS). A dose of 5 mL L-glutamine, 5 mL penicillin- pared with placebo (16). Because TLR4 is 5 mg/kg eritoran was used in vivo (dosage streptomycin and 25 mL fetal bovine also involved in sterile inflammation, eri- adopted from Shimamoto et al. [19]) and serum. RAW 264.7 cells were scraped toran has been tested in models of nonin- 8 ng/mL for in vitro experiments. and resuspended at a concentration of 1 × fectious pathobiology and has been 106 cells/mL per well of a six-well plate. shown to be beneficial (17–21). We hy- Animals Cells were allowed to adhere overnight. pothesized that TLR4 antagonists, such as Male C57BL/6 mice, weighing 20–30 g, Before each experiment, cells were eritoran, would provide a similar protec- were purchased from The Jackson Labora- washed with PBS, and new media were tion after warm hepatic I/R, which is tory (Bar Harbor, ME, USA) and used for placed into the wells. RAW 264.7 cells heavily TLR4 mediated. experiments at the age of 8–12 wks. Hous- were incubated with LPS (1 μg/mL), eri- High-mobility group box protein B1 ing conditions and access to food and toran (8 ng/mL) and/or recombinant (HMGB1) is an important damage- water were the same for all mice. All HMGB1 in the disulfide form (1 μg/mL, associated molecular pattern (DAMP) animal protocols were approved by the donated from Timothy Billiar’s laboratory, molecule found in the nucleus of most Animal Care and Use Committee of the University of Pittsburgh) for 18 h. All eukaryotic cells, displays cytokine like University of Pittsburgh. Experimental treatments were performed in duplicate. activity and mediates sterile inflamma- protocols were followed in strict adher- After the incubation period, the media tion by binding to MD2 in the TLR4 re- ence to the regulations set forth by the Na- and cells were collected for analysis. ceptor complex (22–25). During liver I/R, tional Institutes of Health (NIH) guide- Hepatocytes were cultured in Wil- HMGB1 is released passively from necrotic lines for the use of laboratory animals (27). liams E. Medium containing 10% calf cells and can be actively secreted from serum, 15 mmol/L HEPES, 2 mmol/L multiple cell types (22,26). Once bound to Liver I/R L-glutamine and 100 U/mL penicillin TLR4, HMGB1 activates an intracellular A hepatic warm ischemia and reperfu- and streptomycin. Hepatocytes were iso- signaling cascade that involves recruiting sion model was used as previously de- lated from mice by an in situ collagenase MyD88, IL-1 receptor–associated kinase scribed (28). In brief, a midline laparo- perfusion technique (type VI, Worthing- (IRAK-1), activating MAPK pathways tomy incision was made, the blood ton), as previously described (29). Hepa- and NF-κB translocation (22,26). HMGB1, supply to the left and median lobes of the tocytes were separated from the non- like LPS, promotes inflammation, cell liver were occluded with a microvascular parenchymal cells by two cycles of death and organ injury. We have previ- clamp for 60 min and then 1 or 6 h of differential centrifugation and further ously shown that HMGB1 is a key medi- reperfusion was initiated once the clamp purified over a 30% Percoll gradient ator of liver I/R injury via its interaction was removed. Sham animals had anes- (Sigma-Aldrich, St. Louis, MO, USA). with TLR4 (22). thesia and a laparotomy incision with ex- Cells were allowed to adhere overnight. In our study, we sought to determine posure of the portal triad, without clamp- Before each experiment, cells were whether eritoran blocks TLR4 signaling, ing. After 6 h of reperfusion, the mice washed with PBS and new media placed subsequent inflammatory cascades and were killed. The ischemic portions of the into the wells. Hepatocytes were exposed end-organ damage. We hypothesized liver tissue were collected for Western to hypoxia as previously described (1). In that pharmacological inhibition of TLR4 blot, polymerase chain reaction (PCR), brief, cells were placed in the hypoxia with eritoran would antagonize the in- histological, and immunofluorescent chamber for 18 h, whereas normoxic cells teraction between HMGB1 and TLR4, analysis. Of note, mice received either eri- remained in the incubator. All treatments since HMGB1 binding to TLR4 con- toran (5 mg/kg) or vehicle control in- were performed in duplicate. After the tributes significantly to the injury re- traperitoneally 30 min before ischemia incubation period, the media and cells sponse associated with liver I/R. We and recombinant HMGB1 (25 μL/ mouse) were collected for analysis. found that eritoran is protective in liver injected intraperitoneally before the initi- Of note, the in vitro activity of eritoran I/R through inhibition of HMGB1- ation of reperfusion.