Inhibition of HMGB1 or TLR4 deficiency can protect mice from ischemia-reperfusion-caused liver injury.30 HMGB1 released from acetaminophen-induced necrotic hepatocytes has been demonstrated to be able to induce macrophage activation and cytokine release.28,

31 However, the cellular and molecular immune mechanisms of HMGB1 in liver injury, particularly tissue damage-induced pathogenic inflammation, remain elusive. How the specific innate immune receptor for HMGB1 activates macrophages and induces proinflammatory cytokines and how these cytokines prime the subsequent innate immune response and mediate inflammation are completely unclear. In this study, we demonstrated that the interaction between macrophages and γδ T cells plays an important role in acetaminophen-induced liver inflammation.

Serum HMGB1 released from necrotic hepatocytes Midostaurin molecular weight stimulates the production CCI-779 purchase of IL-23 by hepatic macrophages in a TLR-4-dependent manner, and IL-23 aids in the generation of IL-17A-producing γδ T cells in the liver. IL-17A secreted by γδ T cells then recruits hepatic neutrophils. Thus, the HMGB-TLR4-IL-23-IL17A axis between macrophages and γδ T cells contributes to the accumulation of neutrophils and liver inflammation. ALT, alanine aminotransferase; DAMPs, damage-associated molecular pattern molecules; DMSO, dimethyl sulfoxide; H&E, hematoxylin/eosin; HMGB1, high-mobility group box 1; IFN, interferon; NK, natural killer; NKT, natural killer T; PMA, phorbol-12-myristate-13-acetate; TLR, Toll-like receptor. C57BL/6 males aged 6-8 weeks were purchased from the Shanghai Laboratory Animal Center, Chinese Academy Sciences (Shanghai, China). T cell receptor (TCR)δ−/−mice, IL-23p40−/− mice, and TLR4−/− mice

were kindly provided by professors Z.N. Yin (Nankai University), Z.X. Lian (University of Science and Technology of China), and S.B. Su (Sun Yat-Sen University), respectively. All mice were housed in microisolator cages under humidity- and temperature-controlled specific pathogen-free conditions in the animal facility NADPH-cytochrome-c2 reductase of the School of Life of the University of Science and Technology of China. The mice were maintained on an irradiated sterile diet and provided autoclaved water. All experiments were performed according to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD). Fresh acetaminophen (Sigma-Aldrich, USA) solution was prepared for each experiment by dissolving acetaminophen to a concentration of 10 mg/mL in phosphate-buffered saline (PBS) warmed to 40°C. Mice were fasted for 16 hours and then injected intraperitoneally with PBS or acetaminophen at 400 mg/kg (body weight). At the indicated timepoints, sera were collected and stored at −20°C for measuring alanine aminotransferase (ALT), total bilirubin, and cytokines. At the end of the experiment, the mice were anesthetized, bled, and euthanized.