For neutralization of endogenous IL-17A or IL-23, 0.2 mg of neutralizing rabbit antimouse IL-17A (Clone TC11-18H10.1, BioLegend, USA) or neutralizing rabbit antimouse IL-123p19 (Clone G23-8, eBioscience, USA) was administered intravenously at the time of acetaminophen treatment. Control rabbit IgG was used as an isotype control. For deletion of γδ T cells, NK1.1+ cells, or CD4+ cells, the mice were injected intravenously with 0.5 mg of an anti-γδ TCR mAb (clone TIB-207, ATCC, Manassas, VA), anti-NK1.1

mAb (clone HB191, ATCC), or anti-CD4 mAb (clone TIB-207, ATCC), respectively, 48 hours before acetaminophen treatment. For inhibition of macrophages, mice were injected intravenously with GdCl3 at 20 mg/kg (body weight, Sigma-Aldrich) GSI-IX research buy at 24 hours before acetaminophen treatment. For inhibition of HMGB1, mice were treated with glycyrrhizin (TCI, Shanghai, China) at 5 mg/mouse 1 hour before acetaminophen

treatment. Acute liver injury was evaluated by serum levels of ALT and total bilirubin. They were measured using diagnostic kits (Rongsheng, Shanghai, China). Total RNA was isolated from frozen liver tissue using total RNA purification solutions (Invitrogen, USA). Two μg of total RNA was reverse-transcribed at 25°C for 15 minutes, 42°C for 50 minutes, and 70°C for 10 minutes using reverse transcription kits (Sangon Biotech, Shanghai, China). Complementary DNA (cDNA) fragments were amplified using the following gene-specific primers: IL-17A (sense 5-GCTCCAGA AGGCCCTCAG-3; antisense 5-CTTTCCCTCGCA oxyclozanide Selleckchem Y 27632 TTGACA-3); IL-23p19 (sense 5-AGCGGGACATAT GAATCTACTAAGAGA-3; antisense 5-TCCTAGT AGG GAGGTGTGAAGTTG-3); IL-23p40 (sense 5-TCCACCAAACTCCCCAGACA-3; antisense 5-CTG TGCATGCTCTTTGGTTGAT-3); and β-actin (sense 5-TGGAATCCTGTGGCATCCATGAAA-3; antisense 5-TAAAACGCAGCTCAGTAACAGTCC-3).

Quantitative RT-PCR was performed to measure the messenger RNA (mRNA) expression of IL-17A, IL-23p19, and IL-23p40 using commercially available SYBR Premix Ex Taq (TaKaRa Biotechnology, Dalian, China) and specific primers in a reaction with an optimal number of cycles at 95°C for 10 seconds, then 60°C for 30 seconds in a Corbett Rotor-Gene 3000 real-time PCR system (Corbett Research). The gene expression levels were calculated relative to the housekeeping gene β-actin. Liver specimens from mice exposed to different treatments were fixed in 4% paraformaldehyde, dehydrated with a graded series of alcohol, and embedded in paraffin. Six-micron tissue sections were prepared and stained with H&E. At each indicated timepoint, sera were harvested for measurement of IL-17A, IL-23, IL-23p40, and HMGB1. Hepatic mononuclear cells were stimulated in vitro with IL-1β (50 ng/mL, PeproTech, USA), IL-23 (50 ng/mL, Miltenyi Biotec, USA) or the combination for 48 hours.