A toll-like receptor 9 agonist sensitizes mice to mitochondrial dysfunction-induced hepatic apoptosis via the Fas/FasL pathway

  • Binbin Song
  • Shigeki Aoki
  • Cong Liu
  • Kousei ItoEmail author
Molecular Toxicology


Early hepatocyte death occurs in most liver injury cases and triggers liver inflammation, which in combination with other risk factors leads to the development of liver disease. However, the pathogenesis of early phase hepatocyte death remains poorly understood. Here, C57BL/6J mice were treated with the hepatotoxic drug flucloxacillin (FLUX) and the toll-like receptor 9 agonist CpG oligodeoxynucleotide (ODN) to reproduce the early phase of drug-induced hepatotoxicity and investigate its pathogenesis. C57BL/6J mice were treated with FLUX (100 mg/kg, gavage) alone or in combination with ODN (40 μg/mouse, intraperitoneally). Plasma alanine aminotransferase (ALT) level was measured as a marker of hepatotoxicity. FLUX or ODN alone was insufficient to induce ALT elevation, whereas combination treatment with FLUX and ODN increased ALT levels 24 h after FLUX treatment and upregulated Fas ligand in natural killer T (NKT) cells and Fas in hepatocytes. FLUX induced mitochondrial permeability transition (MPT), and pretreatment with ODN sensitized mitochondria to FLUX-induced MPT. The increase in ALT levels induced by ODN and FLUX co-treatment was suppressed in Fas ligand (gld/gld)-deficient mice and in mice deficient in a component of MPT pore opening (cyclophilin D-knockout mice). These results suggested that ODN activated the Fas/Fas ligand-mediated pathway in NKT cells and hepatocytes, which may predispose to FLUX-induced mitochondrial dysfunction and lead to early phase hepatocyte apoptosis. Taken together, these findings elucidate a potentially novel mechanism underlying drug-induced early phase hepatocyte death related to the Fas/Fas ligand death receptor pathway and mitochondrial dysfunction.


Flucloxacillin NKT Liver injury Innate immunity CpG-ODN 



Drug-induced liver injury




Human leukocyte antigen


Alanine aminotransferase


Aspartate transaminase


Total bilirubin


Natural killer T


Natural killer


Toll-like receptor 9


High-mobility group box-1


CpG oligodeoxynucleotide


Intraperitoneal injection


Fas ligand


Liver mononuclear cell


T cell receptor


Mitochondrial permeability transition


Fluorescence-activated cell sorting


TNF-related apoptosis-inducing ligand



Cyp D

Cyclophilin D


Cyclosporin A


Reactive oxygen species



This work was supported by the Japan Society for the Promotion of Science [JSPS KAKENHI, Grant No. 15H04661 to K.I.].

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interests in association with this manuscript.

Supplementary material

204_2019_2454_MOESM1_ESM.pptx (270 kb)
Supplementary material 1 (PPTX 270 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Binbin Song
    • 1
  • Shigeki Aoki
    • 1
  • Cong Liu
    • 1
  • Kousei Ito
    • 1
    Email author
  1. 1.Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical SciencesChiba UniversityChibaJapan

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