Authors
C. MINSART (1), C. LIEFFERINCKX (1), S. RORIVE (2), A. LEMMERS (3), C. DRESSEN (4), E. QUERTINMONT (5), E. TRÉPO (6), C. MORENO (6), J. DEVIÈRE (7), S. GORIELY (8), I. LECLERCQ (9), R. MOREAU (10), T. GUSTOT (7) / [1] Université Libre de Bruxelles Faculté de Médecine, Brussels, Belgium, Laboratory of Experimental Gastroenterology , [2] Erasme Hospital, Anderlecht, Belgium, Department of Pathology , [3] Erasme Hospital, Anderlecht, Belgium, Department of Gastroenterology, HepatoPancreatology and Digestive Oncology, [4] Université Libre de Bruxelles Faculté de Médecine, Brussels, Belgium, Laboratory of Physiology and Pharmacology, [5] Université Libre de Bruxelles Faculté de Médecine, Brussels, Belgium, Laboratory of Experimental Gastroenterology, [6] Erasme Hospital, Brussels, Belgium, Department of Gastroenterology, HepatoPancreatology and Digestive Oncology, [7] Erasme Hospital, Brussels, Belgium, Department of Gastroenterology, HepatoPancreatology and Digestive Oncology , [8] Université Libre de Bruxelles Faculté de Médecine, Brussels, Belgium, Institute for Medical Immunology, [9] Université catholique de Louvain (UCL), Brussels, Belgium, Laboratory of Hepato-Gastroenterology, [10] Hôpital Beaujon, Service d'Hépatologie, INSERM U-481, Clichy, France, Centre de recherche sur l'Inflammation (CRI)
Introduction
Voluntary or accidental acetaminophen (N-acetyl-p-aminophenol, APAP) overdose can induce a hyperacute form of liver failure potentially responsible for multiple organ failures and death. The mechanisms of this hepatotoxicity are incompletely understood. In "normal" conditions, high-mobilty group box 1 (HMGB1) is a small nuclear protein who binds DNA and regulate many transcriptional events by modulating transcription factor-DNA interactions. In case of overdose, APAP induces hepatocytes necrosis and thus HMGB1 is released. Extracellular HMGB1 acts like damage-associated molecular pattern (DAMPs) and contributes to APAP-induced liver injury but the mechanisms associated with this activity are incompletely understood or controversial.
Aim
The aim of the present study was to investigate the early effects of HMGB1 in APAP-induced liver injury, its direct effect on hepatocytes and its role in the propagation of necrosis process.
Methods
APAP hepatotoxicity was induced in vivo by intraperitoneal injection in C57Bl/6 mice and in vitro on cultured HepaRG cells. HMGB1 was quantified by ELISA or immuno-staining. Cell death was determined by MTT, ALT, LDH and caspase-3 assays. Glycyrrhizin (GL) and ethyly pyruvate (EP) was used to inhibit HMGB1. Liposomal clodronate was administrated to mice to deplete Kupffer cells (KC). Expression of HMGB1 receptors was assessed by RT-PCR and flow cytometry. Expression of proteins who participate to necroptotic process was demonstrated by western blot. Dabrafenib and necrostatin-1was used to inhibit receptor-interacting protein (RIP)3 and RIP1 respectively.
Results
We confirmed that, in APAP-challenged mice, inhibition of HMGB1 by glycyrrhizin improved survival and reduced further HMGB1 release. Depletion of Kupffer cells by liposomal clodronate in mice exacerbated APAP-induced hepatocyte necrosis and HMGB1 release suggesting that HMGB1 did not act through Kupffer cells activation. Based on these results, we hypothesized that a feed-forward circuit between HMGB1 and hepatocytes exist. In vitro, addition of APAP on cultured HepaRG cells induced cell necrosis characterized by lactate dehydrogenase release without caspase-3 activation, and HMGB1 release. Inhibition of HMGB1 by glycyrrhizin or ethyl pyruvate reduced APAP-induced HepaRG cell necrosis and further HMGB1 release. Exposure of HepaRG cells to recombinant human HMGB1 (rhHMGB1) resulted in cell death, supporting the hypothesis that HMGB1 acts directly on hepatocytes. Inhibition of RIPK3 by dabrafenib prevented APAP- and rhHMGB1-induced HepaRG cell death but inhibition of RIPK1 by Necrostatin-1 did not, suggesting the contribution of necroptosis. Moreover, inhibition of TRIF by Pepinh- TRIF reduced rhHMGB1-induced HepaRG cell death and Trif mutant mice were partially protected against APAP-induced liver injury.
Conclusions
In conclusion, these data support the hypothesis that HMGB1 contributes to the amplification of APAP-induced liver injury through feed-forward circuit with hepatocytes. This pathway seems to be independent of resident macrophages and, at least partially, dependent of TRIF/RIPK3 necrosis pathway resulting in the propagation of the liver injury.
