Hepatitis B virus HBx protein impairs liver regeneration through enhanced expression of IL-6 in transgenic mice Ivan Quétier 1,2,3 , Nicolas Brezillon 1,2,3 , Marion Duriez 1,2,3 , Hélène Massinet 1,2,3 , Eric Giang 1,2,3 , James Ahodantin 1,2,3 , Céline Lamant 1,2,3 , Marie-Noëlle Brunelle 1,2,3 , Patrick Soussan 1,2,3,4,5 , Dina Kremsdorf 1,2,3,⇑ 1 Inserm, U845, Pathogenèse des hépatites virales B et immunothérapie, Paris, France; 2 Université Paris Descartes, Sorbonne Paris-Cité, Faculté de Médecine Necker, Paris, France; 3 Institut Pasteur, Département de Virologie, Paris, France; 4 Service de Virologie, Hôpital Tenon, Paris, France; 5 Université Pierre et Marie Curie, Paris, France Background & Aims: Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatec- tomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. Methods: PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutral- izing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. Results: Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phos- phorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neu- tralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. Conclusions: HBx is able to induce delayed hepatocyte prolifera- tion after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microen- vironment, HBx may participate in cell cycle deregulation and progression of liver disease. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction The liver has a unique capacity to regenerate after injury or partial hepatectomy (PH). It has been demonstrated that the evolution of liver diseases is dependent on repeated rounds of hepatocyte apoptosis or the constitutive expression of pro- proliferative genes, and that hepatocyte proliferation is responsible for liver regeneration following a two-thirds PH. Liver regeneration after PH is a multi-step process that first involves an initiation phase, where multiple genes and pathways are acti- vated to mediate the exit of hepatocytes from a quiescent state and progression through the cell cycle in a synchronous manner [1]. In mice, hepatocytes reach S phase peak around 40 h after PH [2]. Multiple factors can influence the regeneration process by supplying stimulatory and inhibitory signals for hepatocyte pro- liferation. Tumour necrosis factor alpha (TNF-a) and interleukin- 6 (IL-6), together with their downstream transcription factors STAT3 and nuclear factor kappa B, are the most important initiat- ing factors in the regenerative response [1,2]. Concerning IL-6, its role in hepatocyte proliferation seems to be dose-dependent. Indeed, liver regeneration is impaired in IL-6 knockout mice, while injection of IL-6 in wild type mice inhibits DNA synthesis after PH [3–5]. Hepatitis B virus (HBV) chronic infection is associated with hepatic lesions of variable severity, ranging from a practically normal liver to severe lesions of active chronic hepatitis that can develop into cirrhosis (for review see: [6]). HBV chronic infection may also be associated with the development of hepa- tocellular carcinoma (HCC) [7]. However, the onset of HCC is principally linked to chronic inflammation due to the host immune response to infected liver cells (90% of HCC develop on a cirrhotic liver) [7]. There is also evidence of a direct role for HBV in HCC development through integration of the viral genome in the host genome and expression of viral proteins [7]. In particular, hepatitis B virus X protein (HBx) is thought to play a major role in HCC [8]. Indeed, HBx expression is found in tumors and anti-HBx antibodies are present in sera of chronic carriers with liver disease [9,10]. Moreover, HBx sequences are frequently conserved in integrated viral DNA [11,12]. Journal of Hepatology 2013 vol. 59 j 285–291 Received 19 June 2012; received in revised form 19 February 2013; accepted 19 March 2013; available online 28 March 2013 ⇑ Corresponding author. Address: Inserm U845, CHU Necker, 156, rue de Vaugirard, 75015 Paris, France. Tel.: +33 1 40615343; fax: +33 1 40615581. E-mail address: dina.kremsdorf@inserm.fr (D. Kremsdorf). Abbreviations: PH, partial hepatectomy; HBV, hepatitis B virus; HCC, hepatocel- lular carcinoma; HBx, hepatitis B virus X protein. Research Article