Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models

Peter Jianrui Liu,Peter A C Wing,Paul Klenerman,Emanuele Marchi,Jane A Mckeating,Andrea Magri,Ana Maria Ortega-Prieto,Marcus Dorner,David Durantel,Valentina D’Arienzo,James M Harris,Thomas Michler,Ulrike Protzer,Maarten Van De Klundert,Jochen Wettengel,Efstathios S Giotis

doi:10.1038/s41598-020-70865-7

Abstract

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

Highlights

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide
To determine whether there is any association between hypoxic responsive transcription and chronic hepatitis B (CHB), we performed Gene Set Enrichment Analysis (GSEA) on microarray data from a cohort of chronic HBV infected patients (n = 90) that were free of cirrhosis or HCC and uninfected control subjects[41]
To further validate these results, we analysed the acute transcriptional response of primary human hepatocytes (PHHs)[45] cultured under 1% oxygen for 4 h and identified 113 upregulated genes (FC > 2; FDR < 0.05) and GSEA showed an enrichment in CHB (Supplementary Fig. 2a)

Summary

Introduction

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. Liver cirrhosis is a major risk factor for developing HCC, 10–20% of HBV infected patients that develop HCC are non-cirrhotic, highlighting a role for HBV to promote carcinogenesis via direct and indirect inflammatory mechanisms[7]. Recent single-cell RNA sequencing analysis of the mouse liver highlights a major role for hypoxic and Wnt signalling pathways to shape liver zonation profiles in the normal healthy liver with an enrichment of hypoxic gene expression in the pericentral area[13]. This oxygen gradient is readily perturbed in pathological states such as infection, inflammation and cirrhosis[14]. HIF signalling could have an important role in progressive liver disease and HCC d evelopment[14]

Methods

Results

Conclusion