Introduction

HBV is a global health problem with more than 250 million people chronically infected and at least 780,000 deaths/year from HBV-related liver diseases such as liver cirrhosis and hepatocellular carcinoma (HCC)1,2. HBV replicates in hepatocytes within the liver and current anti-viral treatments suppress viral replication but are not curative, largely due to the persistence of the viral covalently closed circular DNA (cccDNA) reservoir3. Chronic hepatitis B (CHB) is a virus-associated, inflammatory liver disease and one of the leading causes of HCC4, one of the fastest rising and fourth most common cause of cancer related-death world-wide5. Curative therapies (tumour ablation, resection or liver transplantation) are dependent on early detection, however, the majority of HBV and non-viral associated HCC cases are diagnosed at a late stage often resulting in a poor prognosis6. Despite significant advances in our understanding of the HBV replicative life cycle, the mechanisms underlying HCC pathogenesis are not well defined7.

Although liver cirrhosis is a major risk factor for develo** 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 mechanisms7. Three major and non-exclusive viral-dependent pathways have been proposed: (i) integration of viral DNA into the host genome; (ii) expression of viral oncogenic proteins and (iii) viral-driven changes in host gene transcription (reviewed in8). The viral encoded regulatory hepatitis B X protein (HBx) has been reported to promote the expression of both viral and selected host genes, where a recent study reported HBx binding to > 5,000 host genes with diverse roles in metabolism, chromatin maintenance and carcinogenesis9. There is clearly an urgent need to increase our understanding of HBV mediated carcinogenesis to support the development of tools to identify CHB patients at risk of HCC development.

The liver receives oxygenated blood from the hepatic artery and oxygen-depleted blood via the hepatic portal vein, resulting in an oxygen gradient of 4–8% across the pericentral and periportal areas, respectively10. This oxygen gradient has been reported to associate with liver zonation, a phenomenon where hepatocytes show distinct functional and structural heterogeneity across the parenchyma11,12. 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 area13. Importantly, this oxygen gradient is readily perturbed in pathological states such as infection, inflammation and cirrhosis14. One of the best studied oxygen sensing mechanisms is the hypoxia inducible factor (HIF) pathway15. As HIF-signalling pathways are altered in many diseases, including cancer and inflammatory conditions, pharmacological approaches to modulate HIF activity offer promising therapeutic opportunities16,17. When oxygen is abundant, newly synthesised HIFα subunits, including HIF-1α and HIF-2α isomers, are rapidly hydroxylated by prolyl-hydroxylase domain (PHD) proteins and targeted for poly-ubiquitination and proteasomal degradation. In contrast when oxygen is limited these HIFα subunits translocate to the nucleus, dimerize with HIF-β and positively regulate the transcription of a myriad of host genes involved in cell metabolism, proliferation, angiogenesis and immune regulation. Dai et al. reported that increased HIF-1α mRNA and protein expression in HCC are prognostic for more advanced disease stages and poor overall survival post-surgical tumour resection18. Furthermore, ** of HIF-binding sites by ChIP-seq. Blood 117, e207-217. https://doi.org/10.1182/blood-2010-10-314427 (2011)." href="/article/10.1038/s41598-020-70865-7#ref-CR43" id="ref-link-section-d67971006e837">43 and none of the MSigDB signatures were liver-derived (Supplementary Table 1), we analysed an RNA-sequencing (RNA-seq) transcriptome of human hepatoma derived HepG2 cells44 (0.5% oxygen for 16 h). We identified 80 hypoxic upregulated genes (greater than twofold change, FDR < 0.05) (Supplementary Table 2) and GSEA showed an enrichment (FDR = 0.077) in the CHB cohort (Fig. 1b). 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). Since PHHs can rapidly de-differentiate and lose hepatocyte-specific function in vitro46,47, it was reassuring to observe an overlap of hypoxic regulated genes in HepG2 and PHHs (Supplementary Fig. 2b).

Figure 1
figure 1

Increased hypoxia gene expression in CHB. Hypoxia upregulated gene signatures from Molecular Signatures Database were assessed in the CHB cohort, 19 significantly upregulated gene signatures identified (FDR < 0.05) and ranked by Normalized Enrichment Score (NES) (a). GSEA shows a significant enrichment of HepG2 defined hypoxic genes in CHB cohort (FDR = 0.077). The gene set was based on Fold Change > 2, and FDR < 0.05; 80 genes satisfied these criteria and are listed in Supplementary Table 2 (b). CHB cohort was grouped by peripheral ALT activity, with subjects > 40 IU/L (n = 57) or < 40 IU/L (n = 25). GSEA shows a significant enrichment of HepG2 defined hypoxic genes in patients with elevated ALT (FDR = 0.110) (c). HepG2 hypoxic gene set was enriched (FDR = 0.006) in HCV infected patients with cirrhosis (n = 41) compared to normal liver controls (n = 19) (d). MSigDB hallmark gene sets identified the most upregulated pathways in the CHB cohort: 28 gene sets were significantly enriched (FDR < 0.05) and are ranked by NES (e). All GSEA was performed using GSEA_4.0.371.

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Figure 2
figure 2

Effect of HBx on HIF expression and transcriptional activity in HepaRG cells. HepaRG cells encoding HBx were incubated with Tet (50 µM) for 24 h and HBx protein and Smc6 expression detected by western blot, uncropped blots are available in Supplementary Fig. 2 (a). Differentiated HepaRG cells encoding WT or mutated HBx (STOP) were treated or not with tetracycline (1 µM) and infected with HBVWT or HBVX- and 13 days later infection assessed by measuring total viral RNAs. The data is normalised for each cell line relative to HBVWT infection without Tet and represent the mean of 3 independent experiments; 2-way-ANOVA with Bonferroni correction was applied with p < 0.05 deemed as significant (b). HepaRG cells encoding WT or mutated HBx (STOP) were incubated with or without Tet (50 μM, 24 h) and cultured under 20% or 1% oxygen conditions for 24 h. Cells were lysed and expression of HIF-1α, HIF-2α, Carbonic anhydrase IX (CAIX) and housekee** gene B-actin assessed by western blotting, uncropped blots are available in Supplementary Figs. 3 and 4 (c) and mRNA levels of HIF-1α, HIF-2α and several HIF target genes (CAIX, BNIP3, VEGFA and GLUT1) quantified by qPCR (d). HepaRG cells encoding wild type HBx were incubated with Tet (50 µM, 24 h) and cultured at 20% or 1% oxygen for 24 h. The hypoxic cultures were returned to 20% oxygen. After 10 or 20 min, cells were lysed and screened for HIF-1α or HIF-2α and housekee** gene β-actin expression by western blot, uncropped blots are available in Supplementary Fig. 5 (e). The data is shown from a single experiment and is representative of three independent experiments and represents mean ± standard deviation. Normality distribution was assessed by D'Agostino-Pearson test; 2-way-ANOVA with Bonferroni correction was applied with p < 0.05 deemed as significant.

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CHB reflects a dynamic interaction between virus infected hepatocytes and immune cells and periods of active hepatitis, as measured by the elevated activity of the liver enzyme alanine aminotransferase (ALT), associate with increased viremia48. Grou** our CHB cohort by ALT activity showed a significant enrichment (FDR = 0.110) of the hypoxic HepG2 derived gene set in patients with elevated ALT (ALT > 40 IU/mL) (n = 57) compared to those with normal ALT (n = 25) (Fig. 1c). To evaluate whether hypoxic gene expression is observed in other inflammatory liver disease we studied a cohort of hepatitis C virus (HCV) infected patients with cirrhosis (n = 41)49 and observed a significant enrichment of the hypoxic HepG2 gene signature (FDR = 0.006) in the infected group compared to normal controls (n = 19) (Fig. 1d). Analysing the leading edge genes in the CHB (n = 30) and HCV (n = 23) cohorts identified 10 common genes, however, the majority of genes (57%) were unique to their respective cohorts.

We hypothesized that the hypoxic gene signature in CHB was mediated via inflammatory pathways. To evaluate this assumption we studied enriched pathways in the CHB liver using the hallmark gene sets from MSigDB. This analysis identified genes associated with allograft rejection as the most significantly upregulated gene set in CHB. Interestingly HIF-1α was one of the leading-edge genes in this subset; contributing significantly to the core enrichment score. We noted increased HIF-1α mRNA levels in the CHB patients compared to control subjects (Log2 FC = 2.648, p = 0.005). Moreover, we observed a significant increase in inflammatory signaling pathways in CHB liver: TNF-α signaling via NF-κB’, ‘Inflammatory Response’ and ‘Interferon Gamma Response’ (Fig. 1e). In summary these data support the conclusion that the hypoxic phenotype in the diseased liver is likely driven by local inflammation, rather than a direct result of viral infection.

Limited evidence for HBx to stabilise HIF-1α or HIF-2α expression or associated transcriptional activity in vitro

As HBx is the major viral encoded transcriptional activator, previously reported to stabilize HIFs32,33,34,35,Full size image

Studying HIF transcriptional activity in HBV infected hepatocytes and human liver chimeric mice

To complement the HBx studies described above we investigated the effect of HBV infection on HIF oxygen sensing pathways in current state-of-the-art in vitro and in vivo models. HepG2-NTCP cells were infected with HBV and cultured under normoxic conditions and sampled after 3 and 9 days to assess HIF-1α or HIF-2α expression. HBV gene expression was confirmed by measuring HBeAg (53.96 ± 2.7 IU/mL) and HBsAg (12.63 ± 4.4 IU/mL), however, we failed to detect either HIF or CAIX expression in the infected or non-infected cells (Fig. 5a). As a control we treated HepG2-NTCP cells with a HIF PHD inhibitor (FG4592 at 30 µM) and demonstrated HIF protein expression (Fig. 5a). Analyzing published RNA-seq data from HBV infected primary human hepatocytes55 showed no evidence of hypoxic gene upregulation (Fig. 5b). To further validate our conclusions we used the chimeric human liver FNRG mouse model56 to assess whether HBV infection would induce HIF signaling in this model. Female FNRG mice56 between 8–12 weeks of age were transplanted with 0.5 × 106 cryopreserved adult human hepatocytes by intrasplenic injection and monitored for engraftment by measuring human albumin levels in the serum (at least 0.1 mg human albumin per mL in peripheral blood). Engrafted animals were infected with 0.5 million genome equivalent (GE) copies of HBV per mouse and were monitored for HBV replication. Once stable viremia was established (minimum 5 × 107 GE mL−1 of serum) the mice were sacrificed and livers harvested from HBV infected (n = 4) and uninfected (n = 3) animals for RNA isolation and RNA-seq. Analyzing these RNA-seq data sets showed minimal evidence for an increase in hypoxic transcriptional activity in the HBV infected livers (Fig. 5b). For comparative purposes, we show that hypoxic genes were upregulated in the CHB cohort41 (Fig. 5b), demonstrating the influence of inflammation on gene regulation and highlighting the limitations of current HBV replication models to model CHB.

Figure 5
figure 5

Comparing hypoxia gene signatures in HBV infected hepatocytes and humanized liver chimeric mice. Mock or HBV-infected HepG2-NTCP cells (MOI 200) were harvested after 3 or 9 days, lysed and assessed for HIF-1α, HIF-2α or CAIX expression and the housekee** gene B-actin by western blotting. As a positive control HepG2-NTCP cells were treated with the HIF PHD inhibitor FG4592 (FG, 30 μM) for 24 h and protein lysates analysed by western blotting, uncropped blots are available in Supplementary Fig. 9 (a). Induction of hypoxic genes (Supplementary Table 2) in transcriptomic data of HBV infected primary human hepatocytes55, HBV infected human liver chimeric mice and a CHB cohort (b). Fold change was calculated for each of the 80 genes in HBV infection against the healthy controls, where the dotted line represents a twofold change. For the CHB cohort, fold change was calculated from the raw Affymetrix, differential expression was tested using multiple t-tests and significance determined by (adjusted p value < 0.05).

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