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

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.

www.nature.com/scientificreports/ HBx binding to > 5,000 host genes with diverse roles in metabolism, chromatin maintenance and carcinogenesis 9 . 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, respectively 10 . This oxygen gradient has been reported to associate with liver zonation, a phenomenon where hepatocytes show distinct functional and structural heterogeneity across the parenchyma 11,12 . 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 area 13 . Importantly, this oxygen gradient is readily perturbed in pathological states such as infection, inflammation and cirrhosis 14 . One of the best studied oxygen sensing mechanisms is the hypoxia inducible factor (HIF) pathway 15 . As HIF-signalling pathways are altered in many diseases, including cancer and inflammatory conditions, pharmacological approaches to modulate HIF activity offer promising therapeutic opportunities 16,17 . 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 resection 18 . Furthermore, Xiang et al. and Zheng et al. showed that HIF-1α protein expression is predictive of HCC lymph node metastasis and vascular invasion 19,20 . Thus, HIF signalling could have an important role in progressive liver disease and HCC development 14 .
In this study, we report a significant upregulation of hypoxic gene expression in a cohort of chronic HBV infected patients 41 . Our studies to investigate the underlying mechanism using state-of-the-art in vitro and in vivo HBV transgenic mice and human liver chimeric mice models show limited evidence of hypoxic gene expression. These studies highlight a major role of liver inflammation and a complex interplay between HBV and HIF signalling in the chronic infected liver that is not recapitulated by current infection-competent model systems. Collectively, our data show clear evidence of hypoxia-driven gene expression in CHB in the absence of cirrhosis or HCC development that may play a role in driving hepatocarcinogenesis.

Results
Increased hypoxia gene signature in chronic hepatitis B. To determine whether there is any association between hypoxic responsive transcription and 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 (healthy, n = 6) 41 . We used a panel of 43 hypoxia gene signatures obtained from the Molecular Signatures Database (MSigDB v 7.0) 42 , that included hypoxia upregulated genes from diverse tissues and cancer types. We observed a significant enrichment (FDR < 0.05) of 19 gene sets in the CHB cohort (Fig. 1a). Since hypoxic-transcriptional responses can vary between cell and tissue types 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 cells 44 (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 vitro 46,47 , it was reassuring to observe an overlap of hypoxic regulated genes in HepG2 and PHHs ( Supplementary Fig. 2b).
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 viremia 48 . Grouping 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 www.nature.com/scientificreports/ (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 HIFs 32-40 , we used the bipotent HepaRG cell line expressing HBx (HepaRG-HBx WT ) under a tetracycline (Tet) inducible promoter 50,51 to study HBx-HIF interplay. HBx promotes viral transcription by degrading the host structural maintenance of chromosomes (Smc) complex Smc5/6 52 and we confirmed that Tet induced HBx expression, loss of Smc6 expression (Fig. 2a) and restored replication of a defective HBV mutant lacking HBx 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 housekeeping 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.
Scientific RepoRtS | (2020) 10:14101 | https://doi.org/10.1038/s41598-020-70865-7 www.nature.com/scientificreports/ in differentiated HepaRG-HBx WT cells (Fig. 2b). Thus we confirm that HBx in this model system is functionally active. As a control for these experiments we generated HepaRG cells encoding HBx with three nonsense mutations (HepaRG-HBx STOP ). To assess whether HBx can promote or stabilize HIF expression we treated HepaRG-HBx WT or HepaRG-HBx STOP cells with Tet and cultured at 1% oxygen, a typical oxygen concentration used to model hypoxia ex vivo, or standard 'normoxic' laboratory conditions of 20% oxygen for 24 h. HBx had minimal impact on HIF-1α or HIF-2α protein (Fig. 2c) or mRNA levels ( Fig. 2d) in HepaRG cells cultured at 20% oxygen. Culturing HepaRG-HBx WT or HepaRG-HBx STOP cells under 1% oxygen confirmed HIF-1α or HIF-2α expression and importantly showed a negligible effect of HBx on either HIF isoform (Fig. 2c). To assess whether HBx altered HIF transcriptional activity we quantified the mRNA levels of four HIF-regulated host genes (CAIX, BNIP3, VEGFA or GLUT1) (Fig. 2d) and CAIX protein expression (Fig. 2c) and observed no differences. Under normoxic conditions HIFs are hydroxylated by the oxygen-dependent PHDs and targeted for proteosomal degradation. Oxygen reperfusion of hypoxic cells results in a time-dependent loss of HIFs and we assessed whether the presence of HBx could alter the kinetics of HIF expression. A comparable decrease in HIF-1α and HIF-2α proteins was seen after 10-20 min of oxygen reperfusion in both Tet treated and untreated cells (Fig. 2e), demonstrating that HBx has a negligible effect on the kinetics of HIF degradation. In summary, we demonstrate that HepaRG cells are responsive to low oxygen and show a significant increase in hypoxia-associated gene transcription, this effect was not impacted by the co-expression of HBx.
To further investigate a role for HBx to stabilize HIF-1α, we used an adenoviral vector engineered to express HBx (Ad-HBx) and showed HBx expression and Smc6 degradation. Transducing HepG2-NTCP cells with Ad-HBx restored the replication of HBV X-further demonstrating its functional activity (Fig. 3a). HepG2-NTCP cells transduced with Ad-HBx or Ad-OVA (adenoviral vector expressing ovalbumin) were cultured at 20% or 1% oxygen and cells harvested over a 48 h period. We confirmed HBx expression 24 h post-transduction (Fig. 3b) and observed expression of HIF-1α after 8 h at 1% O 2 . Comparable expression levels of HIF-1α were noted in both Ad-HBx and Ad-OVA transduced cells, demonstrating a negligible effect of HBx on HIF-1α induction. These results further highlight a minimal role of HBx in regulating HIF-1α or HIF-2α mRNA or protein expression. www.nature.com/scientificreports/ Studying HIF transcriptional activity in HBV transgenic mice. Since HBV can only infect humans and hominoid primates, no immune competent animal models are available that support natural HBV infection. One of the most-widely used murine models for studying CHB are transgenic mice expressing HBV from a single integrated genome (HBVtg). HBVtg mice have been reported to develop HCC that show similar chromosomal aberrations and gene expression patterns to human HBV-associated HCC 53 . To study the effect of HBV on HIF transcriptional activity in this model system, HBVtg mice were treated with lipid nanoparticle complexed, liver-targeted siRNAs designed to silence all HBV transcripts (siHBV) 54 or with an unspecific control siRNA (siCtrl). The HBV-specific siRNA led to effective HBV silencing with greater than 95% reduction in HBeAg in the serum (Fig. 4a) and viral transcripts in the liver (Fig. 4b). However, silencing HBV mRNAs and antigens had no impact on HIF regulated gene transcripts (CAIX, VEGFA, GLUT1 and PHD2) (Fig. 4b). These studies suggest a minimal role of HBV encoded proteins or RNAs in promoting HIF transcriptional activity.

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 hepatocytes 55 showed no evidence of hypoxic gene upregulation (Fig. 5b). To further validate our conclusions we used the chimeric human liver FNRG mouse model 56 to assess whether HBV infection would induce HIF signaling in this model. Female FNRG mice 56 between 8-12 weeks of age were transplanted with 0.5 × 10 6 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 × 10 7 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 www.nature.com/scientificreports/ upregulated in the CHB cohort 41 (Fig. 5b), demonstrating the influence of inflammation on gene regulation and highlighting the limitations of current HBV replication models to model CHB.

Discussion
In this study we identified increased hypoxia gene signatures in a CHB cohort in the absence of cirrhosis or HCC. We confirmed hypoxic gene expression using MSigDB derived gene sets and recently reported signatures from HepG2 44 and PHH data sets 45 . We noted an increase in HIF-1α mRNA levels, consistent with their transcriptional regulation by inflammatory mediators such as TNFα. Given previous reports that HBx can stabilize HIFs 32-40,57 , we investigated whether functionally active HBx could regulate endogenous HIF-1α and HIF-2α mRNA, protein and transcriptional activity in vitro. We found minimal evidence for HBx regulation of HIFs in three independent model systems: an inducible HepaRG-HBx cell line; an Ad-HBx transduced cell, and in de novo infection of HepG2-NTCP or PHHs. Our study is distinct from earlier reports on at least three counts. Firstly, due to the technical difficulties in visualizing HBx by western blotting or immunofluorescent imaging, many of the earlier studies did not confirm HBx expression. Secondly, the majority of studies did not validate the functional activity of the expressed HBx protein. Finally, several studies assessed HBx stabilization of HIF-1α using transient plasmid transfection systems with hypoxia reporter constructs, rather than directly measuring HIF expression and HIF target gene modulation. Given our current knowledge that HBx degrades Smc6 that silences episomal DNA transcription, the interpretation of these earlier plasmid based systems 58 is now uncertain. Since we have directly confirmed expression and function of HBx in our in vitro models and quantified endogenous HIF transcriptional activity under normoxic or hypoxic conditions we are confident that HBx does not modulate HIF expression or transcriptional activity in the model systems used. Guerrieri et al. identified and validated a role for HBx in regulating genes involved in endocytosis, predominantly members of the Ras-related in brain (Rab) family 9 . Anti-HBx chromatin immunoprecipitation studies  Supplementary Fig. 9 (a). Induction of hypoxic genes (Supplementary Table 2) in transcriptomic data of HBV infected primary human hepatocytes 55 , 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). www.nature.com/scientificreports/ identified HBx binding sites that included RAB1A, RAB2B and RAB5B promoters and none of the validated Rab genes were listed in our hypoxic gene set (Supplementary Table 2). Furthermore, GSEA of the CHB cohort or screening reactome gene sets showed only a modest enrichment in the 'Transferrin Endocytosis' pathway ( Fig. 1e), suggesting a minimal overlap between HBx and HIF regulated genes.
Our results support a model where HBV infection associated inflammatory responses promote HIF expression and these complex virus-cell interactions are not recapitulated by simple in vitro culture systems, HBV transgenic mice or immunodeficient SCID human liver chimeric mouse models. This conclusion is supported by our observation of increased hypoxic gene expression in a cohort of HCV infected patients with cirrhosis (Fig. 1d). Analyzing the leading edge genes from the HBV (n = 30) and HCV (n = 23) cohorts identified 10 common genes, suggesting some gene specific differences between these chronic infections. Our bio-informatic analysis identified 25 hypoxia upregulated genes in chronic HBV infected patients, including LOXL2, SMIM3, TNS1, and IGFBP1. Notably, LOXL2 overexpression in HCC was previously associated with high tumour grade, metastasis, and poor patient overall and disease-free survival 59 . LOXL2 was shown to mediate its pathogenic effects in HCC angiogenesis via vasculogenic mimicry signalling, cytoskeleton reorganization, and bone-marrow derived cell recruitment 59,60 . In fact, hypoxia and HIF-1α signalling have been identified as key regulators of LOXL2 and driver of its pathogenesis, consistent with our observations 60,61 . Another significantly upregulated gene in chronic HBV patients, IGFBP1, was recently reported to be a HIF-2α regulated gene in vitro and in vivo model systems 62 . Furthermore, IGFBP1 is a known NF-κB target gene and is induced by HBV infection 63 . These data suggest co-regulation of IGFBP1 by inflammatory pathways including NF-κB and oxygen sensing mechanisms such as HIF signalling, which is consistent with our observation of inflammatory gene enrichment associating with hypoxia gene signature in CHB.
Our observation of increased hypoxic gene signature expression in CHB patients offers an important insight into HBV disease stage stratification and suggest areas for bio-marker discovery for early HCC detection. This is in agreement with previous studies that have associated higher HIFα mRNA and protein expression in HCC with worse prognostic outcomes for HCC patients [18][19][20] . Moreover, as the liver is a naturally physiologically low oxygen environment, future investigations exploring how oxygen sensing pathways regulate HBV replication and pathogenesis may identify novel therapeutic targets.

Materials and methods
Cell lines and reagents. HepaRG cells expressing HBx under the control of a Tetracycline inducible promoter were cultured in Williams E medium supplemented with 10% FBS, 50 IU penicillin/streptomycin mL −1 , 5 μg human insulin mL −1 and 5 × 10 −7 M hydrocortisone hemisuccinate (Sigma). As a control we generated HepaRG cells expressing an inactive HBx null mutant (HepaRG-HBx STOP ) where three point nonsense mutations (relative to EcoRI site: C to A, 1393nt; C to A, 1396nt and C to T, 1397nt) were introduced to generate three stop codons (respectively, TGA, 1393nt; TGA, 1396nt; TAA, 1397nt) in HBV genotype D. HepG2-NTCP cells 64 were maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM Sodium Pyruvate, 50 IU penicillin/streptomycin mL -1 and non-essential amino acids (Life Technologies, UK). HIF PHD inhibitor FG4592 was purchased from Cambridge Biosciences, UK. Cells were incubated under hypoxia in an atmosphere-regulated chamber with 1% O 2 : 5% CO 2 : balance N 2 (Invivo 400, Baker-Ruskinn Technologies). The Ad-HBx and Ad-Ova express the HBV genotype D HBx gene and chicken ovalbumin gene under control of the Transthyretin (TTR) promoter. Promoter and insert were inserted into the E1 region of adenovirus (Ad5ΔE1/E3) backbone plasmid pAd/PL-DEST through Gateway recombination following the manufacturer's instructions (Gateway System; Invitrogen, Karlsruhe, Germany). Adeno virus stocks were titrated using the cytopathic effect in HepG2 cells as previously described 65 .
HBV genesis and infection. HBV was purified from a HepAD38 producer line as previously reported 64 .
Briefly, virus was purified using centrifugal filter devices (Centricon Plus-70 and Biomax 100.000, Millipore Corp., Bedford, MA) and stocks with a titre between 3 × 10 9 and 3 × 10 10 viral genome equivalents (vge) per mL stored at − 80 °C. HBV-X-virus was purified from a HepG2 based cell line containing a HBV 1.3 × overlength integrated viral genome where both 5′ and 3′ HBx genes were knocked out by a point mutation that changes the eight amino acid to a stop codon (CAA-to-TAA) as previously described 51 . HepG2-NTCP cells were treated with 2.5% dimethyl sulphoxide (DMSO) for 3 days and inoculated with HBV at an MOI of 200 in the presence of 4% polyethylene glycol 8,000. After 18-20 h the inoculum was removed by washing with PBS and the cells cultured in the presence of 2.5% DMSO. Secreted HBe and HBs antigen were quantified by ELISA (Autobio, China).
Gene set enrichment analysis. GSEA was performed with GSEA_4.0.3 71 using publicly available Affymetrix microarray datasets 41,49 from Gene Expression Omnibus (GEO) (accessions: GSE83148, GSE14323). Gene sets were derived from HepG2 RNA-seq data 44 downloaded from GEO (GSE120886) or PHH RNA-Seq accessed from the published Supplementary Material 45 . To determine whether HBV infection induces hypoxia-responsive genes, we interrogated the mRNA expression patterns of the liver chimeric mice RNA-Seq dataset for the top 80 hypoxia-induced genes as identified in HepG2 hepatic cells 44 . In-house datasets were compared with RNAseq 55 from HBV-infected primary human hepatocytes. Data were retrieved from GEO (accessions: GSE120886, GSE93153, GSE118295). For consistency, all datasets were re-analysed with the same Partek Flow bioinformatic pipeline. Microarray analysis was performed with Partek Genomics Suite (v6.6) as previously described 70 and data presented using Graphpad Prism 8.

Statistical analyses.
All analyses were performed using Prism 8 (GraphPad, La Jolla, CA). Data are shown as means ± SD, probabilities are indicated by *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001, with Bonferroni corrections for multiple testing when appropriate. www.nature.com/scientificreports/ Data availability RNA-seq data from HBV infected mice are deposited in the GEO archive at NCBI, with the accession number GSE145835 and entitled: Transcriptional profiling of hepatocytes isolated from chronically HBV-infected human liver chimeric mice. Our in-house data was compared with RNA-seq 55 from HBV-infected primary human hepatocytes and data retrieved from GEO (accessions: GSE120886, GSE93153, GSE118295).