- 期刊:Good
- Index Factor: 24.5
- Publication time: 2023.11
Dr. Mai has something to say
Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes to promote the production of hepatitis B surface antigen (HBsAg) and the development of liver cancer, however, the in situ distribution and extent of HBV integration are poorly understood in patients with chronic HBV infection, or even in patients with HBsAg clearance. This article mainly explores whether transcriptionally active HBV integration events spread throughout liver tissue at different stages of chronic HBV infection, especially in patients with HBsAg clearance.
Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, promote the production of hepatitis B surface antigen (HBsAg) and lead to liver cancer. In the present study, we aim to explore whether transcriptionally active HBV integration events spread throughout liver tissue at different stages of chronic HBV infection, especially in patients with HBsAg loss.
Overview of the study
1. Characteristics of the study population
The authors collected liver biopsy tissue from 18 patients with chronic HBV infection and conducted a population profile survey. The population included untreated patients at the time of liver biopsy (n=9), treated (OT) patients without HBsAg loss (n=6), and patients with HBsAg loss (n=3).
Of the nine untreated patients, 4 were HBeAg-positive (EP) patients, denoted as EP1-4, and 5 were HBeAg-negative (EN) patients, denoted as EN1-5. In the OT group, 6 CHB patients were receiving antiviral therapy for more than 1 year at the time of liver biopsy, which was recorded as OT1~6. The remaining three patients who had achieved HBsAg disappearance (SL) after long-term antiviral therapy were named SL1-3. At the time of liver biopsy, patients SL1 and SL2 were still receiving NUC therapy, while patient SL3 had discontinued antiviral therapy. At the time of inclusion in this study, patients OT5 and OT6 had undergone a liver biopsy prior to antiviral therapy, while the other patients had not.
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Table 1: Patient characteristics survey
2. Spatial heterogeneity of intrahepatic cells by spatial transcriptome
Spatial transcriptome (ST) sequencing was used to analyze intrahepatic cell heterogeneity. Each section contains liver biopsy tissue from two patients. After data processing, 13,059 spots from 18 patients were obtained, and these spots in different slices were combined for dimensionality reduction and clustering, and a total of 7 clusters were divided. The distribution of clusters is presented in the UMAP/tSNE projection space (Figure 1A) and the organizational physical space (Figure 1B), and these clusters are spatially intertwined.
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Figure 1. Spatial heterogeneity in patients with chronic HBV infection
The authors employed a marker-based strategy to score the cell type enrichment in each Spot and compared the scores between the clusters. Normal hepatocyte fractions were higher in cluster 3 and lower in cluster 4, with little difference in the rest of the clusters. Monocytes as well as tissue-resident macrophages were significantly enriched in cluster 5 (Figure 2A). Fibroblasts, hepatic endothelial cells, and T cell subtypes scored higher in cluster 4, and neutrophils scored higher in cluster 6. Notably, the composition of the cell cluster varied between patients, with a significantly higher abundance of cluster 5 in EP2 and OT6 patients (Figure 2B), reflecting different levels of stromal cell and immune cell infiltration.
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Figure 2. Cluster comparison and cell type scoring
3. HBV integration-derived virus-host chimeric mRNA expression
To obtain high resolution of the spatial distribution of transcriptionally active HBV integration, the authors used the ChimericSeq package to capture virus-host chimeric reads in 10 × Visium ST data. Overall, 13 154 virus-host chimeric reads were detected in liver biopsy tissues from all patients, and the spatial distribution of Spots containing transcriptionally active viral integrations was subsequently analyzed. The results showed that transcriptionally active HBV integration events were present in 7.86% of liver tissue spots, all of which were highlighted in red (Figure 3A). The median percentage of Spots with transcriptionally active viral integration in each patient was 3.17%, with the highest percentage being 35.50% of EN2 in HBeAg-negative and treatment-naïve patients. The percentage of Spots containing viral integration was significantly lower in patients who were receiving or had received antiviral therapy than in patients who were not treated. The percentage of Spots with transcriptionally active viral integration in patients receiving antiviral therapy was lower than in patients who were not receiving treatment. Spots with transcriptionally active viral integration are almost undetectable in patients with HBsAg loss (Figure 3B).
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Figure 3. Cluster comparison and cell type scoring
In addition, these Spots containing viral integration showed different chimeric read densities, with the maximum number of total chimeric reads in Spots from patient EP2 being 441 (Figure 3D). The number of total chimeric reads, the number of unique chimeric reads, and the number of unique viral integration events were significantly but not strongly correlated with serum HBsAg levels (Figure 4). There was no significant correlation between the number of chimeric readouts and patient age, known duration of infection, grade of inflammation, or stage of fibrosis. The number of different viral integration events was also correlated with serum HBV DNA levels.
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Figure 4. Correlation of transcriptionally active HBV integration events with serum HBsAg and HBV DNA levels
4. Differential localization of transcriptionally active HBV integration events
Similarly, a total of 5498 unique chimeric reads were found using the ChimericSeq package, of which 90.81% (4993/5498) of the viral breakpoint sites were localized to the nucleotide position between 1590 and 1840 of the HBV sequence, corresponding to a viral genome region spanning direct repeat sequence 2 (DR2) and direct repeat sequence 1 (DR1), which contains enhancer II., which can upregulate the expression of the viral genome. (Figure 5A) and found that these transcriptionally active HBV integration events were randomly distributed in cellular chromosomes, mapped to 349 different human genes (Figure 5B).
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Figure 5. Visualization of transcriptionally active HBV integration sites on human chromosomes and viral genomes
5. Heterogeneously expressed HBsAg and HBcAg in the liver
In order to verify whether the transcriptionally active viral integration events were consistent with HBsAg positivity in liver sections, the relative distribution of HBsAg was analyzed by IHC, and the IHC signal of HBcAg was also detected to more accurately reflect the cccDNA-directed HBV expression. It was found that although EP3 patients were widely HBsAg and HBcAg positive, only 9.4% of the viral integration spots appeared, indicating that the viral integration event did not occur in all infected cells. OT3 was widely expressed HBsAg and HBcAg was highly expressed, while only 2.76% of the virus-integrated Spots were available, suggesting that HBsAg may be mainly derived from intrahepatic cccDNA. EN2 contained 35.5% viral integration spots, and HBsAg was strongly positive and HBcAg expression was negative, suggesting that transcriptionally silenced cccDNA may be present in infected hepatocytes. SL1 and SL2 are HBsAg negative, but a low proportion of viral integration spots are present, suggesting that these viral integrations may be silent for HBsAg expression.
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Figure 6. Spatially resolved metabolomics highlighted the anatomical heterogeneity of human kidneys, with heterogeneous expression of intrahepatic HBsAg and HBcAg
6. 肝内HBV cccDNA 促进HBsAg表达
Quantitative analysis of intrahepatic total HBV DNA and cccDNA was performed in 12 of the 18 patients with a second biopsy. Intrahepatic cccDNA levels were found to be well correlated with the percentage of integrated spots, total intercalated reads, unique chimeric reads, and different integration events, while intrahepatic HBV total DNA levels were not significantly correlated with the number of chimeric reads and different integration events, but were correlated with serum HBsAg levels (Fig. 7).
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Figure 6. Quantification of intrahepatic levels of total HBV DNA and cccDNA and their correlation with transcriptionally active HBV integration events
Results discussed
In summary, the authors' spatial transcriptome sequencing found that 7.86% of the liver tissue points were found in all patients, including three patients with HBsAg loss. There were 13,154 virus-host chimeric reads. These HBV integration sites are randomly distributed on chromosomes and can be mapped to host genes involved in the development of liver cancer, such as ALB, CLU, and APOB. Patients who are receiving or have received antiviral therapy have a significantly lower percentage of dots containing viral integration and significantly fewer mosaic reads compared to those who have not received treatment. There was a good correlation between intrahepatic cccDNA levels and viral integration events. Transcriptionally active HBV integration occurs at different stages in patients with chronic HBV infection, including patients with HBsAg loss. Antiviral therapy is associated with a decrease in the number and extent of transcriptionally active viral integration, which means that early therapeutic intervention may further reduce the number of viral integration events.