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Chronic hepatitis B virus infection imbalances short-chain fatty acids and amino acids in the liver and gut via microbiota modulation

Gut Pathogens volume 17, Article number: 18 (2025) Cite this article

Abstract

The commensal microbiota is closely related to HBV infection and HBV-related liver diseases; however, how HBV and viral components dynamically affect the targeted organ liver microbiota is not well-known. In this study, an HBV-carrier mouse model established by HBsAg+ hepatocyte replacement in Fah−/− recipient mice, named HBs-HepR mice, was used to analyze the microbiota and metabolomics at the time of triggering the specific anti-HBV CD8+ T cell response in the liver. The composition and relative abundance of microbiota were both altered in the gut and liver of HBs-HepR mice. Particularly, increased Muribaculaceae and Alloprevotella, and decreased Lachnospiraceae-NK4A136 and Rikenella were observed in the gut; while increased Ralstonia and Geobacillus were observed in the liver of HBs-HepR mice. Furthermore, changes in microbial functions were revealed. There were no significant differences in the levels of SCFAs in fecal and serum; however, decreased propionic acid and acetic acid were detected in the livers of HBs-HepR mice, which was negatively related to the abundance of Geobacillus in the liver. Significantly decreased levels of 9 kinds of amino acids were detected in the feces of HBs-HepR mice, which was positively related to decreased Rikenella in the gut. A significant increase in L-glycine was observed in the liver and serum, positively related to the abundance of Geobaillus in the livers of HBs-HepR mice. In conclusion, chronic HBV infection imbalanced SCFA and amino acid metabolism by modulating microbiota in the liver, unlike in the gut, which was involved in the immune activation phase.

Background

Hepatitis B virus (HBV)-induced liver immune tolerance and immune activation are among the most important mechanisms for chronic infection and disease progression. The liver immune microenvironment and its regional immune response are significantly regulated by the gut-liver axis [1,2,3]. The gut is rich in a large number of commensal microbiota, which play important roles in the development, differentiation, and functional homeostasis of immune cells in the body and are closely related to the development and progression of a series of liver diseases, including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), liver cirrhosis and cancer [4,5,6]. Noticeably, there is microbiota in liver tissue under certain circumstances, which may better explain tissue-specific mechanisms underlying liver disease severity [7, 8]. However, how HBV and viral components dynamically affect the liver microbiota of targeted organ during HBV infection is unknown, which limits our understanding of microbial associations with HBV-related liver diseases.

Previous studies have reported that age-related HBV clearance depends on the gut microbiota since young mice are prone to inducing HBV immune tolerance, while adult mice exhibit effective anti-HBV immune responses to clear HBV due to the establishment of the gut microbiota in the body [9, 10]. The exact cellular and molecular mechanisms by which the gut microbiota regulates anti-HBV immune responses are not yet clear, but are speculated to be related to the load of the gut microbiota [11]. On the other hand, HBV infection can alter the structure and composition of the gut microbiota, with significant dynamic changes in the proportions of Firmicutes and Bacteroidetes in the gut. Lactobacillus and Bifidobacterium significantly differ between acute and chronic HBV infection models [12]. Analysis of clinical samples from patients with chronic HBV infection and related liver disease confirmed that compared with healthy controls, asymptomatic HBV carriers had a significant decrease in the ratio of gut Bifidobacterium/Enterobacteriaceae (B/E), and chronic hepatitis B (CHB) patients and liver cirrhosis patients had a more significant decrease in the ratio of gut B/E. These findings suggest that an imbalance in the gut microbiota is correlated with the progression of HBV-related liver disease, and intervention with the probiotic Bifidobacterium may have potential therapeutic effects on HBV-related liver disease [13, 14]. Fecal microbiota transplantation (FMT) can restore the balance of the gut microbiota and is considered a potential strategy for the treatment of HBV-related liver diseases [15, 16]. To date, there have been almost no reports on the liver microbiota during HBV infection in humans.

Alterations in the composition and structure of the commensal microbiota cause functional changes in the levels of metabolites, such as short chain fatty acids (SCFAs) and amino acids. SCFAs, mainly acetic acid, propionic acid, and butyric acid, have important physiological functions, which can provide energy, regulate electrolyte balance, protect the mucosal barrier of intestine and promote nutrient absorption. Gut commensal microbiota plays a critical role in host immune homeostasis by the production of SCFAs [17]. However, the changes of SCFAs and amino acids during chronic HBV infection are not yet clear. HBx transgenic mice fed SCFAs showed significantly delayed development of HCC, indicating some of the underlying mechanisms of SCFAs that are relevant to HBV-related liver disease [18]. Whether the gut and liver microbiota regulate anti-HBV immunity and relate to these metabolites deserves investigating.

HBV can only naturally infect the hepatocytes of humans and chimpanzees; thus, research on the targeted organ has been limited. A novel HBV-carrier mouse model named HBs-HepR mice was generated by adoptively transferring HBsAg+ hepatocytes via spleen injection into Fah−/− recipient mice in our laboratory [19]. This novel HBV mouse model (HBs-HepR mice) can better simulate the pathological process of HBV infection in humans and provides a useful platform for the research on microbiota imbalances in the liver. In this study, the microbiota and metabolome were analyzed in HBs-HepR mice and control mice at 18 weeks after hepatocyte transfer, the time at which specific anti-HBV immune responses are triggered. The results revealed the interaction between HBV and the microbiota in the liver and gut, which compensates for the limitations of obtaining liver samples from clinical patient cohorts, and provided an important theoretical basis for regulating the host microbiota and promoting the clearance of HBV and disease control in humans.

Methods

Animals

As reported [19], we established a chronic HBV-carrier mouse model (HBs-HepR mice) by using Fah−/− mice as recipients and adoptively transferring HBsAg+ hepatocytes from HBV transgenic C57BL/6 J-TgN (Albl-HBV) 44Bri (HBs-Tg) mice (Department of Laboratory Animal Science of Peking University, Beijing, China) to replace the recipient hepatocytes. WT hepatocytes from C57BL/6 J mice were used to establish a control mouse model (B6-HepR mice). Fah−/− mice (129S4 background) were provided by Dr. Xin Wang (Inner Mongolia University, Hohhot, China), which were used to backcrossed with C57BL/6 J mice (Shanghai Experimental Animal Center, Shanghai, China) to obtain Fah−/− mice (B6 background). Fah−/− mice were treated with 2-(2-nitro-4-tifluoro-methylbenzyol)−1, 3-cyclohexanedione (NTBC) in their drinking water at the concentration of 7.5 mg/L. All mice were fed with irradiated mouse feed (S0010, WQJX BIO-TECHNOLOGY, Wuhan, China) and maintained under specific pathogen-free (SPF) controlled conditions (22 °C, 55% humidity, and 12 h day/night) in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The animal experiments were ethically approved by the Animal Use and Management Committee of University of Science and Technology of China (approval number USTCACUC25120122086).

For B6-HepR and HBs-HepR mice, NTBC was withdrawn from the drinking water after hepatocyte transfer immediately, and NTBC was added for 3 days on 25 days after hepatocyte transfer and then withdrawn enduringly. The mouse model was successful 12 weeks after hepatocyte reconstruction. The HBs-HepR mice were in a state of HBV immune tolerance at the time of 12 weeks after HBsAg+ hepatocyte transfer, and chronic hepatitis was observed in HBs-HepR mice beginning 14 weeks after HBsAg+ hepatocyte transfer, demonstrated by the increased serum levels of ALT and AST. At 18 weeks after hepatocyte transfer, HBsAg-specific CD8+ T cells were generated in the liver and mediated hepatocyte apoptosis and hepatitis [19]. Thus, HBs-HepR mice and control mice at 18 weeks after hepatocyte transfer were used to analyze the microbiota and metabolome.

Hepatocyte transfer intrasplenically

Isolated mouse hepatocytes were obtained as described previously [20]. Hepatocytes (1.0 × 106) resuspended in serum-free Dulbecco’s modified Eagle’s medium (DMEM) were transferred into Fah−/− mice via intrasplenic injection. After hepatocyte transfer, the NTBC-treated water was withdrawn. Twenty-five days after hepatocyte reconstruction, NTBC-treated water was supplied again for 3 days, and then withdrawn. The chronic HBV-carrier mouse model (HBs-HepR mice) and the control mouse model (B6-HepR mice) were deemed to be successful 12 weeks after hepatocyte reconstruction [19].

Sample collection

Eighteen weeks post hepatocyte transfer, fecal, serum and liver samples were harvested from HBs-HepR, B6-HepR and Fah−/− mice for microbiota analysis and metabolite analysis. As for the stool samples, by using a tweezer pinch and gently squeeze from the proximal end of the colon (near the cecum) towards the distal end (towards the anus). Stool samples were freshly collected into sterile tubes and stored at − 80 °C before use.

Microbiota analysis

16S rRNA sequencing was performed for the gut microbiota, and 2bRAD-M sequencing was performed for the liver microbiota (OE Biotech Co. Ltd., Shanghai, China). The microbial community structure and relative abundance were analyzed. Microbiota alpha diversity was calculated and analyzed using Chao1. Microbiota beta diversity was calculated and analyzed using permutational multivariate analysis of variance (PERMANOVA). Principal coordinate analysis (PCoA) by binary-jaccard was shown. PICRUSt2 (2.3.0b0) was used to predict the potential functional content of pathways based on Clusters of Orthologous Groups of proteins (COG) and pathways of Kyoto Encyclopedia of Genes and Genomes (KEGG).

Metabolite analysis

Short-chain fatty acids (SCFAs), including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, pentanoic acid, and hexanoic acid, were detected in the serum, fecal and liver samples by liquid chromatography‒mass spectrometry (LC‒MS) (Shanghai Luming Biotechnology Co., Ltd., Shanghai, China). Amino acids, including L-4-hydroxyproline, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, were detected in the serum, fecal and liver samples by GC‒MS/MS (Shanghai Luming Biotechnology Co., Ltd., Shanghai, China). Gas chromatography‒mass spectrometry (GC‒MS) (TSQ9000, Thermo Scientific, MA, USA) was used.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. Pearson correlation analysis was performed between the microbiota and metabolites. All data are shown as the mean ± SEM and were analyzed using analysis of variance (ANOVA) as appropriate. P < 0.05 was considered significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Results

Gut microbiota composition and abundance altered by chronic HBV infection

16S RNA sequencing was used to compare the microbiota profiles of HBs-HepR mice, control B6-HepR mice and recipient Fah−/− mice. The numbers of Amplicon Sequence Variant (ASV) in each group and the common and unique ASVs among the three groups were presented in Fig. 1A. Analysis of beta diversity revealed significant differences in the distribution of microbiota communities among these three groups according to permutational multivariate analysis of variance (PERMANOVA) (Fig. 1B). A bar plot of the number of species annotated at the phylum, family, genus, and species levels in each group indicated a decreased number of species in the fecal samples of HBs-HepR mice (Fig. 1C). Compositions of the gut microbiota community at the phylum level in each group included Bacteroidota (64.6%, 62.2% and 71.1% in Fah−/−control mice, B6-HepR mice and HBs-HepR mice, respectively) and Firmicutes (26.2%, 30.0% and 24.6% in Fah−/− control mice, B6-HepR mice and HBs-HepR mice, respectively). The ratio of Firmicutes to Bacteroidota (F/B ratio) was slightly decreased in HBs-HepR mice (Fig. 1D). The top 20 ASVs with high abundance in the three groups, as shown by the Circos diagram, illustrated the relationships between the fecal samples and species, and that there were significant differences among these three groups, mainly in the Bacteroidota (Fig. 1E).

Fig. 1
figure 1

Gut microbial diversity and composition of HBs-HepR mice compared with those of control B6-HepR mice. Fecal samples were harvested from HBs-HepR (n = 3), B6-HepR (n = 6) and Fah−/− control mice (n = 4). 16S RNA sequencing was used to analyze the commensal microbiota. A Venn diagram of ASVs. The numbers of ASVs in each group and the unique and shared ASVs among the three groups are shown. B Beta diversity was evaluated using PCoA with binary Jaccard. A dot represents one sample. C Community bar plot for the numbers of species annotated at the phylum, family, genus, and species levels in each group. D Compositions of the microbiota community at the phylum level in each group. The ratio of Firmicutes to Bacteroidota was calculated. E Circos diagram illustrating the relationships between samples and species. The top 20 ASVs with high abundance were plotted using the circle package in R

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Furthermore, the top 15 compositions of microbial community at the genus level among the HBs-HepR, B6-HepR and Fah−/− control groups were shown in Fig. 2A. Decreased levels of Clostridia-UCG-014, Roseburia and Parasutterella were observed in both the HBs-HepR and B6-HepR groups compared with the Fah−/− control group, indicating that hepatocyte transfer and reconstruction induced alterations in the gut microbiota. When comparing B6-HepR mice with Fah−/− control mice, increased relative abundances of Lachnospiraceae-NK4A136, Lactobacillus and Colidexibacter and decreased relative abundances of Prevotellaceae-UCG-001, Bacteroides and Lachnoclostridium were observed, indicating increased probiotic microbiota in B6-HepR mice. However, these results were not observed for HBs-HepR mice. Increased levels of ASF356, Muribaculaceae and Alloprevotella and decreased levels of Allistipes, Helicobater and Rikenella were observed in HBs-HepR mice, indicating that these alterations might be related to HBsAg+ hepatocytes (Fig. 2A). Among these alterations, Alloprevotella was the most enriched genus with a significantly increased abundance in the gut of HBs-HepR mice (Fig. 2B). Analysis of key microbiota at the genus level by random forest indicated that Muribaculaceae, Alloprevotella, Rikenella and Lachnospiraceae-NK4A136 accounted for the greatest differences among the HBs-HepR, B6-HepR and Fah−/− control groups (Fig. 2C). These results indicated that the gut microbiota composition and abundance were altered by chronic HBV infection, especially increasing Muribaculaceae and Alloprevotella and decreasing Lachnospiraceae-NK4A136 and Rikenella.

Fig. 2
figure 2

Differential fecal microbial communities at the genus level in HBs-HepR mice. A Heatmap of the compositions of the microbial community (top 15) at the genus level in the HBs-HepR, B6-HepR and Fah−/− control groups. B The differential microbial community at the genus level (top 10) among the three groups. C Dot plot of key microbiota at the genus level by random forest analysis

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Changes in the liver microbiota composition and abundance caused by chronic HBV infection

Liver samples were subjected to 2bRAD-M sequencing to analyze the microbiota. The number of ASVs in each group and the number of common ASVs among the three groups were shown (Fig. 3A), and alpha diversity analysis indicated the degree of sample dispersion within each group and the significant differences in indices between HBs-HepR mice and B6-HepR mice and between HBs-HepR mice and Fah−/− control mice (Chao1, p value < 0.01), which indicated a greater degree of species richness in HBs-HepR mice (Fig. 3B). Beta diversity showed significant differences in the distribution of microbiota communities among these three groups (Fig. 3C). Compositions of the liver microbiota community at the phylum level in each group showed decreased Chlamydiota and increased Bacillota and Pseudomonadota abundances in HBs-HepR mice (Fig. 3D). The top 20 ASVs with high abundance in the three groups, as shown by the Circos diagram, illustrated the relationships between liver samples and species, and that there were significant differences among these three groups (Fig. 3E).

Fig. 3
figure 3

Liver microbial diversity and composition of HBs-HepR mice compared with control B6-HepR mice. Liver samples were harvested from HBs-HepR (n = 3), B6-HepR (n = 6) and Fah−/− control mice (n = 4). 2bRAD-M sequencing was used to analyze the microbiota. A Venn diagram of ASVs. The number of ASVs in each group and the number of shared ASVs among the three groups are shown. B Alpha diversity was evaluated using the Wilcoxon rank sum test. C Beta diversity was evaluated using PCoA with binary Jaccard. A dot represents one sample. D Compositions of the microbiota community at the phylum level in each group. (E) Circos diagram illustrating the relationships between samples and species

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Furthermore, the 15 most abundant components of the microbial community at the family and genus levels were shown in Fig. 4A. Differential microbiota were observed in HBs-HepR mice compared with B6-HepR mice, especially Ralstonia, Geobacillus and Cytobacillus, which markedly increased in HBs-HepR mice. Ralstonia and Geobacillus, which are important indicator species, were determined by indicator analysis, and these genera exhibited high relative abundances and indicator values (Fig. 4B). By random forest analysis, 30 key microbiota at the genus level were identified. Ralstonia and Geobacillus were ranked as the top 3 by importance, which could distinguish differences between groups (Fig. 4C). These results indicated that chronic HBV infection significantly increased the relative abundance of Ralstonia and Geobacillus in the liver.

Fig. 4
figure 4

Differential liver microbial communities at the family and genus levels in HBs-HepR mice. A Heatmap of the compositions of the microbial community (top 15) at the family and genus levels in the HBs-HepR, B6-HepR and Fah−/− control groups. B Map of indicative species by indicator analysis. The bar chart represents the relative abundance of each species. The bubble size represents the indicator value of each species in the three groups. C Dot plot of key microbiota at the genus level by random forest analysis. The top 30 most abundant genera were selected, and the R package randomForest was used to construct the map

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Changes in microbial functions in the gut and liver of HBs-HepR mice

Based on 16S rRNA sequencing data from fecal samples, analysis of clusters orthologous groups of proteins (COGs) between HBs-HepR mice and B6-HepR mice revealed 15 clusters, and the differential clusters included increased levels of putative alpha-1,2-mannosidase (COG3537), periplasmic ferric-dicitrate binding protein FerR, regulates iron transport through sigma-19 (COG3712), N-acetyl-beta-hexosaminidase (COG3525), and decreased levels of DNA-binding transcriptional regulator IscR family (COG1959), ABC-type amino acid transport/signal transduction system, periplasmic component/domain (COG0834) and ABC-type transport system, periplasmic component (COG0747) in HBs-HepR mice (Fig. 5A). Differential Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment at level 3 between HBs-HepR mice and B6-HepR mice indicated increased levels of phosphonate and phosphinate metabolism and decreased levels of meiosis-yeast, platinum drug resistance, mineral absorption, dioxin degradation and aminobenzoate degradation in HBs-HepR mice (Fig. 5B). These results indicated that alterations in the composition and abundance of the gut microbiota exhibited functional changes induced by chronic HBV infection.

Fig. 5
figure 5

COG and KEGG functional prediction based on fecal 16S RNA sequencing. A Heatmap of differential clusters of orthologous groups of proteins (COGs) between HBs-HepR mice and B6-HepR mice (top 15). A bar showing the diffserential clustering of COG and mean proportions are shown for HBs-HepR mice and B6-HepR mice. B Heatmap of differential pathways of Kyoto Encyclopedia of Genes and Genomes (KEGG) at level 3 between HBs-HepR mice and B6-HepR mice (top 15). The bars of differential pathways and the mean proportions are shown. PICRUSt2 (2.3.0b0) was used. Statistical analysis was performed with the Wilcoxon signed-rank test

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Based on liver 2bRAD-M sequencing data, KEGG functional prediction indicated that pathways (level 1), including “Organismal Systems”, “Environmental Information Processing”, “Cellular Processes”, “Genetic Information Processing”, “Human Diseases”, and “Metabolism”, were significantly enriched in the livers of HBs-HepR mice (Fig. 6A). Furthermore, 44 differentially expressed pathways (level 2) were identified, as shown in the heatmap (Fig. 6B). The pathways enriched in the liver of HBs-HepR mice were involved in signaling molecules and interaction, lipid metabolism, cancers, immune diseases, immune system, and amino acid metabolism.

Fig. 6
figure 6

KEGG function prediction based on liver 2bRAD-M sequencing. Heatmaps of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways differentially expressed between HBs-HepR mice and B6-HepR mice at level 1 (A) and level 2 (B) are shown

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Decreased propionic acid and acetic acid were negatively related to Geobacillus in the livers of HBs-HepR mice

Samples of fecal, serum and liver tissue were harvested from HBs-HepR, B6-HepR and Fah−/− control mice, and the levels of seven kinds of short-chain fatty acids (SCFAs), including acetic acid, propionic acid, butyric acid, pentanoic acid, hexanoic acid, isobutyric acid and isovaleric acid, were detected by UPLC-ESI–MS/MS. There were no significant differences in the levels of these SCFAs among the fecal and serum samples respectively (sFigure 1A and B). However, the level of propionic acid was significantly lower and the level of acetic acid was slightly lower in the liver samples of HBs-HepR mice than in those of B6-HepR mice and Fah−/− control mice (Fig. 7A). The levels of isobutyric acid and isovaleric acid were decreased in the livers of both HBs-HepR mice and B6-HepR mice compared with those in the livers of Fah−/− control mice, and there were no significant differences between HBs-HepR mice and B6-HepR mice, indicating probably no direct relation to HBV.

Fig. 7
figure 7

Decreased propionic and acetic acid were correlated with the liver microbiota in HBs-HepR mice. Samples of liver tissue were harvested from HBs-HepR (n = 3), B6-HepR (n = 6) and Fah−/− control mice (n = 4), and then the levels of short-chain fatty acids (SCFAs) were detected by UPLC-ESI–MS/MS. A Liver SCFA levels. The data are shown as the mean ± SEM. Comparisons were performed by ANOVA. B Correlation analysis of microbial diversity and metabolomics. A heatmap (left) and network (right) are shown. Pearson correlation was performed

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Furthermore, correlation analysis of microbial diversity and metabolomics indicated that 7 microbiotas at the level of genus negatively related to the level of propionic acid in the liver, including Geobacillus, Brevibacillus-D, Paenibacillus-G, Streptomyces, Sphingobium, Vibrio and Bordetella-B, and 2 microbiotas at the level of genus negatively related to the level of acetic acid in the liver, including Geobacillus and Paenibacillus-G (Fig. 7B). As described above (Fig. 4), chronic HBV infection significantly increased the relative abundance of Geobacillus in the liver, which might account for the reduced levels of propionic acid and acetic acid in the liver.

Amino acid metabolism was modulated by microbiota in the gut and liver differently in HBs-HepR mice

Samples of fecal, serum and liver tissue were harvested from HBs-HepR, B6-HepR and Fah−/− control mice, and then the levels of 19 kinds of amino acids were detected by GC‒MS/MS. There was no significant differences in these 10 kinds of amino acids (sFigure 2); while significantly decreased levels of 9 kinds of amino acids were observed in the fecal samples of HBs-HepR mice, including L-4-hydroxyproline, L-glutamic acid, L-isoleucine, L-cysteine, L-leucine, L-methionine, L-proline, L-threonine and L-serine (Fig. 8A). The levels of these amino acids were positively related to the relative abundances of Ruminococcaceae, Rikenella and Ureaplasma (Fig. 8B). No significant differences in the relative abundances of Ruminococcaceae or Ureaplasma were detected (data not shown). As described above in Fig. 2A, significantly decreased abundance of Rikenella was observed in the gut microbiota of HBs-HepR mice, which might be responsible for the decreased levels of amino acids in the gut.

Fig. 8
figure 8

Reduced levels of amino acids in the gut correlated with the gut microbiota of HBs-HepR mice. Fecal samples were harvested from HBs-HepR (n = 3), B6-HepR (n = 6) and Fah−/− control mice (n = 4). The levels of 19 amino acids, including L-4-hydroxyproline, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, were detected by GC‒MS/MS. A Levels of 9 differential amino acids. These Comparisons were performed by ANOVA. B Correlations between differential amino acids and the gut microbiota community

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The serum levels of amino acids differed from those in the gut, indicating that alterations in fecal samples were regional effects of the gut microbiota. A significantly increased level of L-glycine was observed in the serum and liver of HBs-HepR mice but not in that of B6-HepR mice (Fig. 9A). Decreased levels of L-tyrosine were observed in the serum and liver of both HBs-HepR mice and B6-HepR mice (sFigure 3A and B). These results indicated that chronic HBV infection upregulated the level of L-glycine in the liver and serum. The level of L-glycine was positively related to the abundance of Geobaillus and Paenibaillus-G in the liver (Fig. 9B). As shown in Fig. 4, significantly increased Geobacillus was observed in the liver of HBs-HepR mice, which modulated the amino acid metabolism in the liver particularly.

Fig. 9
figure 9

Increased L-glycine was positively correlated with Geoballius and Paeniballius in the liver of HBs-HepR mice. Liver and serum samples were harvested from HBs-HepR (n = 3), B6-HepR (n = 6) and Fah−/− control mice (n = 4). The levels of 19 amino acids were detected by GC‒MS/MS. A Levels of L-glycine in the serum and liver tissue. Comparisons were performed by ANOVA. B Correlation between differential amino acids and the liver microbiota community

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Discussion

In this study, a novel HBV-carrier mouse model named HBs-HepR mice was used, generated by adoptively transferring HBsAg+ hepatocytes via spleen injection into Fah−/− recipient mice. Microbiome and metabolome analysis of the gut and liver were performed 18 weeks after hepatocyte transfer, a state of chronic HBV infection with CD8+T cell-mediated hepatitis, and showed imbalanced SCFA and amino acid metabolism by modulating the microbiota, especially in the targeted organ liver. Significantly decreased propionic acid, slightly decreased acetic acid, and significantly increased L-glycine were observed in the livers of HBs-HepR mice, which was related to the abundance of Geobaillus (Fig. 10).

Fig. 10
figure 10

Imbalanced SCFAs and amino acids via microbiota modulation in the liver and gut of HBs-HepR mice. The results in this study were summarized schematically. By using the HBs-HepR mice 18 weeks after hepatocyte transfer, a state of chronic HBV infection with CD8+ T cell-mediated hepatitis, microbiome and metabolome analysis of the gut and liver were performed. Differential microbiota at the genus level and related metabolites SCFAs and amino acid were shown

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Chronic HBV infection altered the gut microbiota composition and abundance, especially increasing Muribaculaceae and Alloprevotella and decreasing Lachnospiraceae-NK4A136 and Rikenella (Figs. 1, 2). Multiple studies have explored the composition and abundance of the microbiota in various liver diseases and have suggested that a critical role in the pathogenesis of HBV-related liver diseases is associated with a reduction in beneficial microbiota and an increase in pathogenic microbiota [21,22,23]. At the phylum level, the ratio of Firmicutes to Bacteroidetes was greater in patients with resolved HBV infection, CHB, and advanced liver disease than in healthy individuals [23]. In our study, at the time of triggering the specific anti-HBV CD8+ T cell response, the ratio of Firmicutes to Bacteroidota (F/B ratio) was slightly decreased in HBs-HepR mice (Fig. 1D), suggesting dynamic alterations in the gut microbiota during the progression of HBV infection and related liver diseases. However, the gut microbiota was only analyzed 18 weeks after hepatocyte transfer, lacking the analysis on 12 weeks the time of the successful hepatocyte reconstruction. The vertical comparison is very important and necessary, which should be investigated in future work. The intrahepatic immune cells were examined by single-cell RNA analysis in HBs-HepR mice 12 and 18 weeks after hepatocyte transfer, indicating the dramatically activated CD8+ T cells at 18 weeks post-transfer [19]. The relationship among HBV, gut microbiota and immune responses has not been fully studied.

The abundance of the genus Muribaculaceae in the gut was positively correlated with the number of peripheral circulating CD3+, CD4+ and CD8+ T cells [24]. Here, marked increase in Muribaculaceae was observed in the gut of HBs-HepR mice, which was consistent with the generation of HBsAg-specific CD8+ T cells [19]. Whether the increased microbiota actually influence the HBsAg-specific CD8+ T cell responses and hepatitis should be further studied by antibiotic treatments. In our pervious study, we demonstrated that microbiota maintained anti-HBV immunity by affecting the functions of effector CD4+ T cells including IFN-γ secretion in the hydrodynamical HBV transfection mouse model [11]. Alloprevotella was predominant in HBV-infected patients with a low viral load compared to healthy controls [6, 25], which is also consistent with our findings in HBs-HepR mice (Fig. 2). Lachnospiraceae-NK4A136 and Rikenella are potential probiotic microbiota; however, their alterations in the gut microbiota during HBV infection are poorly known. Here, our study further indicated markedly fewer gut Rikenella in chronic HBV-carrier HBs-HepR mice (Fig. 2A), and Lachnospiraceae-NK4A136 was markedly increased in the control B6-HepR mice but not in the HBs-HepR mice (Fig. 2A). These results help to elucidate the association between HBV infection and the gut microbiota comprehensively.

There are few studies on the liver microbiota during HBV infection and HBV-related liver diseases. In this study, at the genus level, significant changes in the abundances of the Ralstonia and Geobacillus bacterial groups in the liver were revealed, which was unlike the changes in the gut. Ralstonia was significantly decreased in the gut microbiota by drug-induced liver injury and was positively correlated with cortodoxone, prostaglandin I1, bioprostaglandin E2 and anacardic acid; however, the microbiota in the liver was not investigated [26]. The abundance of the genus Geobacillus was significantly greater in the biliary microbiota of extrahepatic cholangiocarcinoma patients, suggesting its involvement in disease pathogenesis [27]. Thus, it was speculated that Ralstonia and Geobacillus in the liver are involved in the pathogenesis of chronic HBV infection.

In this study, no significant differences in SCFAs by gut microbiota were detected, while a significant decrease in propionic acid and a slight decrease in acetic acid were induced by the microbiota in the liver of HBs-HepR mice compared with those in the controls (sFigure 1 and Fig. 7). Propionate was positively associated with liver fibrosis in patients with metabolic dysfunction-associated steatotic liver disease; however, the potential underlying mechanisms were not elucidated [28]. Intestinal microbiota-derived propionic acid plays a protective role in lung injury, and supplementation with propionic acid is a potential therapeutic strategy [29]. Acetic acid was reported to be associated with the early recurrence of HBV-related HCC since acetic acid derived from the gut microbiota supplies energy for tumor cell growth and proliferation in the liver [30]. Potential metabolite biomarkers have been reported for distinguishing between the immune tolerance and immune clearance phases during chronic HBV infection, including 3-cycloheptene-l-acetic acid, which significantly increases in the immune clearance phase compared with the immune tolerance phase [31]. Thus, the precise roles of decreased propionic acid and acetic acid in the liver during chronic HBV infection deserve further investigation.

Several kinds of amino acids were decreased in the gut of HBs-HepR mice compared with those of the controls (Fig. 8), and only was L-glycine significantly increased in the liver of HBs-HepR mice compared with that of the controls (Fig. 9). L-Glycine, an anti-inflammatory, immunomodulatory and cytoprotective amino acid, can diminish liver injury caused by hepatic toxicants and drugs [32,33,34]. Glycine can inactivate Kupffer cells and decrease their production of TNF-α, which protects partial liver grafts from ischemia‒reperfusion injury mediated by Kupffer cells, but has no negative effects on hepatocyte regeneration [35]. Kupffer cells are involved in immune activation and immune tolerance to HBV [36, 37]. Therefore, it is speculated that the specific immune response to HBV in the livers of HBs-HepR mice may be regulated by metabolites such as glycine derived from the local microbiota.

Conclusions

By using a novel mouse model, it was found that key microbiota in the liver led to significant changes in SCFAs and amino acids, unlike in the gut, which was involved in the immune activation phase of chronic HBV infection. The exact roles of propionic acid, acetic acid and L-glycine in the progression of chronic HBV infection and HBV-related liver diseases deserve further exploration, which will be important to regulating the host microbiota and promoting the clearance of HBV and disease control in humans.

Availability of data and materials

The data that support the findings of this study are available in the article or from the corresponding author upon reasonable request.

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Acknowledgements

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Funding

This work was supported by National Key R&D Program of China (2022YFC2304502); Natural Science Foundation of China (82388201, 82071764).

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Authors and Affiliations

  1. Key Laboratory of Immune Response and Immunotherapy, the Institute of Immunology, Biomedical Sciences and Health Laboratory of Anhui Province, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, #443 Huangshan Road, Hefei, 230027, Anhui, China

    Wendi Zhang, Yuwei Wu, Min Cheng, Haiming Wei, Rui Sun, Hui Peng, Zhigang Tian & Yongyan Chen

  2. Department of Geriatrics, Gerontology Institute of Anhui Province, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China

    Min Cheng

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Contributions

W. Zhang contributed the experimental animal and manuscript preparation. M. Cheng contributed the performance of experiments and the figures. Y. Wu contributed the animal experiments. H. Wei contributed the experiment designments. R. Sun contributed the data analysis. H. Peng contributed the experiment designments. Z. Tian contributed the data analysis and manuscript writing. Y. Chen contributed the designment and performance of all the experiments, data analysis and manuscript preparation.

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Zhang, W., Wu, Y., Cheng, M. et al. Chronic hepatitis B virus infection imbalances short-chain fatty acids and amino acids in the liver and gut via microbiota modulation. Gut Pathog 17, 18 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13099-025-00695-3

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Gut Pathogens

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