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Longitudinal multi-omics analyses of the gut–liver axis reveals metabolic dysregulation in hepatitis C infection and cirrhosis

Nature Microbiology volume 8, pages 12–27 (2023)Cite this article

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Abstract

The gut and liver are connected via the portal vein, and this relationship, which includes the gut microbiome, is described as the gut–liver axis. Hepatitis C virus (HCV) can infect the liver and cause fibrosis with chronic infection. HCV has been associated with an altered gut microbiome; however, how these changes impact metabolism across the gut–liver axis and how this varies with disease severity and time is unclear. Here we used multi-omics analysis of portal and peripheral blood, faeces and liver tissue to characterize the gut–liver axis of patients with HCV across a fibrosis severity gradient before (n = 29) and 6 months after (n = 23) sustained virologic response, that is, no detection of the virus. Fatty acids were the major metabolites perturbed across the liver, portal vein and gut microbiome in HCV, especially in patients with cirrhosis. Decreased fatty acid degradation by hepatic peroxisomes and mitochondria was coupled with increased free fatty acid (FFA) influx to the liver via the portal vein. Metatranscriptomics indicated that Anaerostipes hadrus-mediated fatty acid synthesis influences portal FFAs. Both microbial fatty acid synthesis and portal FFAs were associated with enhanced hepatic fibrosis. Bacteroides vulgatus-mediated intestinal glycan breakdown was linked to portal glycan products, which in turn correlated with enhanced portal inflammation in HCV. Paired comparison of patient samples at both timepoints showed that hepatic metabolism, especially in peroxisomes, is persistently dysregulated in cirrhosis independently of the virus. Sustained virologic response was associated with a potential beneficial role for Methanobrevibacter smithii, which correlated with liver disease severity markers. These results develop our understanding of the gut–liver axis in HCV and non-HCV liver disease aetiologies and provide a foundation for future therapies.

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Fig. 1: Study design.
Fig. 2: Longitudinal evaluation revealed reduced hepatic metabolism in peroxisomes and mitochondria alongside higher circulatory levels of corresponding metabolites in HCVi compared with SVR.
Fig. 3: Fibrosis was linked to decreased hepatic FA metabolism, and direct portal pressure to increased portal FFAs independent of HCV, n = 22.
Fig. 4: Only portal metabolomics clustered patients with HCVi into early and advanced fibrosis. HCVi disease severity was linked to increased microbial FA synthesis and glycan degradation driven by transcriptionally active A. hadrus and B. vulgatus.
Fig. 5: In HCVi, transcriptionally active A. hadrus and B. vulgatus were directly linked to portal FFA and glycan products, respectively. Microbially derived portal signals and disease-associated microbial functions correlated with enhanced circulatory and hepatic pathways of inflammation.
Fig. 6: A beneficial role of Methanobrevibacter and methane metabolism reduced in fibrosis after SVR, n = 23.

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Data availability

Please note that the microbial and liver transcriptome sequence and microbial 16S rRNA sequence dataset has been made available in the BioProject repository. The accession number for this repository is PRJNA727609. The serum metabolomics data have been uploaded as source data (SourceData_Metabolites_IndividualCohorts and SourceData_Metabolites_PairedCohorts). Additional minimum input data necessary to interpret the figures and findings have been provided as source data where appropriate. Homo sapiens hg38 reference genome was sourced from https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/. Source data are provided with this paper.

Code availability

There was no custom code or mathematical algorithm utilized in this study.

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Acknowledgements

We thank the patients for participation; staff for support; M. W. Krause, J. E. Balow and T. J. Liang for institutional support; J. H. Hoofnagle, J. Hanover and J. Lack for critical revision of the manuscript; and the institutional review board for approving the protocol. Financial support was provided by the intramural programmes of the National Institute of Diabetes and Digestive and Kidney Diseases (DK054514) (T.H.), National Cancer Institute and Clinical Center of the National Institutes of Health. In addition, the project was funded by an intramural NIH Bench to Bedside and Back Program Award: Mechanisms of microbial translocation in hepatitis C related liver disease 2014 (T.H.).

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Authors and Affiliations

  1. Translational Hepatology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Rabab O. Ali, Gabriella M. Quinn, James A. Haddad, Grace Y. Zhang, Elizabeth C. Townsend, Lisa Scheuing, Kareen L. Hill, Meital Gewirtz, Ohad Etzion & Theo Heller

  2. Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Regina Umarova & Christopher Koh

  3. Immunology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

    Shakuntala Rampertaap & Sergio D. Rosenzweig

  4. Cardiovascular and Pulmonary Branch of the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Alan T. Remaley

  5. Computational Medicine Section, Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Jung Min Han & Vipul Periwal

  6. Clinical Mass Spectrometry Core, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Hongyi Cai & Peter J. Walter

  7. Center for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Bethesda, MD, USA

    Elliot B. Levy

  8. Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    David E. Kleiner

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Contributions

All authors had a substantial contribution to this work. All authors provided approval for the final submitted version of the manuscript. Acquisition, analysis and interpretation of data were performed by all authors. G.M.Q., R.U., J.A.H., E.C.T., M.G. and O.E. substantively revised the work. O.E. contributed to the design of the work. R.O.A. and T.H. were responsible for the design of the work, the acquisition, analysis and interpretation of data, drafted the initial work and substantively revised it. Additionally, T.H. was responsible for conception of the work.

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Correspondence to Rabab O. Ali or Theo Heller.

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Extended data

Extended Data Fig. 1 Graphical Abstract.

Hepatic metabolism in peroxisomes and mitochondria is decreased in chronic HCV infected patients (HCVi) compared to SVR. Fibrosis and necroinflammation in HCVi were linked to increased transcriptional activity of Anaerostipes hadrus mediated fatty acid synthesis and Bacteroides vulgatus mediated intestinal glycan degradation. Microbial-derived fatty acids and glycan products are elevated in portal circulation and linked to enhanced portal and hepatic inflammation in HCVi. Despite decreased hepatic and portal inflammation six months after SVR, hepatic metabolism and peroxisome function remains decreased in SVR with advanced fibrosis. Methanobrevibacter smithii showed decreased function in SVR fibrosis and may have anti-inflammatory properties.

Extended Data Fig. 2 Biochemical and histological markers of inflammation were elevated in HCVi compared to SVR.

Wilcoxon matched pairs signed rank test, two-sided unadjusted p-value. Compared to SVR, HCVi patients had elevated serum markers of hepatocellular inflammation (ALT, alanine aminotransferase p < 0.0001; AST, aspartate aminotransferase p < 0.0001; GGT, gamma-glutamyl transferase p = 0.0001); histological marker of inflammation (HAI, Hepatic Activity index) p < 0.0001; and elevated serum total bilirubin p = 0.0010. HCVi showed no significant difference in fibrosis p = 0.2483, direct portal pressures p = 0.8175, or alkaline phosphatase p = 0.1172. Scatter plots with bars, data is presented as median values + /- IQR. n = 22.

Extended Data Fig. 3 Decreased hepatic fatty acid degradation in HCVi when compared to SVR.

Graphical representation using GAGE R of the hepatic KEGG functional pathway ‘Fatty Acid Degradation’ significantly downregulated in HCVi compared to SVR (FDRp < 0.1). Within each pathway significant DEGs are highlighted blue for fold-change >0 and red for fold-change<0 in HCVi when compared with SVR (FDRp < 0.1). n = 22.

Extended Data Fig. 4 No significant difference in gut microbial composition at phylum level based on presence of cirrhosis in HCVi or SVR.

Fecal 16 S rRNA analysis was performed on fecal samples. Relative abundance of phyla plotted in pie-charts for patient subgroups based on cirrhosis. a) HCVi-Cirr (n = 13) and HCVi-NC (n = 16). b) SVR-Cirr (n = 9) and SVR-NC (n = 14). Analysis performed using QIIME.

Extended Data Fig. 5 Microbial metatranscriptome analysis revealed distinct microbial functions associated with hepatocellular injury in HCVi.

Within HCVi cohort, AST (a) and GGT (b) significantly correlated with microbial KEGG functional modules for glycan degradation including heparan and dermatan sulfate degradation (SCCbg.adj, two-sided, FDRp<0.1). n = 26.

Extended Data Fig. 6 Serum markers of gut dysbiosis and intestinal dysfunction elevated in HCVi when compared to SVR.

Paired comparison of serum IL18 (marker of dysbiosis) and zonulin (marker of gut epithelial integrity) between HCVi and SVR cohorts (two-sided Wilcoxon matched pairs signed rank test, HCVi vs. SVR). Scatter plots with bars, data is presented as median values + /- IQR. Relative quantification of metabolites per Metabolon protocol. n = 23.

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Video illustration of a patient in the HCVi cohort undergoing portal vein cannulation.

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Ali, R.O., Quinn, G.M., Umarova, R. et al. Longitudinal multi-omics analyses of the gut–liver axis reveals metabolic dysregulation in hepatitis C infection and cirrhosis. Nat Microbiol 8, 12–27 (2023). https://doi.org/10.1038/s41564-022-01273-y

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