Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

  1. Robert N Helsley
  2. Tatsunori Miyata
  3. Anagha Kadam
  4. Varadharajan Venkateshwari
  5. Naseer Sangwan
  6. Emily C. Huang
  7. Rakhee Banerjee
  8. Amanda L Brown
  9. Kevin K Fung
  10. William Massey
  11. Chase Neumann
  12. Orabi Danny
  13. Lucas J Osborn
  14. Rebecca C Schugar
  15. Megan R. McMullen
  16. Annette Bellar
  17. Kyle L. Poulsen
  18. Adam Kim
  19. Vai Pathak
  20. Marko Mrdjen
  21. James T Anderson
  22. Belinda Willard
  23. Craig J. McClain
  24. Mack Mitchell
  25. Arthur J. McCullough
  26. Svetlana Radaeva
  27. Bruce Barton
  28. Gyongyi Szabo
  29. Srinivasan Dasarathy
  30. Jose Carlos Garcia-Garcia
  31. Daniel M. Rotroff
  32. Daniela S Allende
  33. Zeneng Wang
  34. Stanley L Hazen
  35. Laura E Nagy
  36. Jonathan Mark Brown  Is a corresponding author
  1. Cleveland Clinic Lerner College of Medicine, United States
  2. Cleveland Clinic, United States
  3. University of Louisville, United States
  4. The University of Texas Southwestern Medical Center, United States
  5. National Institute of Health, United States
  6. University of Massachusetts Medical School, United States
  7. Beth Israel Deaconess Medical Center, United States
  8. Procter and Gamble, United States
  9. Cleveland Clinic, Lerner Research Institute, United States

Abstract

There is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Data availability

Sequencing data have been deposited in GEO under accession code GSE157681.

The following data sets were generated

Article and author information

Author details

  1. Robert N Helsley

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    Robert N Helsley, reports being a paid consultant for the University of Cincinnati..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5000-3187
  2. Tatsunori Miyata

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  3. Anagha Kadam

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  4. Varadharajan Venkateshwari

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  5. Naseer Sangwan

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  6. Emily C. Huang

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  7. Rakhee Banerjee

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  8. Amanda L Brown

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  9. Kevin K Fung

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  10. William Massey

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2087-6048
  11. Chase Neumann

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  12. Orabi Danny

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  13. Lucas J Osborn

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  14. Rebecca C Schugar

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  15. Megan R. McMullen

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  16. Annette Bellar

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  17. Kyle L. Poulsen

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    Kyle L. Poulsen, Grant support from NIH.
  18. Adam Kim

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  19. Vai Pathak

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  20. Marko Mrdjen

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  21. James T Anderson

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  22. Belinda Willard

    Research Core Services, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  23. Craig J. McClain

    University of Louisville, Louisville, United States
    Competing interests
    No competing interests declared.
  24. Mack Mitchell

    The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  25. Arthur J. McCullough

    Research Core Services, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  26. Svetlana Radaeva

    National Institute of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  27. Bruce Barton

    University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7878-8895
  28. Gyongyi Szabo

    Beth Israel Deaconess Medical Center, Boston, United States
    Competing interests
    Gyongyi Szabo, G.S. reports being a paid consult for Allergan, Alnylam, Arrow, Durcect Corporation, Generon, Glympse Bio, Terra Firma, Quest Diagnostics, Pandion Therapeutics, Surrozen, and Zomagen. G.S. has received grants from Gilead, Genfit, Intercept, Novartis, SignaBlok, and Shire; she also holds intellectual property rights with Up to Date..
  29. Srinivasan Dasarathy

    Pathobiology, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1774-0104
  30. Jose Carlos Garcia-Garcia

    Life Sciences Transformative Platform Technologies, Procter and Gamble, Cincinatti, United States
    Competing interests
    Jose Carlos Garcia-Garcia, is an employee of The Procter & Gamble Co..
  31. Daniel M. Rotroff

    Research Core Services, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    No competing interests declared.
  32. Daniela S Allende

    Department of Anatomical Pathology, Cleveland Clinic, Cleveland, United States
    Competing interests
    Daniela S Allende, Kaiser Permanente (CME lecture sessions)Advisory Board for Incyte (on treatment of cholangiocarcinoma).
  33. Zeneng Wang

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    Zeneng Wang, Z.W. report being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. Z.W. reports being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Zehna Therapeutics, Cleveland Heart Lab, a wholly owned subsidiary of Quest Diagnostics, and Procter & Gamble..
  34. Stanley L Hazen

    Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    Competing interests
    Stanley L Hazen, S.L.H. report being named as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. S.L.H. reports being a paid consultant for Zehna Therapeutics, having received research funds from Zehna Therapeutics, Procter & Gamble, and Roche Diagnostics. S.L.H. report being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Zehna Therapeutics, Cleveland Heart Lab, a wholly owned subsidiary of Quest Diagnostics, and Procter & Gamble..
  35. Laura E Nagy

    IDepartment of Inflammation and Immunity, Cleveland Clinic, Lerner Research Institute, Cleveland, United States
    Competing interests
    No competing interests declared.
  36. Jonathan Mark Brown

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner College of Medicine, Cleveland, United States
    For correspondence
    brownm5@ccf.org
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2708-7487

Funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All mice were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility. All experimental protocols were approved by the institutional animal care and use committee (IACUC) at the Cleveland Clinic.

Human subjects: Patients with AH were classified as moderate (MELD < 20, n=112) and severe (MELD {greater than or equal to}20, n=152) according to the MELD score at admission as part of either of two independent clinical trials (ClincalTrials.gov identifier # NCT01809132 and NCT03224949) or the NOAC biorepository. These studies were approved by the Institutional Review Boards of all 4 participating institutions and all study participants consented prior to collection of data and blood samples. Written informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Boards at Johns Hopkins Medical Institutions.

Reviewing Editor

  1. Hossein Ardehali, Northwestern University, United States

Publication history

  1. Received: December 21, 2021
  2. Accepted: December 31, 2021
  3. Preprint posted: January 1, 2022 (view preprint)
  4. Accepted Manuscript published: January 27, 2022 (version 1)
  5. Accepted Manuscript updated: February 4, 2022 (version 2)
  6. Version of Record published: February 17, 2022 (version 3)

Copyright

© 2022, Helsley et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Robert N Helsley
  2. Tatsunori Miyata
  3. Anagha Kadam
  4. Varadharajan Venkateshwari
  5. Naseer Sangwan
  6. Emily C. Huang
  7. Rakhee Banerjee
  8. Amanda L Brown
  9. Kevin K Fung
  10. William Massey
  11. Chase Neumann
  12. Orabi Danny
  13. Lucas J Osborn
  14. Rebecca C Schugar
  15. Megan R. McMullen
  16. Annette Bellar
  17. Kyle L. Poulsen
  18. Adam Kim
  19. Vai Pathak
  20. Marko Mrdjen
  21. James T Anderson
  22. Belinda Willard
  23. Craig J. McClain
  24. Mack Mitchell
  25. Arthur J. McCullough
  26. Svetlana Radaeva
  27. Bruce Barton
  28. Gyongyi Szabo
  29. Srinivasan Dasarathy
  30. Jose Carlos Garcia-Garcia
  31. Daniel M. Rotroff
  32. Daniela S Allende
  33. Zeneng Wang
  34. Stanley L Hazen
  35. Laura E Nagy
  36. Jonathan Mark Brown
(2022)
Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice
eLife 11:e76554.
https://doi.org/10.7554/eLife.76554

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