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Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis

  • Yeonhee Cho
    Affiliations
    Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA
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  • Terence Ndonyi Bukong
    Affiliations
    Armand-Frappier Sante Biotechnologie Research Center, Institut National de la Recherche Scientifique, Laval, Quebec, Canada
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  • David Tornai
    Affiliations
    Division of Gastroenterology, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
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  • Mrigya Babuta
    Affiliations
    Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA
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  • Ioannis S. Vlachos
    Affiliations
    Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA

    Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Broad Institute of MIT and Harvard, Cambridge, MA, USA
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  • Eleni Kanata
    Affiliations
    Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA

    Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Broad Institute of MIT and Harvard, Cambridge, MA, USA
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  • Donna Catalano
    Affiliations
    Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
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  • Gyongyi Szabo
    Correspondence
    Corresponding author. Address: 330 Brookline Avenue, ST-214B, Boston, MA 02215, USA. Tel.: 1 617 667-9050.
    Affiliations
    Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA

    Broad Institute of MIT and Harvard, Cambridge, MA, USA
    Search for articles by this author
Published:September 02, 2022DOI:https://doi.org/10.1016/j.jhep.2022.08.029

      Highlights

      • Alcohol directly induces neutrophil extracellular trap formation in neutrophils.
      • Alcohol-induced neutrophil extracellular trap production increases low-density neutrophils in AH.
      • Low-density neutrophils in AH have a defective phenotype and exhibit reduced homing and clearance.
      • High-density neutrophils in AH have an activated phenotype.

      Background & Aims

      In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH.

      Methods

      We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics.

      Results

      We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release in vitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs in vivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice.

      Conclusion

      Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH.

      Impact and implications

      In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

      Graphical abstract

      Keywords

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