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
Graphical Abstract
Keywords
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Article info
Publication history
Published online: September 02, 2022
Accepted:
August 17,
2022
Received in revised form:
August 12,
2022
Received:
July 22,
2021
Identification
Copyright
© 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
