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Abstract
Objective Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH.
Design C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated.
Results We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract).
Conclusion Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.
- FIBROSIS
- ALCOHOLIC LIVER DISEASE
- HEPATIC STELLATE CELL
- MACROPHAGES
Data availability statement
All other data relevant to the study are included in the article or uploaded as supplementary information.
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Data availability statement
All other data relevant to the study are included in the article or uploaded as supplementary information.
Footnotes
X @MrigyaBabuta
Contributors MB and GS designed the study. MB performed the experiments, analysed and interpreted results. CM provided scientific inputs for this study and contributed in planning experimental models. MdCR contributed in planning experimental models. CC, DH, CC, JM, YW, AAD and VB processed liver tissues and performed liver protein and RNA isolation. MO-R performed liver perfusions. YZ performed hepatic stellate cell isolations. PTN, YC and RJ performed flow cytometry and analysis. YW performed bioinformatic analysis. IN performed pathological screening of the liver sections. GS is the principal investigator and the guarantor of the study and was responsible for study concept, design, data analysis and interpretation and procuring funds. GS and MB wrote the manuscript. All the authors read the manuscript and approved its submission.
Funding Research reported in this publication was supported by the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under award numbers R01AA017729, R01AA011576 and U01AA026933 to GS. Microscopy and Histopathology Core B of the Harvard Digestive Disease Center at the Beth Israel Deaconess Medical Center received National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, grant P30DK034854.
Competing interests GS reports being a paid consult for Durect Corporation, Cyta Therapeutics, Generon, Terra Firma, Quest Diagnostics, Pandion Therapeutics, Surrozen, Merck, Novartis, Pfizer, Lab Corp, Intercept and Takeda. She has stock options in Glympse Bio, Satellite Bio and Ventyx.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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