Role of myeloid cell transcription factor EB in alcohol-induced liver injury in mice

Abstract

Background and aims: Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality worldwide, with no currently effective treatment. ALD is caused by excessive lipid buildup, which eventually triggers inflammation and fibrosis in the liver. Activation of hepatic Kupffer cells (KCs) and macrophages drives liver inflammation, which can worsen alcohol-induced liver injury. The autophagy-lysosome system is crucial for macrophages to support their innate immune functions. Transcription factor EB (TFEB) is a key regulator of autophagy and lysosomal biogenesis, but the role of macrophage TFEB in ALD development is unknown. The aim of this study was to evaluate the effects of Gao-binge alcohol consumption on myeloid cell TFEB and elucidate the role of myeloid TFEB in ALD.

Methods: Two-to-three-month-old male and female LysM Cre^- (WT) and LysM Cre⁺ Tfeb ^{Flox/Flox (f/f)} (myeloid-Tfeb KO) mice were subjected to chronic alcohol feeding plus an acute binge following the Gao-binge model. Serum alanine aminotransferase, aspartate aminotransferase, triglycerides, and cholesterol content were determined using biochemical assays. Total hepatic protein content and messenger RNA (mRNA) levels of autophagy-related proteins and inflammatory markers were determined using immunoblotting, immunohistochemistry, and real-time quantitative polymerase chain reaction (RT-qPCR). Isolated hepatic infiltrating macrophages and KCs from mice given intragastric ethanol infusions were analyzed by Western blot for TFEB and autophagy-related protein content. Raw 264.7 macrophages were treated with ethanol, lipopolysaccharide (LPS), and LPS plus ethanol to examine nuclear TFEB translocation using immunofluorescence.

Results: We found that TFEB levels were higher in macrophage/KC cells than in hepatocytes and cholangiocytes. While ethanol feeding increased serum alanine aminotransferase and aspartate aminotransferase levels, as well as hepatic triglyceride levels, no significant differences were observed between WT and myeloid-Tfeb KO mice. The number of F4/80-positive KCs/macrophages was similar in all four experimental groups, but hepatic neutrophil infiltration increased in alcohol-fed myeloid-Tfeb KO mice. LPS or ethanol alone induced nuclear TFEB translocation only moderately in Raw 264.7 macrophages.

Conclusions: Our findings suggest that myeloid TFEB is dispensable for alcohol-induced liver injury in mice.

Keywords

Autophagy / Hepatitis / Inflammation / Lysosome / Steatosis / Transcription factor EB (TFEB)

Authors’ contributions

Sha Neisha Williams: Writing - original draft, Validation, Investigation, Formal analysis, Data curation. Kafayat Yusuf: Investigation, Formal analysis, Data curation. Xiaojuan Chao: Formal analysis, Data curation. Hong-Min Ni: Resources, Meth-odology, Data curation, Conceptualization. Wen-Xing Ding: Writing - review & editing, Validation, Supervision, Funding acquisition, Formal analysis, Conceptualization.

Data availability statement

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Declaration of competing interest

Wen-Xing Ding is an associate editor for Liver Research and was not involved in the editorial review or the decision to publish this article. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was partly supported by the National Institutes of Health funds R37 AA020518, R21 AA030617, and R01AA031230 (WXD). Sha Neisha Williams was supported by NIH/NIAAA F31AA031623. Migrated vs. resident hepatic macrophages isolated from the mouse models were provided by the Cell Bank Repository of the NIAAA-supported Integrative Liver Cell Core (R24AA012885) of the University of Southern California.

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