Neutrophil-to-hepatocyte communication via LDLR-dependent miR-223–enriched extracellular vesicle transfer ameliorates nonalcoholic steatohepatitis
Yong He, … , George Kunos, Bin Gao
Yong He, … , George Kunos, Bin Gao
Published December 10, 2020
Citation Information: J Clin Invest. 2021;131(3):e141513. https://doi.org/10.1172/JCI141513.
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Research Article Hepatology

Neutrophil-to-hepatocyte communication via LDLR-dependent miR-223–enriched extracellular vesicle transfer ameliorates nonalcoholic steatohepatitis

Abstract

Neutrophil infiltration around lipotoxic hepatocytes is a hallmark of nonalcoholic steatohepatitis (NASH); however, how these 2 types of cells communicate remains obscure. We have previously demonstrated that neutrophil-specific microRNA-223 (miR-223) is elevated in hepatocytes to limit NASH progression in obese mice. Here, we demonstrated that this elevation of miR-223 in hepatocytes was due to preferential uptake of miR-223–enriched extracellular vesicles (EVs) derived from neutrophils as well other types of cells, albeit to a lesser extent. This selective uptake was dependent on the expression of low-density lipoprotein receptor (LDLR) on hepatocytes and apolipoprotein E (APOE) on neutrophil-derived EVs, which was enhanced by free fatty acids. Once internalized by hepatocytes, the EV-derived miR-223 acted to inhibit hepatic inflammatory and fibrogenic gene expression. In the absence of this LDLR- and APOE-dependent uptake of miR-223–enriched EVs, the progression of steatosis to NASH was accelerated. In contrast, augmentation of this transfer by treatment with an inhibitor of proprotein convertase subtilisin/kexin type 9, a drug used to lower blood cholesterol by upregulating LDLR, ameliorated NASH in mice. This specific role of LDLR and APOE in the selective control of miR-223–enriched EV transfer from neutrophils to hepatocytes may serve as a potential therapeutic target for NASH.

Authors

Yong He, Robim M. Rodrigues, Xiaolin Wang, Wonhyo Seo, Jing Ma, Seonghwan Hwang, Yaojie Fu, Eszter Trojnár, Csaba Mátyás, Suxian Zhao, Ruixue Ren, Dechun Feng, Pal Pacher, George Kunos, Bin Gao

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Figure 3

Fatty acid (e.g., palmitic acid [PA]) promotes neutrophil transfer of miR-223 to hepatocytes.

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Fatty acid (e.g., palmitic acid [PA]) promotes neutrophil transfer of mi...
(A) After pretreatment with PA (0.3 mM) for 18 hours, AML12 cell medium was replaced with fresh serum-free medium and the cells cocultured with neutrophils for another 6 hours. miR-223 levels in AML12 cells were measured by RT-qPCR. (B and C) AML12 cells were pretreated with vehicle or PA (0.3 mM) for 18 hours, followed by incubating with DiD-labeled neutrophil-derived EVs for 24 hours. Representative images of DiD fluorescence (red) and nuclei (DAPI, blue) are shown in panel B, and mean fluorescence intensity per cell is quantified in panel C. Scale bars: 20 μm. (D) AML12 cells were treated with vehicle or PA (0.3 mM) for 3 hours, and analyzed by RT-qPCR for several endocytosis-related genes. (E) AML12 cells were pretreated with vehicle or PA (0.3 mM) for 18 hours, followed by incubating with the DiD-labeled neutrophil-derived EVs for 24 hours and staining with an anti-LDLR antibody. Representative images of DiD fluorescence (red), LDLR immunofluorescence (green), and nuclei (DAPI, blue) are shown. Scale bars: 20 μm. Values represent means ± SEM from 3–4 independent experiments. **P < 0.01, ***P < 0.001. Significance was determined by 1-way ANOVA followed by Tukey’s post hoc test for multiple groups (A) and a 2-tailed Student’s t test for comparing 2 groups (C and D). ND, not detectable.