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 2

Neutrophils transfer miR-223 to hepatocytes via EVs.

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Neutrophils transfer miR-223 to hepatocytes via EVs. (A–C) WT mice were...
(A–C) WT mice were fed HFD for 2.5 months, and then daily intraperitoneally injected with isotype antibody or combined (Combo) antibodies (rat anti–mouse Ly6G antibody and anti-rat secondary antibody). Liver, circulating blood leukocytes, and serum samples were collected 24 hours after the last injection (n = 5 in isotype group, n = 7 in combined group). (A) The percentage of circulating neutrophils (CD11b+Gr-1+/CD45+ cells), and serum miR-223 levels were quantified. (B) The percentage of liver neutrophils (CD11b+Gr-1+/CD45+ cells) and the percentage of miR-223+ hepatocytes (which were detected by miR-223 in situ hybridization) were quantified. (C) RT-qPCR analyses of inflammatory, fibrogenic, and miR-223 target genes in the liver samples. (D) Mouse hepatocyte line AML12 cells were cocultured with WT or miR-223KO neutrophils for 6 hours, and then miR-223 in AML12 cells was measured. (E) Primary WT or miR-223KO hepatocytes were cocultured with neutrophils for 6 hours, and miR-223 in hepatocytes was then measured. (F) AML12 cells were cocultured with neutrophils in the presence of vehicle or EV release inhibitor GW4869 (20 nM) for 6 hours, and miR-223 levels in AML12 cells were then measured. (G) Neutrophilic EVs were lysed by using 0.1% Triton X-100 for 30 minutes at room temperature. Neutrophil-derived EVs (250 μg/mouse) pretreated with or without 0.1% Triton were intravenously injected into miR-223KO mice for 4 hours. Hepatic miR-223 was detected by in situ hybridization. Representative images of miR-223 (green), phalloidin (red), and nuclei (DAPI, blue) are shown. Scale bars: 10 μm. Values represent means ± SEM from 3–4 independent experiments. *P < 0.05, **P < 0.01. Significance was determined by a 2-tailed Student’s t test for comparing 2 groups (A and B) or 1-way ANOVA followed by Tukey’s post hoc test for multiple groups (D and F). ND, not detectable.