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.

Keywords: Hepatology; Obesity.

Figure 1. Immune cell–derived miR-223 is selectively transferred into the liver (hepatocytes), ameliorating NASH.

(A) C57BL/6J mice were fed HFD or CD for 3 months. Serum and different organ samples were collected for the measurement of miR-223 levels (n = 3–4). (B–F) WT and miR-223KO mice were transplanted with WT or miR-223KO mouse bone marrow (BM). Two months later, these mice were subjected to HFD feeding for 3 months (n = 4–6). (B) Serum and liver tissue samples were collected for miR-223 measurement. (C) Frozen liver tissue sections from HFD-fed mice were analyzed by miR-223 in situ hybridization along with immunofluorescence staining of neutrophil marker MPO and cell cytoskeleton marker F-actin that was detected by using Alexa Fluor–phalloidin. Representative images of miR-223 expression (green), MPO (yellow), phalloidin (red), and nuclei (DAPI, blue) are shown. White arrows indicate MPO⁺ neutrophils (left panel) or miR-223⁺MPO⁺ neutrophils (right panel). Red arrows indicate miR-223⁺ hepatocytes. Scale bars: 10 μm. (D) Serum ALT was measured (left panel). RT-qPCR analyses of liver Ly6g and F4/80 mRNA levels (right panel). (E) Representative images of H&E staining (scale bars: 200 μm), Oil red staining (scale bars: 100 μm), Sirius red staining (scale bars: 200 μm), and Masson’s trichrome staining (scale bars: 100 μm) of liver tissue sections are shown. (F) Oil red⁺ area and fibrotic area per field were quantified. Values represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by 2-tailed Student’s t test for comparing 2 groups (A, B, D, and F). ND, not detectable. The superscript characters shown for transplanted mice indicate the donor mouse BM.

Figure 2. Neutrophils transfer miR-223 to hepatocytes via EVs.

(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.

Figure 3. Fatty acid (e.g., palmitic acid [PA]) promotes neutrophil transfer of miR-223 to hepatocytes.

(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.

Figure 4. Neutrophil-derived, miR-223–enriched EV uptake in lipotoxic hepatocytes is partially dependent on LDLR.

(A) After pretreatment with vehicle or PA (0.3 mM) for 18 hours, WT and Ldlr-KO hepatocytes were cultured with fresh serum-free medium and incubated with DiD-labeled neutrophil-derived EVs, followed by performing immunofluorescence staining. Representative images of DiD (red), LDLR (green), and nuclei (DAPI, blue) are shown, and mean fluorescence intensity per cell was quantified. Scale bars: 20 μm. (B) DiD-labeled neutrophil-derived EVs were intravenously injected into WT and Ldlr-KO mice for 4 hours. Representative images of DiD (red), LDLR (green), and nuclei (blue) in the liver are shown, and relative fluorescence intensity per field was quantified. Scale bars: 10 μm. (C) In the left panel, after pretreatment with vehicle or PA (0.3 mM) for 18 hours, WT and Ldlr-KO hepatocyte cell medium was replaced with fresh serum-free medium and the cells cocultured with or without neutrophils for another 6 hours. miR-223 levels in hepatocytes were measured by RT-qPCR. In the right panel, AML12 cells were transfected with control or Ldlr siRNA for 24 hours, followed by treatment with vehicle or PA for 18 hours. AML12 cell medium was then replaced with fresh serum-free medium and the cells cocultured with or without neutrophils for another 6 hours. miR-223 levels in AML12 cells were measured by RT-qPCR. (D and E) WT and Ldlr-KO mice were fed CD or HFD for 3 months. Liver tissue samples were collected for the measurement of miR-223 (D) (n = 3–6). Serum samples were collected for EV isolation, and miR-223 in EVs and EV numbers were measured (E). Values represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Significance was determined by a 2-tailed Student’s t test for comparing 2 groups (A–D) or 1-way ANOVA followed by Tukey’s post hoc test for multiple groups (E).

Figure 5. Restoration of hepatic miR-223 reverses NASH in HFD-fed Ldlr-KO mice.

Ldlr-KO mice were fed HFD for 3 months with the intravenous injection of adenovirus-Gfp (Ad-Gfp) (n = 5) or Ad-miR-223 (n = 5) starting at 2 months for a total of 2 times (once every 2 weeks). Liver and serum samples were collected. (A) Representative images of H&E staining (scale bar: 200 μm), Sirius red staining (scale bar: 200 μm), and α-SMA staining (scale bar: 100 μm) of liver tissue sections are shown. Fibrotic area per field was quantified. (B) RT-qPCR analyses of several genes. Values represent means ± SEM. *P < 0.05, ***P < 0.001, as determined by 2-tailed Student’s t test for comparing 2 groups (A).

Figure 6. PA induces neutrophils to release APOE-enriched EVs.

Bone marrow neutrophils were cultured in serum-free medium and stimulated with vehicle or PA (0.3 mM) for 6 hours. (A) miR-223 levels in neutrophils and neutrophil-derived EVs were measured. (B) Apoe and Pu.1 mRNA levels in neutrophils were measured. (C) Western blot analyses of APOE in bone marrow neutrophils and EV marker proteins in neutrophil-derived EVs from WT and Apoe-KO mice. (D) Colocalization of APOE with an early endosome marker (EEA1), EV marker (CD63), or late endosome marker (RAB7) was determined. Representative images of these markers (red), APOE (green), and nuclei (DAPI, blue) are shown. Scale bars: 5 μm. (E) AML12 cells and primary mouse hepatocytes were incubated with DiD-labeled EVs that were derived from serum-free–cultured neutrophils. Representative images of DiD (red) and nuclei (blue) are shown, and mean fluorescence intensity per cell was quantified (bottom). Scale bars: 20 μm. Values represent means ± SEM. *P < 0.05, **P < 0.01, as determined 2-tailed Student’s t test for comparing 2 groups (A, B, and E).

Figure 7. Apoe deficiency in immune cells worsens NASH partially due to less transfer of miR-223 to hepatocytes.

WT mice were transplanted with WT or Apoe-KO mouse bone marrow (BM). The superscript characters indicate the donor mouse BM. Two months later, these mice were subjected to CD or HFD feeding for 3 months. Serum and liver tissue samples were collected (n = 3 in CD-fed group, n = 9 in HFD-fed group). (A) miR-223 in the liver was measured by RT-qPCR. (B) Serum ALT was measured. (C) Representative images of H&E staining (scale bars: 200 μm), Sirius red staining (scale bars: 200 μm), and α-SMA staining (scale bars: 100 μm) of liver tissue sections are shown. Fibrotic area per field was quantified (bottom). (D) RT-qPCR analyses of several genes in the liver tissues from HFD-fed mice. Values represent means ± SEM. *P < 0.05, **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 (B and C).

Figure 8. Administration of the PCSK9 inhibitor alirocumab enhances LDLR-dependent miR-223 transfer.

(A) AML12 cells were pretreated with the PCSK9 inhibitor alirocumab or control antibody (10 μg/mL) for 24 hours, and then the cell medium was replaced with serum-free medium. LDLR expression in hepatocytes was measured by Western blotting (upper panel). These hepatocytes were cocultured with neutrophils for another 6 hours, and miR-223 levels in hepatocytes were measured by RT-qPCR (lower panel). (B–E) C57BL/6J mice were also fed an MCD diet or control diet (CD) for 6 weeks. After 2-week MCD diet feeding, these mice were subcutaneously injected with alirocumab or control antibody at the dose of 10 mg/kg once per week for another 4-week MCD diet feeding. Serum and liver samples were collected (n = 3 in CD-fed group, n = 8 in MCD-fed group). (B) Liver LDLR protein was measured. (C) Frozen liver tissue sections from MCD-fed mice were analyzed by miR-223 in situ hybridization along with immunofluorescence staining of the hepatocyte marker albumin. Representative images of miR-223 expression (green), albumin (red), and nuclei (DAPI, blue) are shown. Scale bars: 10 μm. (D) miR-223⁺ hepatocytes were quantified in MCD-fed mice. (E) miR-223 target genes were measured by RT-qPCR in MCD-fed mice. Values represent means ± SEM. *P < 0.05, **P < 0.01. 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 (D and E).

Figure 9. Administration of the PCSK9 inhibitor alirocumab ameliorates MCD-induced NASH by promoting LDLR-dependent miR-223 transfer.

After 2-week MCD diet feeding, C57BL/6J mice were subcutaneously injected with alirocumab or control antibody at the dose of 10 mg/kg once per week for another 4-week MCD diet feeding. Serum and liver samples were collected (n = 8). (A) Representative images of H&E staining (scale bars: 200 μm), MPO staining (scale bars: 100 μm), F4/80 staining (scale bars: 200 μm), and Sirius red staining (scale bars: 200 μm) of liver tissue sections are shown. MPO⁺ cells per field, F4/80⁺ area, and fibrotic area per field were quantified. (B) RT-qPCR analyses of several genes in the liver. (C) Serum ALT levels and NAS were analyzed. Values represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by 2-tailed Student’s t test for comparing 2 groups (A and C).

Figure 10. Scheme depicting a selective LDLR- and APOE-dependent EV transfer of neutrophilic miR-223 into hepatocytes and its role in the progression of NAFLD.

In obesity, free fatty acids (FFAs) elevate miR-223 expression in neutrophils by regulating APOE/PU.1 signaling and miR-223 subsequently forms a feedback loop to prevent NASH progression by amplifying the preferential uptake of neutrophil-derived and antiinflammatory miR-223/APOE-enriched EVs in hepatocytes. This selective uptake is dependent on the expression of LDLR on hepatocytes and APOE on neutrophil-derived EVs. Upregulation of LDLR by treatment with the PCSK9 inhibitor alirocumab ameliorates NASH partially due to the augmentation of this miR-223 transfer.