MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages - PubMed (original) (raw)

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

Banishree Saha et al. J Biol Chem. 2016.

Abstract

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.

Keywords: alcoholic hepatitis; cell signaling; exosome (vesicle); extracellular vesicles; liver injury; phagocytosis.

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Figures

FIGURE 1.

Ethanol treatment increases total number of EVs in a dose- and time-dependent manner in primary human monocytes and THP-1 cells. Primary human monocytes were isolated from healthy individuals and treated with different doses of ethanol (0–100 m

) for 48 h. EVs were quantified in the culture supernatant by NanoSight. A, number of EVs, exosomes, and MVs were quantified in the culture supernatant by NanoSight. B, primary monocytes were exposed to 50 m

ethanol for 0–48 h, and the frequency of vesicles was determined. C, THP-1 cells were treated with 0–50 m

ethanol for 48 h or were left untreated. Total number of EVs, exosomes, and MVs, was determined. D, kinetics of vesicle secretion from the THP-1 cells in the presence of 50 m

ethanol is shown as a line graph. The results represent three independent experiments. (* indicates p < 0.05 versus control.)

FIGURE 2.

Characterization of EVs from THP-1 human monocytes. In human monocytic cells, THP-1 cells were treated with ethanol for 48 h or left untreated. A, EM image of untreated THP-1 cells (control); B, ethanol-treated THP-1 cells are shown (×3000 magnification). The blown up image of the selected region (×7500 magnification) in the ethanol-treated THP-1 cells show the presence of EVs on the surface of the cells. C, isolated EVs from the THP-1 monocytes express the exosomal marker CD63. D, THP-1-derived EVs have an average size of 131 nm. Three-dimensional graph represents particle size versus intensity versus concentration (particles/ml) of THP-1-derived EVs. The data are representative of three independent experiments.

FIGURE 3.

Alcohol-induced THP-1 or primary monocyte-derived EVs promote naive monocytes to differentiate into macrophages with M2 phenotype. EVs were derived from alcohol-treated (EtOH EVs) and untreated THP-1 cells or primary monocytes after 48 h of alcohol/no alcohol treatment. Primary human monocytes were treated with control and EtOH EVs for 5 days. The cells were stimulated with 25 μl of EVs at a concentration of 25 μg/ml on day 1 and day 3. Macrophage markers were assessed by flow cytometry. A, histogram shows the MFI of CD14 expression. The MFI value for each experimental condition is shown below the histogram plot. B, percentage of CD14+CD68+ expression in monocytes untreated or treated with control and EtOH EVs is shown. C and D, M1 macrophage markers CD16 and CD86 expression in monocytes cultured in the presence of control and EtOH EVs. E and F, M2 macrophage markers CD206- and CD163-expressing cells were determined in monocytes treated with control and EtOH EVs. The results represent three independent experiments. (* indicates p < 0.05 versus medium control.).G, frequency of expression of the various macrophage markers in naive monocytes treated with primary monocyte-derived control and EtOH EVs. The results represent three independent experiments. (* indicates p < 0.05 versus control EVs.)

FIGURE 4.

Functional effect of alcohol-induced THP-1 and primary monocyte-derived EVs on naive monocytes. Primary monocytes were incubated with control and ethanol EVs derived from THP-1 cells for 5 days. Cell culture supernatants were collected, and cytokine levels were determined by ELISA. A, TNFα. B, IL-12. C, IL-10. D, TGFβ levels are shown for the control and EtOH EV-treated monocytes. The results represent three independent experiments. (* indicates p < 0.05 versus control EVs.) E, naive monocytes incubated with primary monocyte-derived EtOH and control EVs were assayed for TNFα, IL-12, IL-10, and TGFβ secretion. The results represent three independent experiments. (* indicates p < 0.05 versus control EVs.) F and G, primary monocytes incubated with THP-1-derived control and ethanol EVs were also tested for their phagocytic capacity. The monocytes were incubated with dextran beads conjugated with FITC. The dot plot analysis shows the dextran FITC+ phagocytic cells at different temperatures for control and EtOH EV-treated monocytes. F, phagocytosis index is calculated as percentage of FITC+ cells multiplied by MFI of FITC-positive phagocytic cells. The results represent three independent experiments. (*indicates p < 0.05 versus control EVs.)

FIGURE 5.

miR-27a-enriched EVs promote naive monocytes to differentiate into macrophages. A, total RNA was isolated from THP-1 cells treated with 50 m

ethanol for 48 h, and RT-PCR was performed to study the levels of miR-27a, miR-146a, and miR-9 using TaqMan® miRNA assay. The results represent three independent experiments. (* indicates p < 0.05 versus EtOH.) B, similarly levels of miR-27a, miR-146a, and miR-9 were determined in control and EtOH EVs from THP-1 cells. The results represent three independent experiments. (* indicates p < 0.05 versus control EVs.) C, miR-27a mimic or scrambled mimic was loaded into THP-1-derived EVs as described under “Experimental Procedures.” The graph shows the overexpression of miR-27a in the EVs loaded with various concentrations of miR-27a mimic. D, EVs containing scrambled mimic and miR-27a mimic (10–75 pmol) were added to naive monocyte cells and incubated for 5 days. The EVs were added on day 1 and day 3 at a concentration of 25 μg/ml. Macrophage markers were assessed by flow cytometry. The results represent three independent experiments, where * indicates p < 0.05 versus scrambled mimic.

FIGURE 6.

Functional effect of miR-27a-containing EVs on the cytokine secretion and phagocytosis of macrophages derived from naive monocytes. Primary human monocytes were incubated with miR-27a mimic (10–75 pmol) or scrambled mimic (75 pmol) loaded into THP-1-derived EVs. Cell culture supernatants were collected, and cytokine levels were determined by ELISA. A, TNFα and IL-12. B, IL-10 and TGFβ levels are shown. The results represent three independent experiments. (* indicates p < 0.05 versus control EVs.) C and D, primary monocytes were incubated with control and ethanol EVs for 5 days, and phagocytosis assay was performed. C, percent of phagocytic cells was determined by FITC+ cells. D, phagocytosis index is calculated as percentage of FITC+ cells multiplied by mean fluorescence intensity of FITC-positive phagocytic cells. The results represent three independent experiments. (* indicates p < 0.05 versus scr mimic.) E and F, primary monocytes were pretreated with control, mannan, anti-IgG1, and anti-CD206 and then incubated with EVs loaded with scrambled mimic and miR-27a mimic. Phagocytosis assay was performed as described previously using dextran-FITC beads. The data are representative of three independent experiments, where * indicates p < 0.05.

FIGURE 7.

Increased level of miR-27a in alcoholic hepatitis patients is associated with increased IL-10 and TGF-β secretion from immune cells. A, number of EVs was measured in the plasma of healthy controls and AH patients using Nanosight. Exosomes were defined as EVs with diameter less than 150 nm, and microvesicles were the EVs with a diameter of more than 150 nm and above. B, level of miRNA-27a was quantified in the plasma-derived EVs isolated from healthy controls and AH patients by TaqMan qPCR assay. C and D, IL-10 and TGF-β secretion was determined from plasma samples of healthy controls and AH patients. The data are represented as mean ± S.E. (n = 6–8 individuals), where * indicates p < 0.05. E–G, EVs were isolated from the plasma of AH patients and healthy controls. Primary monocytes were co-cultured with the EVs for 5 days. E, cell surface expression of various markers; F, IL-10 secretion; and G, phagocytosis measured for the monocytes. The data are represented as mean ± S.E. (4–5 individuals).

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MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages - PubMed (original) (raw)