Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor

Abstract

Objective: Obesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion.

Methods: Plasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays.

Results: Circulating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity.

Conclusion: Our results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.

Keywords: Adipokines; Exosomes; Extracellular vesicles; MIF; Microvesicles; Obesity.

Graphical abstract

Figure 1

Plasma MV and EXO concentrations are significantly increased in obesity. A. Schematic protocol used to isolate EV subtypes, including MVs and EXOs, from plasma samples. Aliquots of platelet-free plasma (PFP), and successively depleted of MVs (PFP w/o MVs) then EXOs (PFP w/o EVs) were conserved for multiplex analysis. B–C. Plasma MV and EXO concentrations in control, overweight, and obese patients. Total circulating MVs (B) were quantified on PFP by flow cytometry and presented as MV number per μl of plasma sample. EXO concentration (C) was estimated by NTA analysis on isolated EXO fraction. Control (n = 21), overweight (n = 7) and obese patients (n = 20). *p < 0.05, **p < 0.005, ***p < 0.001. D. Circulating MV distribution in plasma from control patients. Percent (%) of circulating MVs derived from platelets (CD41⁺), endothelial cells (CD146⁺), erythrocytes (CD235a⁺), leukocytes (CD45+), monocytes (CD11b⁺), or with procoagulant activity (annexin V⁺) estimated by flow cytometry. E–J. Circulating MV concentration levels derived from platelets (B), endothelial cells (C), erythrocytes (D), leukocytes (E), monocytes (F), or with procoagulant activity (G) were measured in control (n = 20), overweight (n = 12) and obese patients (n = 21). MV concentrations were expressed as events detected by flow cytometry per μl of plasma. K. Plasma EXO mean size (nm) was measured by NTA on EXO isolated from control (n = 20), overweight (n = 9) and obese (n = 17) patients. L–M. Circulating plasma MV and EXO concentrations (L) and size (M) measured using NTA analysis in lean and obese mice (n = 4 per group). Significant differences (p < 0.05) between groups using a non-parametric ANOVA test corrected for multiple comparisons by Tukey's test are indicated by *.

Figure 2

Proteome profiling of EV subtypes reveals the presence of numerous soluble factors. Protein arrays incubated with human plasma MVs or EXOs reveal that EVs convey many soluble factors. Results presented are mean of signal intensity (expressed as mean pixels per μg of protein) from two independent MV or EXO preparations prepared from two different obese patients, respectively.

Figure 3

MIF specifically uses MVs as a secretory pathway. A. MIF concentration significantly decreased upon removal of plasma MVs. MIF concentration, successively measured in PFP, PFP w/o MVs, and PFP w/o EVs is presented in control patients (left, n = 19), overweight patients (middle, n = 19), and obese patients (right, n = 19). B. MV-associated MIF ratios are unchanged with obesity. Percent of EV-associated fractions and soluble MIF fraction was calculated based on MIF concentrations measured in PFP, PFP w/o MV, and PFP w/o EV. MVs carry around half of circulating MIF in control, overweight and obese patients. C–D. Western blots of EV preparations confirmed specific MIF association with MVs. MV and EXO preparations isolated from plasma patients (C) or from 3T3-L1 adipocytes and T-CEM lymphocytes supernatants (D) were analyzed for MIF presence. MIF specifically associated with MVs, whatever EV cellular origin. β-actin and CD9 were respectively used as MV and EXO protein markers. E. MIF is located within MVs. Proteinase K treatment did not modify MV-associated MIF signal by comparison to untreated MVs excluding that MIF is exposed on MVs. Combined treatment of proteinase K and Triton X100 led to MIF signal disappearance, establishing that MIF is packed within MVs. The transmembrane protein CD9 and the cytoplasmic spindle-oriented caveolin are respectively used as positive controls of this proteinase K protection assay.

Figure 4

MV-associated MIF transduces ERK activation in macrophages. A–B. Production of MVs depleted or not of MIF from mouse embryonic fibroblasts (MEFs) derived from WT and knockout MIF embryos. MEF-derived EVs (B) were isolated from cell supernatants of MEFs expressing (WT) or not (MIF^−/−) MIF protein (A). β-actin and CD9 were respectively used as MV and EXO protein markers. C. MIF presence within MVs does not influence MV internalization. WT MVs and MIF^−/− MVs (20 μg/ml each) were both labeled with PKH26 membranous dye and MV internalization was imaged following different time of MV incubation (0, 0.5, 1, 2 and 4 h) on recipient cells. The absence of MIF does not influence the ability of recipient cells to internalize MVs. Mean Fluorescent Intensity (MFI) within 10 cells on three independent images was measured to quantify PKH26-labeled MV internalization. D. MV-associated MIF induces ERK phosphorylation via MIF tautomerase activity. MV-associated MIF (20 μg/ml) incubation induces a robust ERK1/2 phosphorylation in RAW cells, similar to the one observed following 200 ng/ml recombinant MIF (recMIF) treatment. Of note, MIF-depleted MVs (MIF^−/− MVs) are unable to mediate ERK1/2 activation. Addition of the inhibitor of MIF tautomerase activity, Iso-1 (50 μM), during MV incubation time, abolished the ability of WT MVs to induce ERK1/2 phosphorylation. Results presented were quantified from at least three independent experiments. E. Incubation of WT MVs with MIF-neutralizing antibodies does not modify their ability to induce ERK1/2 phosphorylation in macrophages. Treatment with neutralizing MIF antibodies or their control IgG isotypes (80 μg/ml each) was performed during MV incubation on RAW cells. Results presented were quantified from three independent experiments.