Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function

Abstract

Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Keywords: dendritic cells; extracellular vesicles; immunomodulation; mesenchymal stromal cells; microRNA.

Figure 1

Characteristics of MSC-derived EVs. MSC-EVs were isolated from MSC conditioned medium by differential ultracentrifugation. Isolated EVs were assessed for (A) particle concentration, (B) particle size distribution, and (C) morphology using nanoparticle-tracking analysis and transmission electron microscopy, respectively. Flow cytometry was used to comfirm the expression of EV signature markers (D). The scale bar represents 100 nm. The error bars represent mean ± SEM of four independent experiments.

Figure 2

MSC-EVs preferentially target DCs. Representative images from confocal microscopy showing that EV-PKH26⁺ labeled MSC-EVs (red) are closely associated with DCs (green). No uptake of MSC-EVs by CD3⁺ T cells (blue) was detected. The scale bars represent 50 and 20 μm.

Figure 3

MSC-EVs impair antigen uptake by immature DCs. FITC dextran uptake was used to assess antigen uptake by immature, mature and MSC-EV treated immature DCs. (A) Representative histograms and (B) cumulative data of FITC dextran uptake as assessed by flow cytometry. Pale gray histograms represent isotype control. Results express mean ± SEM of four independent experiments (*p < 0.005, **p < 0.01).

Figure 4

MSC-EVs modulate DC maturation and cytokine secretion. (A) Cumulative (top) and representative (bottom) data showing expression of surface markers CD38, CD83, CD80, CD86 and HLA-DR as determined by flow cytometry on immature, mature and MSC-EV treated mature DCs. (B) Cell culture supernatants collected from iDC, mDC and mDC-EVs were analyzed for the levels of IL-6, IL-12p70, TGF-β and IL-10. (C) Supernatants collected from the co-culture of CD3⁺ T cells with either mDCs or mDCs-EVs were analyzed for the levels of IFN-γ, IL-6, TNF-α and IL-2. T cells cultured with medium alone served as a control. (D) Representative (left) and cumulative (right) data showing allogeneic CD3⁺ T cell proliferation stimulated with mDCs and mDC-EVs as assessed by flow cytometry analysis of CFSE dilution. Data represent mean ± SEM of 4–6 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ND, not detectable.

Figure 5

MSC-EVs impair DC migratory via suppression of CCR7 expression (A) Representative histograms (left) and cumulative levels (right) of CCR7 expression by mDCs and mDC-EVs. Live cells were selected prior to histograms and levels of expression were compared to isotype controls. (B) Migration efficiency of mDCs and mDC-EVs. Mature DCs migrated toward the medium without CCL21 served as a background control. Results were expressed as mean ± SEM of 4–6 independent experiments. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 6

MSC-EVs encapsulate immunomodulatory microRNAs. (A,B) The profile of RNAs derived from MSCs and MSC-EVs as analyzed using Bioanalyzer. (C) Principal component analysis plot of microRNA profile of MSCs (blue) and MSC-EVs (red). (D) Heatmap showing the 79 differentially expressed microRNAs between MSCs and MSC-EVs. Heatmap colors represent relative microRNA expression as indicated by the color key, i.e., under-expressed (blue) and over-expressed (red). Each row is representative of one microRNA and each column one sample. RStudio™ was used to plot the results. (E) RT-qPCR validation of the immunomodulatory microRNAs enriched in MSC-EVs. The graphs depict relative expression levels conveyed as 2dCT and results were expressed as mean ± SEM of 3 independent experiments. **p < 0.01.

Figure 7

Transfection of DCs with miR-21-5p partially mimics the function of MSC-EV treated DCs. (A) In silico analysis of miR-21-5p targets revealed that this microRNA directly targets CCR7 and IL12A genes. Targets for miR-21-5p were queried using miRSystems and graphic representations and complementarity information of miR-21-5p with CCR7 and IL12A genes were obtained from the online database http://www.targetscan.org/vert_72/. (B) Migratory capacity of non-transfected DCs and DCs transfected with MiR-21-5p mimics or scramble controls, as detected using a transwell system. DCs migrated toward medium without the chemoattractant served as a background control. (C) CCR7 expression was analyzed by flow cytometry. Live cells were selected prior to histograms and levels of expression were compared to the isotype control. Results were expressed as mean ± SEM of 4-6 independent experiments. *p < 0.05.