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Aug 12, 2015 - ferred by EV than monocytes or immature dendritic cells indicating that the differentiation status influences the efficiency of EV uptake.
EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12371 ..................................................................................................................................................................

The Uptake of Extracellular Vesicles is Affected by the Differentiation Status of Myeloid Cells L. Czernek, A. Chworos & M. Duechler

Abstract Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland

Received 8 May 2015; Accepted in revised form 12 August 2015 Correspondence to: M. D€uchler, Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 112, Sienkiewicza Street, 90-363 Lodz, Poland. E-mail: [email protected]

Intercellular communication includes the exchange of various membrane vesicles including exosomes. Exosomes are intraluminal nanovesicles generated from multivesicular bodies, a late endosomal compartment. Cancer cells release exosomes that influence their proximate and distant environment to facilitate angiogenesis, metastatic dissemination and immune escape. Cancer-derived vesicles may also trigger an anti-tumour response by transferring tumour antigens to immune cells. We wanted to investigate whether differentiation and maturation of myeloid cells changes their capacity to take up cancer-derived extracellular vesicles (EV). We compared the efficiency of vesicle uptake by monocytes, macrophages and dendritic cells. To visualize and quantify the cellular uptake, EV were labelled with two different dyes, carboxyfluoresceine diacetate succinimidyl-ester (CFSE), or DSSN+, a water soluble distyrylstilbene oligoelectrolyte which preferentially intercalates into the cell membrane. With the help of cytokines, THP-1 monocytes were differentiated into immature or mature dendritic cells, or macrophages. EV uptake was monitored by flow cytometry and immunofluorescence microscopy. The results show that macrophages and mature dendritic cells acquired stronger fluorescence transferred by EV than monocytes or immature dendritic cells indicating that the differentiation status influences the efficiency of EV uptake.

Introduction Cells stay in contact with the environment by receiving and sending signals through secreted factors such as cytokines, hormones, chemokines and through direct cell–cell contacts. In addition, cells release various types of extracellular vesicles like ectosomes, exosomes and occasionally apoptotic bodies [1]. The classification of released vesicles is generally based on their cellular origin. Exosomes are described as 30–110 nm sized vesicles arising from multivesicular bodies (MVBs), a late endosomal compartment, through inward budding [2]. Their lipid bilayer shows the same topological orientation like the cell membrane. Upon fusion of MVBs with the plasma membrane, exosomes are released into the extracellular environment. The released vesicles are taken up by other cells and the intercellular transfer of exosomes is recognized as a targeted mechanism of cell-to-cell communication [3]. Exosomes are secreted by virtually all cell types including immune cells, stem cells, neurons and cancer cells and are present in many different biological fluids [3]. Various routes of ingestion of exosomes by target cells have been proposed which might take place simultaneously.

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Exosome can stimulate clathrin- or caveolin-mediated endocytosis through interaction of their membrane proteins with receptors on target cells. Furthermore, uptake can occur through direct fusion of exosomes and cell membranes, by phagocytosis, or macropinocytosis [4]. The fusion of exosomes with cellular membranes results in the delivery of their cargo to the intracellular space. Exosomes function in intercellular communication by transferring a variety of molecules: proteins, lipids and nucleic acids such as mRNAs and miRNAs. While many exosomal proteins are common to all types of exosomes, some proteins show cell-type specificity [5]. Moreover, the content of exosomal RNA and proteins also differs from the molecular composition inside the cell indicating selective integration of specific molecules into exosomes [6, 7]. The selection mechanisms for packaging the exosomal cargo are not fully defined. It is known that the ‘endosomal sorting complex required for transport’ (ESCRT) is critically involved [1]. ESCRT complexes recognize ubiquitinated proteins and insert them into the budding vesicles after removing the ubiquitin tag. Passive mechanisms of exosomal packaging were also suggested. With the inclusion of miRNAs even control elements for gene

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expression are transported through exosomes, which can regulate many different signalling pathways in the recipient cells [6, 8]. Tumour progression depends on an active crosstalk between the tumour and its microenvironment [7, 9, 10] and exosomes are extensively used by cancer cells to stimulate support from cells in the immediate surrounding or at more distant sites after transport of exosomes through the circulation [11, 12]. Tumour-derived exosomes were described to create a premetastatic niche facilitating the spread of metastatic cells [13]. Importantly, tumour-derived exosomes are also involved in the modulation of anti-cancer immune responses [14]. Both, immunostimulatory and immunosupressive functions were described [15]. Cancer-derived exosomes were shown to induce direct or indirect stimulation of T-cell responses [16]. Membrane vesicles released from tumour cells are a possible source of tumour associated antigens to be taken up and presented by antigen presenting cells (APC) [17–19]. Furthermore, tumour antigen carrying exosomes released by antigen presenting cells including dendritic cells, macrophages, or mast cells can exert immunostimulatory functions and function as cancer vaccines [20–22]. On the other hand, tumour-derived exosomes are able to induce antigenspecific tolerance. Exosomes were described to inhibit T-cell activation and cytotoxicity of CD8+ T cells and NK cells [23–25]. They can prevent dendritic cell (DC) maturation thus keeping them in an immunosuppressive state, which stimulates the development of regulatory T cells [26, 27]. Exosomes may express Fas ligand to directly induce T-cell killing [28] or immunosuppressive cytokines, including TGF-beta [27, 29]. We wanted to compare the capacity of monocytes, macrophages and dendritic cells to take up EV. For that purpose, we transferred tumour-derived EV to the human monocytic leukaemia cell line THP1, which has been commonly used to study monocyte/macrophage functions [30–32]. THP-1 cells can be differentiated into macrophages, immature and mature human DCs [33, 34]. Fluorescently labelled EV transfer the fluorescent dyes into the recipient cells which can be monitored by fluorescence microscopy and flow cytometry.

Materials and methods Cell culture. The human monocytic leukaemia cell line THP1 was kindly provided by Prof. J. Dziadek, Polish Academy of Sciences, Lodz, Poland. THP1 cells were cultured at 5 9 105–1 9 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 lg/ml streptomycin, 0.05 mM b-mercaptoethanol and 1 mM sodium pyruvate in a humidified incubator at 37 °C and 5% CO2 (Binder GmbH, Germany). The human malignant melanoma cell lines A375 and 1205Lu (kindly provided by Prof Malgo-

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rzata Czyz, Medical University, Poland), and the ovarian cancer cell line OvC16 (established in our lab from a primary tumour sample) were maintained in RPMI1640 medium supplemented with 10% FBS and antibiotics. For the elimination of bovine serum-derived EV growth media were subjected to ultracentrifugation at 100.000 9 g for 2.5 h at 10 °C. For EV production, cells were grown in this EV-depleted medium. Isolation and characterization of extracellular vesicles. Tumour cell derived EV were isolated by differential centrifugation. Cell culture supernatants were centrifuged for 4 min at 300 9 g to pellet the cells. For precipitation of cell debris and organelles, the supernatants were centrifuged for 30 min at 10.000 9 g (10 °C), than EV were pelleted by ultracentrifugation at 100.000 9 g for 2.5 h (10 °C). The EV pellet was resuspended in PBS and the last centrifugation step was repeated. The pellet was again resuspended in PBS and the protein concentration was determined by the Bradford assay (Bio-Rad Polska, Warszawa, Poland). The isolation of EV was confirmed through detection of surface markers CD9 and CD63. EV were adsorbed to latex beads, stained with anti-CD63 or anti-CD9 antibodies, and analysed by flow cytometry. Serum-derived EV served as negative control. Labelling of extracellular vesicles. EV were fluorescently labelled by two different dyes, carboxyfluoresceine diacetate succinimidyl-ester (CFSE) (Molecular Probes, InVitrogen, Life Technologies Polska, Warsaw, Poland) and 4,40 -bis(40 (N,N-bis(6″-(N,N,N-trimethyl-ammonium)hexyl)-styryl) stilbene tetra-iodide (DSSN+) synthesized at our institute [35]. The emission spectra for DSSN+ have been published [36]. EV were incubated for 30 min at room temperature at 1 lM or 5 lM concentrations of DSSN+ or CFSE, respectively, and separated from unincorporated dyes by an ultracentrifugation step. As a negative control, the same amount of dyes was centrifuged without the presence of EV to monitor unincorporated dyes carried over after the centrifugation (‘PBS control’). Cell differentiation. THP1 monocytic cells were differentiated using 200 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Poznan, Poland) for 3 days to obtain macrophages. To acquire the properties of immature DCs, THP1 cells were cultured in medium supplemented with 100 ng/ml rhIL-4 (recombinant human interleukin 4) and 100 ng/ml rhGM-CFS (recombinant human granulocyte macrophage colony-stimulating factor) for 5 days. Medium was exchanged every 2 days with fresh cytokinesupplemented medium. Further maturation of immature DCs was achieved by culturing for 3 more days in serumfree medium supplemented with rhIL-4 (100 ng/ml), rhGM-CFS (100 ng/ml), 20 ng/ml rhTNF-a (tumour necrosis factor a), and 200 ng/ml ionomycin. Flow cytometry. Flow cytometric measurements were performed on a FACSCalibur (Becton-Dickinson, Heidelberg, Germany). For each sample, a minimum of 104 cells

508 Extracellular Vesicle Uptake into Myeloid Cells L. Czernek et al. .................................................................................................................................................................. was measured. Results were analysed using the CellQuest software (Becton-Dickinson). THP-1 cell differentiation was confirmed by detection of characteristic surface markers for macrophages, immature and mature DCs (CD11b, CD11c, CD14, CD15, CD40, CD80, CD86, CD163, HLA-DR, HLA-ABC). CFSE or DSSN+ fluorescence was detected in the FL1 channel. For the flow cytometric analysis of EV, the vesicles were adsorbed to aldehyde-sulphate latex beads (3.8 lm size) for 20 min at room temperature. BSA was used to saturate the binding sites of the beads, followed by incubation with glycine (100 mM) in PBS for 20 min at room temperature. The beads were washed twice with FACS buffer (PBS, 1% BSA, 0,1% azide) incubated with FITC-conjugated or PE-conjugated monoclonal antibodies for 1 h in dark, washed again with cold FACS buffer, and analysed. EV transfer experiments. Cells were seeded at a density of 5 9 104 cells/well in 24-well plates and incubated in EVdepleted medium at 37 °C for 24 h with labelled EV or the PBS control. As positive control, cells were directly labelled at concentrations of 1 lM DSSN+ or 10 lM CFSE. After 24 h incubation, cells were washed twice in cold FACS buffer and analysed by flow cytometry. For immunofluorescence microscopy, cells were grown on cover slips, which were washed with PBS after EV incubation. Phase contrast and fluorescence pictures were taken on a Nikon Eclipse Ti-U microscope, recorded on a Nikon Digital sight DS-U2 camera with the help of the NISElements software (instruments and software from Nikon Instruments, Amsterdam, Netherlands). For the direct competition experiments, identical numbers (5 9 104) of either THP-1 monocytes and macrophages, or monocytes and mature dendritic cells were mixed with 10 lg/ml CFSE-labelled EV. After 4 h, cells were stained with CD11c-PE and CD11b-APC to distinguish between monocytes and macrophages, or HLAABC-PE and CD40-APC to separate monocytes from mature dendritic cells. PE and APC fluorescence was used for gating to achieve separate measurement of the CFSE fluorescence transferred by EV for the monocytes and the more differentiated cells.

Results

of the exosomal marker proteins CD9 and CD63 confirmed the successful isolation of EV (Fig. 1A). Serum derived, bovine EV served as negative controls showing no staining for these human surface proteins. The harvested EV were labelled with the fluorescent dyes CFSE or DSNN+. CFSE is highly membrane permeable due to two acetate side chains. Inside the cell, the acetate groups are cleaved by intracellular esterases and the compound becomes immobilized and fluorescent. CFSE potentially reacts with amine-containing molecules, forming highly stable amide bonds. DSSN+ is a water soluble distyrylstilbene oligoelectrolyte which stably intercalates into cell membranes [36]. In the lipid bilayer, the long hydrophobic molecular axis of DSSN+ is localised perpendicular to the plane of the membrane. The intercalation is stabilized by polar groups at the ends of DSSN+ molecules localized at the membrane surface. DSSN+ has not been described for EV labelling so far. After the labelling, EV were diluted about 200-fold and pelleted again at 100 000 9 g. As a negative control, the dyes diluted in PBS at the same concentration as for EV labelling were incubated and centrifuged (‘PBS control’). Positive EV labelling for both dyes is shown in Fig. 1B for EV isolated from A375 and 1205Lu cells. Extracellular vesicles are taken up by melanoma cells

To demonstrate the uptake of EV by target cells, 1205Lu melanoma cells were exposed for 24 h to DSSN+ or CFSElabelled EV at a concentration of 10 lg protein per ml of EV-depleted medium. After the incubation, the cells were washed in PBS and analysed by immunoflourescence microscopy (Fig. 2A), or flow cytometry (Fig. 2B). Cells directly labelled with the fluorescent dyes served as positive control. The results confirmed that cancer-derived EV are taken up by other cancer cells indicating the involvement of EV in intercellular communication. Analysis by immunofluorescence microscopy at higher resolution revealed that the DSSN+ dye transferred by EV was enriched in cellular membranes (Fig. 2C). We further observed that EV derived from A375 cells were taken up by A375 cells with similar efficiency as EV produced in other cancer cell lines (Fig. 2D). This result suggests that in vitro, EV may circulate back into the cells from which they were produced.

EV characterisation and labelling

EV were purified from two melanoma cell lines (A375, 1205Lu) and one ovarian cancer cell line (OvC16). Cancer cell derived EV were produced in culture medium depleted from calf serum EV and collected using established ultracentrifugation methods. The EV pellet was resuspended in PBS, and the protein concentration was measured. We obtained about 1 lg EV protein per ml of cell culture medium. For flow cytometry analyses, EV (20 lg protein) were adsorbed to latex beads. The presence

Uptake of extracellular vesicles by monocytes, macrophages, and immature and mature dendritic cells

The THP1 monocytic cell line was differentiated into macrophages, immature or mature DCs with the help of cytokines (Figure 3A). Typical changes in surface marker expression confirmed the differentiation into macrophages or DCs (Table 1). In comparison to monocytes, the immature DCs showed a moderate upregulation of MHC class II molecules, which was reduced again during further

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B

Figure 1 Flow cytometry analysis of extracellular vesicles. (A) Extracellular vesicles were isolated from calf serum, or cell culture supernatants of 1205Lu, A375, or OvC16 cells, adsorbed to latex beads and stained with FITC-antiCD9 and PE-antiCD63 antibodies. (B) Extracellular vesicles isolated from 1205Lu or A375 cells were labelled by CFSE or DSSN+, adsorbed to latex beads and analysed by flow cytometry (filled peaks). Latex beads incubated with the PBS control are shown as overlay in the histograms.

maturation. Mature DCs showed a strong upregulation of the costimulatory receptors CD40 and CD86, and the MHC class I antigen. CD11b, the Mac-1 a-chain, was also strongly upregulated along differentiation into DCs and macrophages. Differentiation into macrophages was accompanied by a slight upregulation of CD163, CD14 and the costimulatory receptors CD80 and CD86. In experiments comparing the EV uptake by different cell types, the cell number and the amount of added EV were accurately kept at the same level. As in previous

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experiments, the negative PBS control and direct cell labelling were included for each cell type. Incubation was carried out for 24 h at 37 °C with 5 lg EV protein per 2.5 9 105 cells in 0.5 ml. Experiments were performed with both dyes, CFSE and DSSN+ (Fig. 3B and C). The results in Fig. 3 demonstrate that macrophages and mature dendritic cells take up EV more efficiently than monocytes or immature dendritic cells indicating that the cell differentiation status influences the efficiency of EV uptake.

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B

C

D

Competitive EV uptake in mixed cell cultures

The obtained results might have been distorted by the proliferation during the incubation with EV which proceeded with higher speed for less maturated cells. Measurement of the proliferation rates by simple cell

Figure 2 Uptake of fluorescently labelled extracellular vesicles by melanoma cells. (A) Typical immunofluorescence microscope pictures showing the uptake of labelled extracellular vesicles isolated from OvC16 cell culture supernatants by 1205Lu melanoma cells visualized by dye transfer (left half, CFSE; right half, DSSN+). Upper left, phase contrast picture; lower left, the same cells after direct labelling; upper right, cell fluorescence after EV uptake; lower right, PBS control. A Nikon Plan Fluor 209 objective was used; scale bar = 50 lm. (B) Flow cytometric analysis of the fluorescence transferred by EV isolated from A375 cells into 1205Lu melanoma cells (filled peaks). The stainings of directly labelled cells and the negative (PBS) control cells are shown as overlays. (C) The fluorescence of the DSSN+ dye transferred by EV from A375 melanoma cells accumulates in membranes of OvC16 target cells. Pictures were taken using a Nikon Plan Fluor 1009 objective, scale bar = 50 lm. (D) Flow cytometry analyses show that EV produced by A375 cells are taken up by A375 cells at a similar amount than EV derived from 1205Lu or OvC16 cells.

counting revealed doubling times of 34.9 and 38.8 h for monocytes and immature dendritic cells, respectively, while mature dendritic cells and macrophages needed 52.3 and 51.6 h to double the cell number. To avoid unequal dilution of the fluorescent dye due to the variable ongoing proliferation shorter incubation times

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Figure 3 Uptake of fluorescently labelled EV by monocytes, macrophages or dendritic cells. (A) Phase contrast photographs of THP-1 cells: monocytes (undifferentiated), macrophages, immature and mature DCs. Pictures were taken using a Nikon Plan Fluor 209 objective, scale bar = 100 lm. (B) Flow cytometric analysis of the fluorescence transferred to THP-1 cells by EV (filled peaks). For each cell type, the negative (PBS) control is shown as overlay. (C) A summary of six experiments analysing the fluorescence transfer to THP-1 cells by EV. The mean fluorescence intensities (MFI) for MPh (macrophages), iDC (immature dendritic cells) and mDC (mature dendritic cells) are shown relative to THP-1 (monocytes) MFI (set to 100%). Statistically significant differences (P < 0.05) were calculated by the Student’s t-test.

were chosen for the direct competition experiments. Identical numbers of THP-1 monocytes and either macrophages or mature dendritic cells were mixed and incubated with CFSE-labelled EV for 1, 2 and 4 h. Cells were harvested and stained with antibodies to distinguish between monocytes and differentiated cells. As shown in Table 1, macrophages showed stronger staining for CD11b and CD11c than monocytes, while mature dendritic cells could be discriminated by their expression of HLA-ABC and CD40. Gating based on fluorescence channels 2 and 4 allowed to measure the CFSE fluorescence (channel 1)

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separately for monocytes and more differentiated cells. An example comparing the EV uptake in monocytes and macrophages is shown in Fig. 4. In the lower right part of Fig. 4, the summary from three independent experiments is shown for macrophages and mature dendritic cells. The mean fluorescence intensity acquired by macrophages and mature dendritic cells after 4 h was about 14 and 2.7 times higher, respectively, as compared to monocytes. The results obtained from the competition experiments ideally confirmed the more efficient EV uptake by the more mature myeloid cells.

512 Extracellular Vesicle Uptake into Myeloid Cells L. Czernek et al. .................................................................................................................................................................. Table 1 Phenotypic analysis of THP-1 cell differentiation.

CD11b CD11c CD14 CD15 CD40 CD80 CD86 CD163 HLA-A,B,C HLA-DR

Monocytes

Macrophages

Immature DC

Mature DC

29.2 10.4 63.0 23.4 23.7 13.6 13.4 9.0 57.3 63.8

205.1 71.5 112.4 16.1 42.7 36.4 88.8 31.0 76.4 75.0

180.3 16.1 101.2 29.0 28.5 20.7 127.2 14.6 50.0 100.6

350.0 32.9 79.2 13.6 225.9 22.5 227.2 18.1 155.2 87.6

THP-1 monocytes were differentiated into macrophages, immature or mature DCs, stained with directly labelled antibodies and analysed by flow cytometry. Mean fluorescence intensities for the indicated antigens are shown. Mean values of at least three experiments are shown, for the sake of better readability, standard deviations are not included (in most cases below 10%).

Discussion Cancer-derived exosomes interact with the host immune system in contrary ways. They can either induce an anticancer immune response, or contribute to immunosuppression [14, 16, 28]. Exosomes have been shown to carry MHC-peptide complexes and antigens that are able to initiate an immune reaction mainly through crosspresentation of antigens by APCs which modulates CD8+ or CD4+ responses in an antigen-specific way [18, 19, 37, 38]. On the other hand, exosomes directly suppress cytotoxic T cells and NK cells through their surface molecules including FasL, TRAIL, TGF-beta, CD73 and NKG2D ligands [24, 28, 39–42]. Furthermore,

tumour-derived exosomes increase the production of VEGF by M2 macrophages through transfer of miR-150 [43] and inhibit DC maturation [26, 27]. Immature DC are incapable of stimulating an efficient T-cell response and promote the development of regulatory T cells [44]. Whether cancer-derived EV including exosomes are routed primarily to mature or immature myeloid cells could have an impact on the balance between immune stimulation and immunosuppression. To compare the capacity of EV internalization by various myeloid cells, tumour-derived EV were added to monocytes, macrophages, immature and mature human DCs utilizing the differentiation capability of the human monocytic leukaemia cell line THP1. The uptake of exosomes by human immune cells was shown previously [45–47]. Our results confirm that EV were transferred to monocytes, macrophages, immature and mature dendritic cells. Macrophages and mature DCs, the ‘professional’ APC, showed increased EV uptake in comparison to the more immature cells. When comparing macrophages and mature DCs, bigger differences were observed when EV were labelled with CFSE than with DSSN+. The reason for this difference is unclear but could be based on the final destination of the fluorescent dyes. CFSE binds covalently to amino groups found for instance in proteins, while DSSN+ intercalates into membranes. One could speculate that the membrane turnover, e.g. through further secretion of EV is higher in mature DCs than in macrophages. Macrophages are highly specialized in removal of dying or dead cells, cellular debris and foreign substances by engulfing them by phagocytosis. Their high phagocytotic capacity may be the reason for their highly efficient internalization of EV. Also, Feng et al. demonstrated that

Figure 4 Competition between monocytes and macrophages or mature dendritic cells to take up fluorescently labelled EV. Identical numbers of either monocytes and macrophages, or monocytes and mDC were mixed together with CFSE-labelled EV. After 1, 2 and 4 h the monocyte/macrophage cell mixture was stained with CD11c-PE plus CD11b-APC (upper left dot plot) to discriminate between the two cell types. The CFSE fluorescence transferred by EV for the gated cell populations is shown in the histograms as indicated (4-h time point). The summary from three independent experiments for monocytes/macrophages and monocytes/ mDC is depicted in the lower right diagram. The difference of the mean fluorescence intensity of the mature cell types relative to the one measured in THP-1 monocytes is shown.

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exosomes are internalized more efficiently by phagocytes than by non-phagocytic cells [48]. They analysed eight cell lines and showed that exosomes attached to the membrane of non-phagocytic cells (like Jurkat T cell or mouse NIH3T3 fibroblasts) but were internalized via phagocytosis by phagocytic cells (like THP-1 monocytes or mouse Raw264.7 macrophages). We found efiicient internalisation of EV also in melanoma cells, which are classified as non-phagocytic cell type indicating that other uptake mechanisms such as endocytosis, macropinocytosis or direct fusion of EV with the plasma membrane are also active. Wahlgren et al. investigated the gene knock down by siRNA through exosome transfer. They found a stronger down-regulation of the targeted genes in monocytes than in lymphocytes that may depend on differences in their ability to absorb and internalize exosomes [47]. In contrast to our findings, Morelli et al. obtained stronger internalization of exosomes by immature (CD86-) bone marrow derived DCs than by mature CD86+ cells [49]. The conflicting results may be due to differences in the human versus the mouse system, or the source of DC (bone marrow isolation versus differentiation of monocytes). In conclusion, our results indicate that the cell differentiation status influences the efficiency of EV uptake.

Acknowledgment This study was supported by grant no. 2012/05/B/NZ2/ 00574 from the National Science Centre (NCN, Poland) and by the statutory funds of the Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz.

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