Detection of miRNAs in exosomes released by mouse immature and mature dendritic cells
ABSTRACT: Dendritic cells (DCs) are the most potent antigen (Ag)-presenting cells. Whereas immature DCs down-regulate T cell responses to induce/maintain immunological tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact and vesicle exchange. Transfer of nanovesicles (<100nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle microRNAs (miRNAs), and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, an hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, as they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and post-transcriptional regulation between DCs. The study has analyzed the microRNA content of 4 samples of immature exosomes, 4 samples of matures exosomes, 2 samples of immature bone-marrow-derived DCs, and 2 samples of mature bone marrow-derived DCs.
Project description:Dendritic cells (DCs) are the most potent antigen (Ag)-presenting cells. Whereas immature DCs down-regulate T cell responses to induce/maintain immunological tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact and vesicle exchange. Transfer of nanovesicles (<100nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle microRNAs (miRNAs), and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, an hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, as they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and post-transcriptional regulation between DCs. Overall design: The study has analyzed the microRNA content of 4 samples of immature exosomes, 4 samples of matures exosomes, 2 samples of immature bone-marrow-derived DCs, and 2 samples of mature bone marrow-derived DCs.
Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates ± SD. [mRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol . The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis (n=4). p0739_E1, p0739_ E2, p0739_E3 and p0739_E4 for the exosomes samples and p0739_C1, p0739_C2, p0739_C3, and p0739_C4 for the HMC-1 cells. [miRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol followed by RNeasy clean-up. The microarray experiments were performed by Exiqon.
Project description:Signalling between endothelial cells, endothelial progenitor cells and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signalling pathways. Exosomes are important mediators of intercellular communication in immune signalling, tumour survival, stress responses and angiogenesis. The ability of exosomes to incorporate and transfer mRNAs encoding for ‘acquired’ proteins or miRNAs repressing ‘resident’ mRNA translation suggests that they can influence the physiological behaviour of recipient cells. We here demonstrate that miR-214, a miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signalling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214 depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of Ataxia Telangiectasia Mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis the angiogenesis stimulatory function of the resulting exosomes. Collectively our data indicate that endothelial cells release miR-214 containing exosomes to stimulate angiogenesis through silencing of Ataxia Telangiectasia Mutated in neighbouring target cells. Gene expression analysis of HMEC endothelial cells exposed to supernatant containing either HMEC derived exosomes (miR-214 high), HMEC derived exosomes depleted of miR-214 (miR-214 low) or containing no exosomes (no exosomes). Each sample was analysed in duplo.
Project description:Microarray analysis of exosomal miRNAs vs the miRNAs of their respective donor cells. To determine the miRNA repertoires of exosomes secreted by immune cells, we isolated exosomes from cell supernatants of the Raji B cell line, the Jurkat-derived J77 T cell line, and primary dendritic cells (DCs) derived from human monocytes. Exosomes were isolated by a series of microfiltration and ultracentrifugation steps
Project description:All experiments are performed on human dendritic cells (DCs) differentiated in GM-CSF and IL-4 from CD14+ cells and bone marrow mesenchimal stem cells (MSCs). We found that MSCs impair active immune synapse formation and we evidenced at electron microscopy two types of contact between MSCs and DCs: gap and adherent junctions. In the same experiments we show that MSC contacts induce a reorganization of DC cytoskeleton by the formation of actin podosomes, structures typical of an immature, tolerogenic state of DCs. These results suggest that MSCs exert a tolerogenic effect on DCs by mechanism mediated by cell-cell contact. This induces DC cytoskeleton reorganization with formation of actin podosomes.
Project description:DC-SIGN is a C-type lectin expressed by dendritic cells (DCs) that binds HIV-1, sequestering it within multivesicular bodies to facilitate transmission to CD4+ T cells. Here we characterize the molecular basis of signalling through DC-SIGN by large-scale gene expression profiling and phosphoproteome analysis. Solitary DC-SIGN activation leads to a phenotypically disparate transcriptional program from Toll-like receptor (TLR) triggering with downregulation of MHC II, CD86, and interferon response genes and with induction of the TLR negative regulator ATF3. Phosphoproteome analysis reveals DC-SIGN signals through the leukemia-associated Rho guanine nucleotide exchange factor (LARG) to induce Rho activity. This LARG activation also occurs on DC HIV exposure and is required for effective HIV viral synapse formation. Taken together HIV mediated DC-SIGN signalling provides a mechanism by which HIV evades the immune response yet induces viral spread. Experiment Overall Design: Circulating monocyte derived DCs were isolated from buffy coats by adherence and culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). DC preparations analyzed were more than 98% pure. At day four 10 million immature DCs were either left unstimulated or stimulated using plate bound anti-DC-SIGN antibody for 2 hr. Three replicates of non-stimulated or stimulated cells were taken and used to extract total RNA.
Project description:Exosomes are small membraneous vesicles secreted into body fluids by tumors. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Further exploration into the molecular profiling of exosomes may increase our understanding of their roles in melanoma progression in vivo, and may have potential application in biomarker studies. In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. Our results indicate that melanoma-derived exosomes have unique gene expression signatures and miRNA profiles that may have important functions in melanoma metastasis and progression. Total RNA from cells and exosomes were isolated using mirVana total RNA isolation kit according to the manufacturer’s guidelines. RNA was quantified using Nanodrop ND-1000. The integrity of these total RNAs was assessed using Agilent 2100 Bioanalyzer. Total high-quality RNA was labelled. The miRNA array profiling was performed by using the Affymetrix GeneChip miRNA Array 1.0. Two different RNA preparations from two cell lines and their exosomes were used, except that only one RNA preparation was used for HEMa-LP exosome miRNA array. Due to the limited number of passages (approximately 10), adequate exosomal RNA and proteins from HEMa-LP cells for multiple analyses was not available.
Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.
Project description:We applied Illumina massively parallel signature sequencing to identify miRNomes in hematopoietic stem cells, bone morrow-derived immature DCs, mature DCs, and IL-10 and NO-producing regulatory DCs.The miRNomes of these DC subsets will contribute to investigate the significance of miRNAs in DC immunobiology. Examination of the miRNome in hematopoietic stem cells, bone morrow-derived immature DCs, mature DCs, and IL-10 and NO-producing regulatory DCs. All four mouse cell types.
Project description:We found that cardiac fibroblasts produce and secrete exosomes. miRNA profiling and TaqMan qRT-PCR experiments identified miR-21 expression to be higher in cardiac fibroblasts compared to those of miR-21*, whereas in exosomes miR-21* expression was higher compared to miR-21. The purpose of the study was to validate these findings by miRNA sequencing in cardiac fibroblasts and fibroblasts-derived exosomes. Neonatal rat cardiac fibroblasts were cultured in DMEM + 1% exosome-depleted FBS for 48h. Conditioned medium was collected and exosomes were purified by several centrifugation and filtration steps, following ultracentrifugation. Afterwards total RNA from cardiac fibroblasts and exosomes was isolated for miRNA sequencing.