Inhibition of endolysosome fusion increases exosome secretion.

Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications.

Immune-Associated Proteins Are Enriched in Lung Tissue-Derived Extracellular Vesicles during Allergen-Induced Eosinophilic Airway Inflammation.

Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs' proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.

Extracellular vesicles from mast cells induce mesenchymal transition in airway epithelial cells.

BACKGROUND: In the airways, mast cells are present in close vicinity to epithelial cells, and they can interact with each other via multiple factors, including extracellular vesicles (EVs). Mast cell-derived EVs have a large repertoire of cargos, including proteins and RNA, as well as surface DNA. In this study, we hypothesized that these EVs can induce epithelial to mesenchymal transition (EMT) in airway epithelial cells. METHODS: In this in-vitro study we systematically determined the effects of mast cell-derived EVs on epithelial A549 cells. We determined the changes that are induced by EVs on A549 cells at both the RNA and protein levels. Moreover, we also analyzed the rapid changes in phosphorylation events in EV-recipient A549 cells using a phosphorylated protein microarray. Some of the phosphorylation-associated events associated with EMT were validated using immunoblotting. RESULTS: Morphological and transcript analysis of epithelial A549 cells indicated that an EMT-like phenotype was induced by the EVs. Transcript analysis indicated the upregulation of genes involved in EMT, including TWIST1, MMP9, TGFB1, and BMP-7. This was accompanied by downregulation of proteins such as E-cadherin and upregulation of Slug-Snail and matrix metalloproteinases. Additionally, our phosphorylated-protein microarray analysis revealed proteins associated with the EMT cascade that were upregulated after EV treatment. We also found that transforming growth factor beta-1, a well-known EMT inducer, is associated with EVs and mediates the EMT cascade induced in the A549 cells. CONCLUSION: Mast cell-derived EVs mediate the induction of EMT in epithelial cells, and our evidence suggests that this is triggered through the induction of protein phosphorylation cascades.

Molecular profiling of driver events in metastatic uveal melanoma.

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments. Here we genomically profile metastatic tumors and infiltrating lymphocytes. BAP1 alterations are overrepresented and found in 29/32 of cases. Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease. One outlier tumor has a high mutational burden associated with UV-damage. CDKN2A deletions also occur, which are rarely present in primaries. A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains. Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant. This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.

Endosomal signalling via exosome surface TGFß-1.

Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor ß-1 (TGFß-1) on their surfaces. The latent form of TGFß-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGFß-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGFß-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGFß-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.

DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology.

Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.

Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy.

Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index ( n ≈ 1.35) compared with the LD EVs ( n ≈ 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing.

Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.

Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling.

BACKGROUND: Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown. METHODS AND RESULTS: Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells. CONCLUSIONS: Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum.

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.