Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences.

Background: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. Objectives: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA. Methods: Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). Results: The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. Conclusions: Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases.

Positive association and future perspectives of mitochondrial DNA copy number and telomere length - a pilot twin study.

INTRODUCTION: Recent experimental and population studies have highlighted the existence of telomere-mitochondria interplay. Besides studies revealing the molecular mechanisms underlying the associations of telomere defects and mitochondrial functions, investigations of mitochondrial DNA copy number (mtDNAcn) and telomere length (TL) in healthy and disease phenotypes have likewise begun, with the aim of gaining more insights about their relationship in humans. MATERIAL AND METHODS: A total of 142 asymptomatic adult twins, comprising 96 monozygotic (MZ) and 46 dizygotic (DZ) twins (mean age: 50.54 ±15.43 years), members of the Hungarian Twin Registry, were included in the analysis. Applying the qPCR standard curve method, we investigated the relationship of mtDNA copy number, telomere length and clinical data, besides assessing co-twin similarities of MZ and DZ twins for their mtDNAcn and TL measures. RESULTS: We found that twins were similar in their intraclass correlation coefficients irrespective of zygosity, suggesting a possibly more important role of common (shared) environmental factors compared to non-shared (unique) environmental and to a smaller degree also individual genetic influences. We confirmed a significant positive association between mtDNAcn and TL (r = 0.28, p < 0.01) in age- and sex-corrected analysis. Following bivariate estimates and correction with significant predictors, the independent positive associations were further verified. CONCLUSIONS: Our results extend the until now modest number of studies investigating mtDNAcn and TL simultaneously in humans. In addition, we are the first to examine the relationship between mtDNAcn and telomere length in MZ and DZ twin subjects.

Platelet-derived extracellular vesicles may contribute to the hypercoagulable state in preeclampsia.

It has previously been shown that preeclampsia is associated with disturbed hemostasis and that extracellular vesicles (EVs) play important role in the regulation of hemostatic homeostasis. Thus, we hypothesized that the altered procoagulant characteristics of circulating platelet-derived EVs may contribute to the disturbed hemostasis in preeclampsia. Using multicolor flow cytometry, we have analyzed both tissue factor expressing procoagulant EVs and platelet-derived EV subpopulations derived from resting and activated thrombocytes by examining them in plasma samples of preeclamptic patients and pregnancy-matched healthy individuals. Compared to pregnancy-matched healthy individuals in preeclamptic patients a significantly (p < 0.05) higher ratio of Annexin-V positive activated platelets and a higher number of CD142+ tissue factor bearing procoagulant EVs were found, whereas the absolute amount of circulating CD41a+ platelet-derived EVs and CD62P+/CD41a+ EVs produced by activated thrombocytes was significantly lower in the plasma of preeclamptic women. In the plasma samples, there was no significant difference in the amount of CD63+ platelet-derived EVs. We propose that increased platelet activation and tissue factor expression of platelet derived extracellular vesicles may contribute to the hypercoagulable state observed in preeclampsia.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Radio-detoxified LPS alters bone marrow-derived extracellular vesicles and endothelial progenitor cells.

Stem cell-based therapies raise hope for cell replacement and provide opportunity for cardiac regenerative medicine and tumor therapy. Extracellular vesicles are a membrane-enclosed intercellular delivery system with the potential to improve the therapeutic efficacy of the treatment of a variety of disorders. As the incidence of breast cancer continues to rise, radiotherapy has emerged as a leading treatment modality. Radiotherapy also increases the risk of coronary heart disease and cardiac mortality. In a chest-irradiated mouse model of cardiac injury, we investigated the effects of local irradiation. We found an increased lethality after 16 Gy irradiation. Importantly, radio-detoxified LPS (RD-LPS) treatment prolonged the survival significantly. By flow cytometry, we demonstrated that upon administration of RD-LPS, the number of bone marrow-derived endothelial progenitor cells increased in the bone marrow and, in particular, in the circulation. Furthermore, mass spectrometry analysis showed that RD-LPS altered the proteomic composition of bone marrow cell-derived small extracellular vesicles (sEVs). RD-LPS treatment increased interferon-induced transmembrane protein-3 (IFITM3) expression markedly both in bone marrow cells and in bone marrow cell-derived small extracellular vesicles. This is the first study to demonstrate that radio-detoxified LPS treatment induces an increase of circulating endothelial progenitor cells (EPCs) in parallel with a reduced radiotherapy-related mortality. While the total number of bone marrow-derived extracellular vesicles was significantly increased 24 h after treatment in the RD-LPS groups, the number of endothelial progenitor cells was reduced in animals injected with GW4896 (a chemical inhibitor of exosome biogenesis) as compared with controls. In contrast to these in vivo results, in vitro experiments did not support the effect of sEVs on EPCs. Our data raise the intriguing possibility that IFITM3 may serve as a marker of the radio-detoxified LPS treatment.

Author Correction: The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Perspective: bidirectional exosomal transport between cancer stem cells and their fibroblast-rich microenvironment during metastasis formation.

Carcinomas are complex structures composed of hierarchically organized distinct cell populations such as cancer stem cells and non-stem (bulk) cancer cells. Their genetic/epigenetic makeup and the dynamic interplay between the malignant cell populations and their stromal fibroblasts are important determinants of metastatic tumor invasion. Important mediators of these interactions are the small, membrane-enclosed extracellular vesicles, in particular exosomes. Both cancer cell and fibroblast-derived exosomes carry a set of regulatory molecules, including proteins and different species of RNA, which cooperatively support metastatic tumor spread. Here, we briefly overview potential links between cancer stem cells and the exosome-mediated fibroblast-enriched metastatic niche formation to discuss their role in the promotion of tumor growth and metastatic expansion in breast carcinoma models.

Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis.

Background: The T-helper 17 (Th17) cells have a prominent role in inflammation as well as in bone and join destruction in both rheumatoid and psoriatic arthritis (RA and PsA). Here, we studied Th17 cell differentiation in RA and PsA. Methods: Blood samples from healthy donors, RA and PsA patients were collected. CD45RO- (naive) and CD45RO+ (memory) T cells were isolated from peripherial blood mononuclear cell by magnetic separation. Naive T cells were stimulated with anti-CD3, anti-CD28, and goat anti-mouse IgG antibodies and treated with transforming grow factor beta, interleukin (IL)-6, IL-1ß, and IL-23 cytokines and also with anti-IL-4 antibody. IL-17A and IL-22 production were measured by enzyme linked immunosorbent assay, RORC, and T-box 21 (TBX21) expression were analyzed by quantitative polymerase chain reaction and flow cytometry. C-C chemokine receptor 6 (CCR6), CCR4, and C-X-C motif chemokine receptor 3 expression were determined by flow cytometry. Cell viability was monitored by impedance-based cell analyzer (CASY-TT). Results: RORC, TBX21, CCR6, and CCR4 expression of memory T cells of healthy individuals (but not RA or PsA patients) were increased (p < 0.01; p < 0.001; p < 0.05; p < 0.05, respectively) compared to the naive cells. Cytokine-induced IL-17A production was different in both RA and PsA patients when compared to healthy donors (p = 0.0000026 and p = 0.0001047, respectively). By contrast, significant differences in IL-22 production were observed only between RA versus healthy or RA versus PsA patients (p = 0.000006; p = 0.0013454, respectively), but not between healthy donors versus PsA patients. Conclusion: The naive CD4 T-lymphocytes are predisposed to differentiate into Th17 cells and the in vitro Th17 cell differentiation is profoundly altered in both RA and PsA.

The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells.

Intercellular communication via extracellular vesicles (EVs) and their target cells, especially immune cells, results in functional and phenotype changes that consequently may play a significant role in various physiological states and the pathogenesis of immune-mediated disorders. Monocytes are the most prominent environment-sensing immune cells in circulation, skilled to shape their microenvironments via cytokine secretion and further differentiation. Both the circulating monocyte subset distribution and the blood plasma EV pattern are characteristic for preeclampsia, a pregnancy induced immune-mediated hypertensive disorder. We hypothesized that preeclampsia-associated EVs (PE-EVs) induced functional and phenotypic alterations of monocytes. First, we proved EV binding and uptake by THP-1 cells. Cellular origin and protein cargo of circulating PE-EVs were characterized by flow cytometry and mass spectrometry. An altered phagocytosis-associated molecular pattern was found on 12.5 K fraction of PE-EVs: an elevated CD47 "don't eat me" signal (p < 0.01) and decreased exofacial phosphatidylserine "eat-me" signal (p < 0.001) were found along with decreased uptake of these PE-EVs (p < 0.05). The 12.5 K fraction of PE-EVs induced significantly lower chemotaxis (p < 0.01) and cell motility but accelerated cell adhesion of THP-1 cells (p < 0.05). The 12.5 K fraction of PE-EVs induced altered monocyte functions suggest that circulating EVs may have a role in the pathogenesis of preeclampsia.

Tight co-twin similarity of monozygotic twins for hTERT protein level of T cell subsets, for telomere length and mitochondrial DNA copy number, but not for telomerase activity.

Our study analyzed lymphocyte subpopulations of 32 monozygotic twins and compared the level of the catalytic reverse transcriptase protein subunit (hTERT) in T lymphocytes (Tly), helper- (Th), cytotoxic- (Tc) and regulatory T cell (Treg) subgroups. Four variables related to telomere and mitochondrial biology were simultaneously assessed, applying multi-parametric flow cytometry, TRAP-ELISA assay and qPCR standard curve method on peripheral blood mononuclear cell (PBMC) samples of genetically matched individuals. Twin data of telomerase activity (TA), hTERT protein level, telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) were analyzed for co-twin similarity. The present study has provided novel information by demonstrating very high intraclass correlation (ICC) of hTERT protein level in T lymphocytes (0.891) and in both Th (0.896), Treg (0.885) and Tc (0.798) cell subgroups. When comparing results measured from PBMCs, intraclass correlation was also high for telomere length (0.815) and considerable for mtDNA copy number (0.524), and again exceptionally high for the rate-limiting telomerase subunit, hTERT protein level (0.946). In contrast, telomerase activity showed no co-twin similarity (ICC 0). By comparing relative amounts of hTERT protein levels in different lymphocyte subgroups of twin subjects, in Treg cells significantly higher level could be detected compared to Tly, Th or Tc cell subgroups. This is the first study that simultaneously analyzed co-twin similarity in MZ twins for the above four variables and alongside assessed their relationship, whereby positive association was found between TL and mtDNAcn.

Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype.

The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct "autophagosome-exosome-like" profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.

Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position Paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology.

Extracellular vesicles (EVs)-particularly exosomes and microvesicles (MVs)-are attracting considerable interest in the cardiovascular field as the wide range of their functions is recognized. These capabilities include transporting regulatory molecules including different RNA species, lipids, and proteins through the extracellular space including blood and delivering these cargos to recipient cells to modify cellular activity. EVs powerfully stimulate angiogenesis, and can protect the heart against myocardial infarction. They also appear to mediate some of the paracrine effects of cells, and have therefore been proposed as a potential alternative to cell-based regenerative therapies. Moreover, EVs of different sources may be useful biomarkers of cardiovascular disease identities. However, the methods used for the detection and isolation of EVs have several limitations and vary widely between studies, leading to uncertainties regarding the exact population of EVs studied and how to interpret the data. The number of publications in the exosome and MV field has been increasing exponentially in recent years and, therefore, in this ESC Working Group Position Paper, the overall objective is to provide a set of recommendations for the analysis and translational application of EVs focussing on the diagnosis and therapy of the ischaemic heart. This should help to ensure that the data from emerging studies are robust and repeatable, and optimize the pathway towards the diagnostic and therapeutic use of EVs in clinical studies for patient benefit.

Mesenteric lymph node stromal cell-derived extracellular vesicles contribute to peripheral de novo induction of Foxp3+ regulatory T cells.

Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.

The Role of Extracellular Vesicle and Tunneling Nanotube-Mediated Intercellular Cross-Talk Between Mesenchymal Stem Cells and Human Peripheral T Cells.

The role of extracellular vesicles (EVs) in mediating the immunosuppressory properties of mesenchymal stem cells (MSCs) has recently attracted remarkable scientific interest. The aim of this work was to analyze the transport mechanisms of membrane and cytoplasmic components between T lymphocytes and adipose tissue-derived MSCs (AD-MSCs), by focusing on the role of distinct populations of EVs, direct cell-cell contacts, and the soluble mediators per se in modulating T lymphocyte function. We found that neither murine thymocytes and human primary T cells nor Jurkat lymphoblastoid cells incorporated appreciable amounts of MSC-derived microvesicles (MVs) or exosomes (EXOs). Moreover, these particles had no effect on the proliferation and IFN-γ production of in vitro-stimulated primary T cells. In contrast, AD-MSCs incorporated large amounts of membrane components from T cells as an intensive uptake of EXOs and MVs could be observed. Interestingly, we found a bidirectional exchange of cytoplasmic components between human AD-MSCs and primary T lymphocytes, mediated by tunneling nanotubes (TNTs) derived exclusively from the T cells. In contrast, TNTs couldn't be observed between AD-MSCs and the Jurkat cells. Our results reveal a novel and efficient way of intercellular communication between MSCs and T cells, and may help a better understanding of the immunomodulatory function of MSCs.

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles.

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

Critical role of extracellular vesicles in modulating the cellular effects of cytokines.

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.