Biological properties of extracellular vesicles and their physiological functions

The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory potential. Still, they will act in conjunction with other released parasite-derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non-vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose-dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co-inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose-dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose-dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.

Section: Introductionmentioning

confidence: 99%

More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection

Pérez-Cabezas

Cecílio

Silva

et al. 2018

“…Extracellular vesicles (EVs) are a heterogeneous population of lipid membrane-bound nano-scale structures that are released from cells through distinct mechanisms [1]. There are three commonly accepted major EV sub-types, exosomes, microvesicles and apoptotic bodies, which are defined by their cellular origin, size and composition.…”

Section: Introductionmentioning

confidence: 99%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

“…This increase in research activity was further cemented by the 2006 finding that EVs contain RNA cargoes [8,9]. The field now enjoys an exponential growth in the number of publications [10] and EVs have now been isolated from an ever-expanding array of biofluids and cell types [1]. …”

Section: Introductionmentioning

confidence: 99%

“…EVs may even play a direct role in evolution as studies show EV-mediated transfer of nucleic acids into sperm cells [24]. Final evidence for the importance of EVs is given by their existence across taxonomic kingdoms, from archaea through bacteria to plants and animals [1]. …”

Section: Introductionmentioning

confidence: 99%

“…The proteomic, transcriptomic and lipidomic analyses of exosomes have revealed enrichments in specific proteins including: tetraspanin, cystoskeletal and heat shock proteins, messenger and micro RNAs, and lipids such as ceramide, phosphatidylserine, cholesterol and sphingolipids. These biomolecular compositions can differ markedly between EVs from different cellular origins and also between healthy and disease states [1]. Combined with the high yield and ubiquity of EVs across all biofluids assayed, this suggests a clinical use for EVs as a source of diagnostic disease markers [26–28].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives

Williams

Royo

Aizpurua‐Olaizola

et al. 2018

Self Cite

206

163

It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed, recent reports show that the presence of glycoconjugates is involved in EV biogenesis, in cellular recognition and in the efficient uptake of EVs by recipient cells. It is clear that a full understanding of EV biology will require researchers to focus also on EV glycosylation through glycomics approaches. This review outlines the major glycomics techniques that have been applied to EVs in the context of the recent findings. Beyond understanding the mechanisms by which EVs mediate their physiological functions, glycosylation also provides opportunities by which to engineer EVs for therapeutic and diagnostic purposes. Studies characterising the glycan composition of EVs have highlighted glycome changes in various disease states, thus indicating potential for EV glycans as diagnostic markers. Meanwhile, glycans have been targeted as molecular handles for affinity-based isolation in both research and clinical contexts. An overview of current strategies to exploit EV glycosylation and a discussion of the implications of recent findings for the burgeoning EV industry follows the below review of glycomics and its application to EV biology.