Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC‐MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 ( http://proteomecentral.proteomexchange.org/dataset/PXD001160 ).     

The key role of extracellular vesicles in the metastatic process

Extracellular vesicles (EVs), including exosomes, have a key role in the paracrine communication between organs and compartments. EVs shuttle virtually all types of biomolecules such as proteins, lipids, nucleic acids, metabolites and even pharmacological compounds. Their ability to transfer their biomolecular cargo into target cells enables EVs to play a key role in intercellular communication that can regulate cellular functions such as proliferation, apoptosis and migration. This has led to the emergence of EVs as a key player in tumor growth and metastasis through the formation of “tumor niches” in target organs. Recent data have also been shown that EVs may transform the microenvironment of primary tumors thus favoring the selection of cancer cells with a metastatic behavior. The release of EVs from resident non-malignant cells may contribute to the metastatic processes as well. However, cancer EVs may induce malignant transformation in resident mesenchymal stem cells, suggesting that the metastatic process is not exclusively due to circulating tumor cells. In this review, we outline and discuss evidence-based roles of EVs in actively regulating multiple steps of the metastatic process and how we can leverage EVs to impair metastasis.     

Immunomodulatory role of microRNAs transferred by extracellular vesicles

The immune system is composed of different cell types localised throughout the organism to sense and respond to pathological situations while maintaining homeostasis under physiological conditions. Intercellular communication between immune cells is essential to coordinate an effective immune response and involves both cell contact dependent and independent processes that ensure the transfer of information between bystander and distant cells. There is a rapidly growing body of evidence on the pivotal role of extracellular vesicles (EVs) in cell communication and these structures are emerging as important mediators for immune modulation upon delivery of their molecular cargo. In the last decade, EVs have been shown to be efficient carriers of genetic information, including microRNAs (miRNAs), that can be transferred between cells and regulate gene expression and function on the recipient cell. Here, we review the current knowledge of intercellular functional transfer of EV‐delivered miRNAs and their putative role in immune regulation.     

Role of hypoxia preconditioning in therapeutic potential of mesenchymal stem-cell-derived extracellular vesicles

The use of mesenchymal stem-cells (MSC) in cell therapy has received considerable attention because of their properties. These properties include high expansion and differentiation in vitro, low immunogenicity, and modulation of biological processes, such as inflammation, angiogenesis and hematopoiesis. Curiously, the regenerative effect of MSC is partly due to their paracrine activity. This has prompted numerous studies, to investigate the therapeutic potential of their secretome in general, and specifically their extracellular vesicles (EV). The latter contain proteins, lipids, nucleic acids, and other metabolites, which can cause physiological changes when released into recipient cells. Interestingly, contents of EV can be modulated by preconditioning MSC under different culture conditions. Among them, exposure to hypoxia stands out; these cells respond by activating hypoxia-inducible factor (HIF) at low O₂ concentrations. HIF has direct and indirect pleiotropic effects, modulating expression of hundreds of genes involved in processes such as inflammation, migration, proliferation, differentiation, angiogenesis, metabolism, and cell apoptosis. Expression of these genes is reflected in the contents of secreted EV. Interestingly, numerous studies show that MSC-derived EV conditioned under hypoxia have a higher regenerative capacity than those obtained under normoxia. In this review, we show the implications of hypoxia responses in relation to tissue regeneration. In addition, hypoxia preconditioning of MSC is being evaluated as a very attractive strategy for isolation of EV, with a high potential for clinical use in regenerative medicine that can be applied to different pathologies.     

The potential role of cofilin-1 in promoting triple negative breast cancer (TNBC) metastasis via the extracellular vesicles (EVs)

Triple negative breast cancer (TNBC) is an aggressive cancer, particularly prone to metastasis and is associated with poor survival outcomes. The key to unravelling the aggressiveness of TNBC lies in decoding the mechanism by which it metastasises. Cofilin-1 is a well-studied member of the cofilin family, involved in actin depolymerisation. Studies have described the diverse roles of cofilin-1 including cell motility, apoptosis and lipid metabolism. Levels of cofilin-1 have been shown to be increased in many different types of malignant cells, with increased cofilin-1 protein levels associated with poor prognosis in patients with TNBC. Extracellular vesicles (EVs) are microvesicles typically around 100 nm in size, found in all biological fluids examined to date (Lötvall et al., 2014). Proteomic studies on extracellular vesicles (EVs) have shown that cofilin-1 is amongst the most frequently detected. Moreover, decreased levels of cofilin-1 potentially inhibit the release of EVs from cells. Additionally, Cofilin-1 is essential for the maturation of EVs and may also play a key role in the establishment of the pre-metastatic niche, thus promoting tumour cell migration. Further work into the exact mechanism by which cofilin-1 advances TNBC metastasis, may potentially prevent disease progression and improve outcomes for patients with TNBC.     

From fundamental biology to the search for innovation: The story of fungal extracellular vesicles

Fungal extracellular vesicles (EVs) have attracted increased attention in recent years. Originated from a serendipitous discovery, the initial observation of fungal EVs resulted in a set of data repetitively rejected by several scientific journals, which raised questions about their authenticity. However, after the most fundamental experimental issues related to their observation were addressed, fungal EVs were characterized in dozens of species and became an emerging field. In this essay, we will discuss these fundamental findings and the potential of fungal EVs for the development of vaccines and antifungals.     

Cyclophilin A regulates secretion of tumour-derived extracellular vesicles

Extracellular Vesicles (EVs) are a heterogenous population of particles that play an important role in cell-cell communication in physiological and pathophysiological situations. In this study we reveal that the peptidyl prolyl isomerase Cyclophilin A (CypA) is enriched in cancer-derived EVs from a range of haematopoietic malignancies. CypA-enriched blood cancer EVs were taken up by normal monocytes independent of EV surface trypsin-sensitive proteins and potently stimulated pro-inflammatory MMP9 and IL-6 secretion. Further characterisation revealed that CypA is intravesicular, however, it is not present in all EVs derived from the haematopoietic cells, instead, it is predominantly located in high density EVs with a range of 1.15–1.18 g/ml. Furthermore, loss of CypA expression in haematological cancer cells attenuates high density EV-induced pro-inflammatory MMP9 and IL-6 secretion from monocytes. Mechanistically, we reveal that homozygous loss or siRNA knockdown of CypA expression significantly reduced the secretion of EVs in the range of 100–200 nm from blood cancer cells under normal and hypoxic conditions. Overall, this work reveals a novel role for CypA in cancer cell EV biogenesis.     

Tackling the effects of extracellular vesicles in fibrosis

Fibrosis is a physiological process of tissue repair that turns into pathological when becomes chronic, damaging the functional structure of the tissue. In this review we outline the current status of extracellular vesicles as modulators of the fibrotic process at different levels. In adipose tissue, extracellular vesicles mediate the intercellular communication not only between adipocytes, but also between adipocytes and other cells of the stromal vascular fraction. Thus, they could be altering essential processes for the functionality of adipose tissue, such as adipocyte hypertrophy/hyperplasia, tissue plasticity, adipogenesis and/or inflammation, and ultimately trigger fibrosis. This process is particularly important in obesity, and may eventually, influence the development of obesity-associated alterations. In this regard, obesity is now recognized as an independent risk factor for the development of chronic kidney disease, although the role of extracellular vesicles in this connection has not been explored so far. Nonetheless, the role of extracellular vesicles in the onset and progression of renal fibrosis has been highlighted due to the critical role of fibrosis as a common feature of kidney diseases. In fact, the content of extracellular vesicles disturbs cellular signaling cascades involved in fibrosis in virtually all types of renal cells. What is certain is that the study of extracellular vesicles is complex, as their isolation and manipulation is still difficult to reproduce, which complicates the overview of their physiopathological effects. Nevertheless, new strategies have been developed to exploit the potential of extracellular vesicles and their cargo, both as biomarkers and as therapeutic tools to prevent the progression of fibrosis towards an irreversible event.     

益生菌胞外囊泡与疾病的相关研究进展

益生菌是对宿主健康有益的活的微生物, 具有广泛的促健康作用。它可以缓解和改善肠道相关炎症性疾病的症状, 降低血清胆固醇, 调节肠道菌群平衡, 对恶性肿瘤的治疗和预防有重要作用。胞外囊泡(extracellular vesicles, EVs)是革兰阴性菌及少数革兰阳性菌释放的含有多种生物活性物质的一种纳米级囊泡结构。EVs通过携带生物信息分子在细菌与细菌或细菌与宿主之间的交流中起着非常重要的作用。虽然近年来研究者们对革兰阳性和阴性病原体来源的EVs的研究越来越多, 但对益生菌产生的EVs的研究却很少。本文综述了近年来益生菌释放的EVs在治疗相关疾病中的研究进展, 并对未来的发展形势进行分析。希望本文能围绕益生菌EVs这一新兴领域的最新进展, 寻找基于EVs的相关疾病的诊断工具和有效治疗方法。

     

Large extracellular vesicles: Size matters in tumor progression

Extracellular Vesicles (EVs) represent a heterogeneous population of particles naturally released from all cells, delimited by a lipid bilayer and able to horizontally transfer their cargos to recipient cells. These features imply the growing interest on EVs in cancer biology as biomarkers and therapeutic targets. In this review, we will highlight the specific process related to biogenesis and release of large EVs (L-EVs) derived from the plasma membrane (PM) compared to the small and well described exosomes, generated through the classical endosome-multivesicular body (MVB) pathway. The control of PM rigidity by cells depends on lipid/protein composition, cytoskeleton dynamics, cytoplasmic viscosity, ions balance, metabolic reprogramming and specific intracellular signaling pathways, all critical determinants of L-EVs biogenesis. We will focus in details on a specific class of L-EVs, named Large Oncosomes (LO), exclusively shed by cancer cells and with a size ranging from 1 μm up to 10 μm. We will examine LO specific cargos, either proteins or nucleic acids (i.e. mRNA, microRNAs, single/double-stranded DNA), as well as their functional role in cancer development and progression, also discussing the mechanisms of L-EVs internalization by recipient cells. Overall we will highlight the potential of LO as specific diagnostic/prognostic cancer biomarkers discussing the associated challenges.     

An emerging interplay between extracellular vesicles and cytokines

Extracellular vesicles (EVs) are small membrane-bound particles that are naturally released from cells. They are recognized as potent vehicles of intercellular communication both in prokaryotes and eukaryotes. Because of their capacity to carry biological macromolecules such as proteins, lipids and nucleic acids, EVs influence different physiological and pathological functions of both parental and recipient cells. Although multiple pathways have been proposed for cytokine secretion beyond the classical ER/Golgi route, EVs have recently recognized as an alternative secretory mechanism. Interestingly, cytokines/chemokines exploit these vesicles to be released into the extracellular milieu, and also appear to modulate their release, trafficking and/or content. In this review, we provide an overview of the cytokines/chemokines that are known to be associated with EVs or their regulation with a focus on TNFα, IL-1β and IFNs.     

A method of separating extracellular vesicles from blood shows potential clinical translation,and reveals extracellular vesicle cargo gremlin-1 as a diagnostic biomarker

Extracellular vesicles (EVs) have potential as minimally invasive biomarkers. However, the methods most commonly used for EV retrieval rely on ultracentrifugation, are time-consuming, and unrealistic to translate to standard-of-care. We sought a method suitable for EV separation from blood that could be used in patient care. Sera from breast cancer patients and age-matched controls (n = 27 patients; n = 36 controls) were analysed to compare 6 proposed EV separation methods. The EVs were then characterised on 8 parameters. The selected method was subsequently applied to independent cohorts of sera (n = 20 patients; n = 20 controls), as proof-of-principle, investigating EVs’ gremlin-1 cargo. Three independent runs with each method were very reproducible, within each given method. All isolates contained EVs, although they varied in quantity and purity. Methods that require ultracentrifugation were not superior for low volumes of sera typically available in routine standard-of-care. A CD63/CD81/CD9-coated immunobead-based method was most suitable based on EV markers'' detection and minimal albumin and lipoprotein contamination. Applying this method to independent sera cohorts, EVs and their gremlin-1 cargo were at significantly higher amounts for breast cancer patients compared to controls. In conclusion, CD63/CD81/CD9-coated immunobeads may enable clinical utility of blood-based EVs as biomarkers.     

Characterization and proteome profiling of extracellular vesicles in a murine model of Staphylococcus aureus endophthalmitis

Endophthalmitis is a vision-threatening complication of intraocular surgery or penetrating injury of which Staphylococcus aureus is an important etiological agent. Extracellular vesicles (EVs) hold a tremendous possibility for developing diagnostic and therapeutic biomarkers due to their role in the pathogenesis of various infections. The aim of this study was to characterise the protein cargo of EVs, isolated from a murine (C57BL/6) model of S. aureus endophthalmitis by LC–MS/MS. Contralateral eye injected with sterile media served as control and both eyes were enucleated after 24 h, followed by extraction of EVs by ultracentrifugation. EVs were characterized by DLS and western blotting with tetraspanin markers, CD9 and CD81 and quantified by ExoCet quantification kit. Proteomic analysis identified 1964 proteins (FDR ≤ 0.01) in EVs from infected mice eyes, of which 40 proteins varied significantly in their amounts in comparison to EVs obtained from control eyeballs (P-value ≤ 0.05). The results of this study provide insight into the global EV proteome of S. aureus endophthalmitis with their functional correlation and differential protein amounts between infected and control set. Annexin A5, cathepsin D and C5a play a pivotal role in disease pathogenesis and could possibly play a role as a prognostic marker in endophthalmitis.     

Generation of mesenchymal stem-like cells for producing extracellular vesicles

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with therapeutic potential against autoimmune diseases, inflammation, ischemia, and metabolic disorders. Contrary to the previous conceptions, recent studies have revealed that the tissue repair and immunomodulatory functions of MSCs are largely attributed to their secretome, rather than their potential to differentiate into desired cell types. The composition of MSC secretome encompasses cytokines and growth factors, in addition to the cell-derived structures known as extracellular vesicles (EVs). EVs are membrane-enclosed nanoparticles that are capable of delivering biomolecules, and it is now believed that MSC-derived EVs are the major players that induce biological changes in the target tissues. Based on these EVs’ characteristics, the potential of EVs derived from MSC (MSC-EV) in terms of tissue regeneration and immune modulation has grown during the last decade. However, the use of MSCs for producing sufficient amount of EVs has not been satisfactory due to limitations in the cell growth and large variations among the donor cell types. In this regard, pluripotent stem cells (PSCs)-derived MSC-like cells, which can be robustly induced and expanded in vitro, have emerged as more accessible cell source that can overcome current limitations of using MSCs for EV production. In this review, we have highlighted the methods of generating MSC-like cells from PSCs and their therapeutic outcome in preclinical studies. Finally, we have also discussed future requirements for making this cell-free therapy clinically feasible.     

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle‐free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.     

Interplay of extracellular vesicles and other players in cerebral malaria pathogenesis

Background

Malaria is a serious parasitic infection affecting millions of people worldwide each year. Cerebral malaria is the most severe complication of Plasmodium infections, predominantly affecting children. Extracellular vesicles are essential mediators of intercellular communication and include apoptotic bodies, microvesicles and exosomes. Microvesicle numbers increase during disease pathogenesis and inhibition of their release can prevent brain pathology and mortality.

Impact of endometriosis on embryo quality and endometrial receptivity in women undergoing assisted reproductive technology

ART is an important treatment method for infertile patients with endometriosis. However, the effects of endometriosis on embryo quality and endometrial receptivity remain unclear. Thus, we aimed to simultaneously investigate the impact of endometriosis and its stage on embryo quality and endometrial receptivity in women undergoing ART. We retrospectively analyzed the data from patients with and without endometriosis who underwent oocyte retrieval and/or high-quality embryos transfer between July 2015 and December 2020, including 1312 IVF cycles and 608 IVF or frozen-thawed embryo transfer (FET) cycles, respectively. The endometriosis group had a lower percentage of good cleavage-stage embryos and fertilization rates than those in the control group (p = 0.038 and 0.008, respectively). The number of retrieved oocytes, MII oocytes, cleavage, blastocysts, and blastulation rates was comparable between two groups. We found no significant difference in clinical pregnancy, implantation, live birth, miscarriage, or multiple pregnancy rates between the two groups among patients who transferred high-quality embryos. Stratification analysis showed that patients with stage III-IV endometriosis had fewer retrieved oocytes than those with stage I-II endometriosis (p = 0.012) and marginally fewer retrieved oocytes than the control group (p = 0.051). The stage I-II group had the lowest percentage of good cleavage-stage embryos, which was significantly lower than that of the control group (p = 0.043). In FET cycles, patients with stage III-IV endometriosis had a higher miscarriage rate than those in the control group (p = 0.023). Our results suggest that endometriosis does not alter endometrial receptivity but affects embryo quality, oocyte fertilization ability, and ovarian response.     

Proteomic identification of the contents of small extracellular vesicles from in vivo Plasmodium yoelii infection

Small extracellular vesicles, including exosomes, are formed by the endocytic pathway and contain genetic and protein material which reflect the contents of their cells of origin. These contents have a role in vesicle-mediated information transfer, as well as physiological and pathological functions. Thus, these vesicles are of great interest as therapeutic targets, or as vehicles for immunomodulatory control. In Plasmodium spp. infections, vesicles derived from the parasite or parasite-infected cells have been shown to induce the expression of pro-inflammatory elements, which have been correlated with manifestations of clinical disease. Herein, we characterised the protein cargo of naturally occurring sEVs in the plasma of P. yoelii-infected mice. After in vivo infections, extracellular vesicles in the size range of exosomes were collected by sequential centrifugation/ultracentrifugation followed by isopycnic gradient separation. Analysis of the vesicles was performed by transmission electron microscopy, dynamic light scattering, SDS–PAGE and flow cytometry. LC-MS analysis followed by bioinformatics analysis predicted parasite protein cargo associated with exosomes. Within these small extracellular vesicles, we identified proteins of interest as vaccine candidates, uncharacterized proteins which may be targets of T cell immunoreactivity, and proteins involved in metabolic processes, regulation, homeostasis and immunity. Importantly, the small extracellular vesicles studied in our work were obtained from in vivo infection rather than from the supernatant of in vitro cultures. These findings add to the growing interest in parasite small extracellular vesicles, further our understanding of the interactions between host and parasite, and identify novel proteins which may represent potential targets for vaccination against malaria.