Journal of Extracellular Vesicles最新文献

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%–3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na⁺/Ca²⁺ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca²⁺ transporter responsible for Ca²⁺ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca²⁺ deposition due to reduced Ca²⁺ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3^−/−) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, μCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3^−/− mice, thus supporting the critical role of NCX3 in facilitating Ca²⁺ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.

Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.

Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EV_VHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EV_LV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EV_LV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.

The excretory–secretory proteome plays a pivotal role in both intercellular communication during disease progression and immune escape mechanisms of various pathogens including cestode parasites like Taenia solium. The cysticerci of T. solium causes infection in the central nervous system known as neurocysticercosis (NCC), which affects a significant population in developing countries. Extracellular vesicles (EVs) are 30–150-nm-sized particles and constitute a significant part of the secretome. However, the role of EV in NCC pathogenesis remains undetermined. Here, for the first time, we report that EV from T. solium larvae is abundant in metabolites that can negatively regulate PI3K/AKT pathway, efficiently internalized by macrophages to induce AKT and mTOR degradation through auto-lysosomal route with a prominent increase in the ubiquitination of both proteins. This results in less ROS production and diminished bacterial killing capability among EV-treated macrophages. Due to this, both macro-autophagy and caspase-linked apoptosis are upregulated, with a reduction of the autophagy substrate sequestome 1. In summary, we report that T. solium EV from viable cysts attenuates the AKT–mTOR pathway thereby promoting apoptosis in macrophages, and this may exert immunosuppression during an early viable stage of the parasite in NCC, which is primarily asymptomatic. Further investigation on EV-mediated immune suppression revealed that the EV can protect the mice from DSS-induced colitis and improve colon architecture. These findings shed light on the previously unknown role of T. solium EV and the therapeutic role of their immune suppression potential.

Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parikh, Simon Spivack, Olivier Loudig

In the originally published article, author Kaushal Parikh's name was misspelled. This has been corrected in the online version of the article.

We apologize for this error.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae’s antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.