Exosome-like Extracellular Vesicles Research Articles

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 mM) or rotenone (0.2-50 mM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI.

207 Anti-aging effect of follicular fluid exosome-like extracellular vesicles on bovine oocytes matured in vitro

207 Anti-aging effect of follicular fluid exosome-like extracellular vesicles on bovine oocytes matured in vitro

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.

Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80–100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36−/− mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

PMON54 Exosomes Isolated from Conditioned Media of Immortalized Kisspeptin Neurons Exert Diverse Effects on Central and Peripheral in Vitro Cell Models.

Abstract Previous work in our laboratory explored the proteomic contents of exosome-like extracellular vesicles (EVs) released into the media in vitro from immortalized Kisspeptin (KP) neuronal cell lines KTaV-3 (derived from female mouse AVPV) and KTaR-1 (derived from KNDy neurons in the ARC). EVs were isolated from conditioned media via ultracentrifugation or filtration kit and validated using a NanoCyte, and LCMS-MS analysis revealed that relative abundance of exosomal cargo varied dependent upon estrogen (E2) exposure in vitro, with ∼150-170 proteins up-regulated and ∼200-220 proteins downregulated by E2 in EVs of KTaR-1 and KTaV-3 KP neurons. Since E2-regulated KP exosomal proteins included candidates implicated in the regulation of synaptic plasticity and signaling (i.e. annexins, semaphorins, connexins), we investigated the effects of exposure to purified EVs on gene expression in immortalized GnRH neurons (GT1-7 cells). Notably, EVs from 24h E2-treated KTaV-3 neurons induced increased expression of kiss1r in GT1-7 cells, in contrast to KTaV-3 neurons not exposed to E2, suggesting that AVPV KP neurons may signal an increase in KP receptivity in GnRH neurons in vivo via non-neuronal communication. Additionally, increases in expression of the synaptic scaffolding protein PSD-95 (dlg4) were seen in GT1-7 cells treated with E2-treated KTaV-3 EVs, suggesting AVPV KP neurons may use extracellular vesicles to modulate GnRH neuronal synaptic plasticity over the estrous cycle. While EVs isolated from KTaR-1 conditioned media did not alter GT1-7 kiss1r or dlg4 levels, treatment (24h) resulted in induction of selective gap junction hemichannel expression in GT1-7 cells. KTaR-1 (but not KTaV-3) EVs increased expression of Cx26 (gjb2) irrespective of E2 exposure, while induction of Cx43 (gja1) expression in GT1-7 cells was only observed following treatment with E2-deprived KTaR-1 EVs. Further, we found that KTaR-1 media and EVs can affect osteoblast function in vitro, including increases in sp7 and runx2 expression, E2-dependent modulation of wnt10b, and formation of bone matrix (evaluated by Alizarin Red assay) in cultured osteoblast lines, supporting recent studies implicating ARC KP neurons in bone remodeling. Lastly, we found significant levels of immunomodulatory pentraxins (PTX3) in KP EVs, with abundance dependent upon prior E2 exposure in KTaR-1 KP neurons, as revealed by ELISA. Together, results from these studies suggest that EVs may represent additional intercellular communication pathways utilized by Kiss-1 neurons to elicit changes in nearby neuronal populations and potentially even in the periphery, affecting inflammation and bone remodeling. Future studies will address mechanisms involved in the E2 regulation of exosomal cargo in these critical neuronal populations. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau.

The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer's disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer's patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.

Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody-coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT-EVs. Immunoblot analysis demonstrated KIT-EVs have a distinct protein profile from KIT-depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT-EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT-EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle- and exosome-like EVs. Further, selective KIT-immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.

Effects of a 6-wk Sprint Interval Training Protocol at Different Altitudes on Circulating Extracellular Vesicles.

This study aimed to investigate the modulation of circulating exosome-like extracellular vesicles (ELVs) after 6 wk of sprint interval training (SIT) at sea level and at 2000, 3000, and 4000 m. Thirty trained endurance male athletes (18-35 yr) participated in a 6-wk SIT program (30-s all-out sprint, 4-min 30-s recovery; 4-9 repetitions, 2 sessions per week) at sea level ( n = 8), 2000 m (fraction of inspired oxygen (F io2 ) 0.167, n = 8), 3000 m (F io2 0.145, n = 7), or 4000 m (F io2 0.13, n = 7). Venous blood samples were taken before and after the training period. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis, and characterized according to international standards. Candidate ELV microRNAs (miRNAs) were quantified by real-time polymerase chain reaction. When the three hypoxic groups were analyzed separately, only very minor differences could be detected in the levels of circulating particles, ELV markers, or miRNA. However, the levels of circulating particles increased (+262%) after training when the three hypoxic groups were pooled, and tended to increase at sea level (+65%), with no difference between these two groups. A trend to an increase was observed for the two ELV markers, TSG101 (+65%) and HSP60 (+441%), at sea level, but not in hypoxia. Training also seemed to decrease the abundance of miR-23a-3p and to increase the abundance of miR-21-5p in hypoxia but not at sea level. A 6-wk SIT program tended to increase the basal levels of circulating ELVs when performed at sea level but not in hypoxia. In contrast, ELV miRNA cargo seemed to be modulated in hypoxic conditions only. Further research should explore the potential differences in the origin of ELVs between normoxic and local and systemic hypoxic conditions.

Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus.

BackgroundTubular damage has a role in Diabetic Kidney Disease (DKD). We evaluated the early tubulointerstitial damage biomarkers in type-1 Diabetes Mellitus (T1DM) pediatric participants and studied the correlation with classical DKD parameters.MethodsThirty-four T1DM and fifteen healthy participants were enrolled. Clinical and biochemical parameters [Glomerular filtration Rate (GFR), microalbuminuria (MAU), albumin/creatinine ratio (ACR), and glycated hemoglobin A1c (HbA1c)] were evaluated. Neutrophil gelatinase-associated lipocalin (NGAL), Hypoxia-inducible Factor-1α (HIF-1α), and Nuclear Factor of Activated T-cells-5 (NFAT5) levels were studied in the supernatant (S) and the exosome-like extracellular vesicles (E) fraction from urine samples.ResultsIn the T1DM, 12% had MAU >20 mg/L, 6% ACR >30 mg/g, and 88% had eGFR >140 ml/min/1.72 m2. NGAL in the S (NGAL-S) or E (NGAL-E) fraction was not detectable in the control. The NGAL-E was more frequent (p = 0.040) and higher (p = 0.002) than NGAL-S in T1DM. The T1DM participants with positive NGAL had higher age (p = 0.03), T1DM evolution (p = 0.03), and serum creatinine (p = 0.003) than negative NGAL. The NGAL-E correlated positively with tanner stage (p = 0.0036), the median levels of HbA1c before enrollment (p = 0.045) and was independent of ACR, MAU, and HbA1c at the enrollment. NFAT5 and HIF-1α levels were not detectable in T1DM or control.ConclusionUrinary exosome-like extracellular vesicles could be a new source of early detection of tubular injury biomarkers of DKD in T1DM patients.

135&#x2003;Effect of different concentrations of follicular fluid exosome-like extracellular vesicles on in vitro oocyte maturation and embryo development in cattle.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Exogenous miRNAs induce post-transcriptional gene silencing in plants

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea. Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399, trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants.

OPTC-5. Molecular signatures of podoplanin expressing glioblastoma cell subsets with putative role in cancer associated thrombosis and microthrombosis

Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating transmembrane protein by transforming events and release from cancer cells into the circulation are of considerable interest. We took advantage of single-cell and bulk GBM transcriptome dataset mining and investigated the pattern of PDPN expression across several databases. Our analysis indicated that PDPN is expressed by distinct (mesenchymal) glioblastoma cell subpopulations and is downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. Additionally, we utilized isogenic and stem GBM cell lines, xenograft models in mice, ELISA assays for PDPN, tissue factor (TF), platelet factor 4 (PF4) and clotting activation markers (D-dimer), and multicolor nano-flow cytometry to show that GBM cells exteriorize PDPN and/or TF as cargo of exosome-like coagulant extracellular vesicles EVs. We also documented an increase of platelet activation (PF4) or coagulation markers (D-dimer) in mice harboring the corresponding PDPN- or TF-expressing glioma xenografts, respectively. While PDPN was a dominant regulator of systemic platelet activation, co-expression of PDPN and TF impacted local microthrombosis. Our work suggests that distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention.

Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci.

PurposeRecently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci.MethodsA. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure.ResultsMolecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans.ConclusionHoney is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilm-modulating strategy in dental caries.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

Hypoxic Induction of Exosome Uptake through Proteoglycan-Dependent Endocytosis Fuels the Lipid Droplet Phenotype in Glioma.

As an adaptive response to hypoxic stress, aggressive tumors rewire their metabolic phenotype into increased malignant behavior through extracellular lipid scavenging and storage in lipid droplets (LD). However, the underlying mechanisms and potential lipid source retrieved in the hypoxic tumor microenvironment remain poorly understood. Here, we show that exosome-like extracellular vesicles (EV), known as influential messengers in the tumor microenvironment, may also serve anabolic functions by transforming hypoxic, patient-derived human glioblastoma cell lines into the LD+ phenotype. EVs were internalized via a hypoxia-sensitive, endocytic mechanism that fueled LD formation through direct lipid transfer, and independently of fatty acid synthase activity. EVs can enter cells through multiple and yet ill-defined pathways. On a mechanistic level, we found that hypoxia-mediated EV uptake depends on increased heparan sulfate proteoglycan (HSPG) endocytosis that preferentially followed the lipid raft pathway. The functional relevance of HSPG was evidenced by the reversal of EV-mediated LD loading by targeting of HSPG receptor function. IMPLICATIONS: Together, our data extend the multifaceted role of EVs in cancer biology by showing their LD-inducing capacity in hypoxic glioma cells. Moreover, these findings highlight a potential function for HSPG-mediated endocytosis as a salvage pathway for EV retrieval during tumor stress conditions.

RNA-binding proteins contribute to small RNA loading in plant extracellular vesicles.

Plants utilize extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called “cross-kingdom RNAi.” It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins (RBPs) in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and Annexins (ANN), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not non-EV enriched sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11rh37 and ann1ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RBPs play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11rh37 and ann1ann2 showed increased susceptibility to Botrytis cinerea, supporting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.

A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that βγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of βγ-CAT. The cellular action of βγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of βγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles

ABSTRACT Hepatitis A virus (HAV), a classic nonenveloped virus, has recently been found to be released mainly in the form of quasi-enveloped HAV (eHAV) by hijacking host endosomal sorting complexes required for transport (ESCRT) complexes. Unlike the nonenveloped virion, eHAV contains the viral protein pX on the surface of the HAV capsid as an extension of VP1. How HAV capsids acquire the host envelope and whether the pX protein is involved in this process were previously unknown. Here, we analyse the role of pX in foreign protein secretion in exosome-like extracellular vesicles (EVs) and the formation of eHAV. Fusion of pX to eGFP guided eGFP into exosome-like EVs through directing eGFP into multivesicular bodies (MVBs), and apoptosis-linked gene 2-interacting protein X (ALIX) release was significantly enhanced. Coimmunoprecipitation (co-IP) demonstrated the interaction between pX and the ALIX V domain. Removal of the C-terminal half of pX abolished eHAV release and reduced the interaction between the HAV virion and ALIX. Finally, the C-terminal half of pX alone was sufficient for loading eGFP into EVs by interacting with ALIX. In conclusion, the C-terminal part of pX is important for eHAV production and may have potential for large protein complex loading into exosome-like EVs for therapeutic purposes.

Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research.

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.