Nanomechanical characterization of exosomes and concomitant nanoparticles from blood plasma by PeakForce AFM in liquid

BackgroundTo date, EVs characterization techniques are extremely diverse. The contribution of AFM, in particular, is often confined to size distribution. While AFM provides a unique possibility to carry out measurements in situ, nanomechanical characterization of EVs is still missing.MethodsBlood plasma EVs were isolated by ultracentrifugation, analyzed by flow cytometry and NTA. Followed by cryo-EM, we applied PeakForce AFM to assess morphological and nanomechanical properties of EVs in liquid.ResultsNanoparticles were subdivided by their size estimated for their suspended state into sub-sets of small S1-EVs (< 30 nm), S2-EVs (30–50 nm), and sub-set of large ones L-EVs (50–170 nm). Non-membranous S1-EVs were distinguished by higher Young's modulus (10.33(7.36;15.25) MPa) and were less deformed by AFM tip (3.6(2.8;4.4) nm) compared to membrane exosomes S2-EVs (6.25(4.52;8.24) MPa and 4.8(4.3;5.9) nm). L-EVs were identified as large membrane exosomes, heterogeneous by their nanomechanical properties (22.43(8.26;53.11) MPa and 3.57(2.07;7.89) nm). Nanomechanical mapping revealed a few non-deformed L-EVs, of which Young's modulus rose up to 300 MPa. Taken together with cryo-EM, these results lead us to the suggestion that two or more vesicles could be contained inside a large one being a multilayer vesicle.ConclusionsWe identified particles similar in morphology and showed differences in nanomechanical properties that could be attributed to the features of their inner structure.General significanceOur results further elucidate the identification of EVs and concomitant nanoparticles based on their nanomechanical properties.     

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.     

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.     

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.     

Interferon gamma gene +874A/T polymorphism and intracellular interferon gamma expression in pulmonary tuberculosis

We investigated whether IFN-γ gene +874(A/T) polymorphism influences intracellular interferon gamma expression in T-cell subsets of normal healthy subjects (NHS) and pulmonary tuberculosis patients (PTB). Peripheral blood mononuclear cells were stimulated with live Mycobacterium tuberculosis (MTB) and the intracellular IFN-γ expression was studied using flow cytometry. Genotyping of IFN-γ gene +874(A/T) was done using allele specific polymerase chain reaction. Significantly increased IFN-γ expressing CD3+CD4+ and CD3+CD8+ T cells were observed in NHS with AA genotype compared to TT genotype in unstimulated (p = 0.0308 and p = 0.0157) and MTB stimulated (p = 0.0494 and p = 0.0287) cultures and this difference was not observed in PTB patients. The present study suggests that the variant genotypes of IFN-γ (+874) may be associated with altered expression of IFN-γ at the intracellular level and play an immunoregulatory role at the site of M. tuberculosis infection.     

Mitochondrial DNA in extracellular vesicles declines with age

The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell‐free mtDNA (ccf‐mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30–400 nm), lipid‐bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf‐mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30–64 years cross‐sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV‐derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age‐dependent manner.     

Proinflammatory,anti-inflammatory cytokines and adiponkines in students with central obesity

Several studies have investigated the correlation between central obesity and inflammatory cytokines and the anti-inflammatory cytokine adiponectin. But, the correlation between central obesity and the anti-inflammatory cytokines IL-4, IL-5 has not been studied yet. Thus, we aimed to study the IL-4 and IL-5 correlation to central obesity in adolescent Egyptian girls among proinflammatory and anti-inflammatory cytokines. The study was carried out on 86 obese adolescent girls (BMI > 95 percentile) divided into two groups according to central obesity. The group I with waist to hip ratio <0.8 as a control and group II with waist to hip ratio >0.8 (central obesity). There was a significant increase in TNF-alpha (p < 0.0001), and IL-1β (p < 0.0001), as proinflammatory cytokines in group II, as compared to their corresponding group I. Group II showed a significant increase in the anti-inflammatory cytokines IL-4 and IL-5 than group I at (p < 0.0001) and (p < 0.0005) respectively. In addition there was a significant decrease in the anti-inflammatory adiponectin and an increase in the inflammatory leptin levels in group II at (p < 0.0001) and (p < 0.0001) respectively in comparison to group I. A high positive correlation has been observed between waist to hip ratio, leptin, TNF-α, IL-1-β, IL-4 and IL-5 at (r = 0.331, p < 0.03), (r = 0.559, p < 0.001), (r = 0.435, p < 0.004), (r = 0.509, p < 0.001), (r = 0.550, p < 0.0015), in group II respectively and a high negative one with adiponectin at (r = ?0.410, p < 0.0001). We concluded that central obesity lowers adiponectin plasma level through increasing proinflammatory adipokines such as TNF-α, IL-1β, leptin. Further studies are needed to explore the positive correlation we found between central obesity and the anti-inflammatory cytokines IL-4 and IL-5 known to be associated with bronchial asthma.     

Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology,Cargo, and Immunostimulatory Properties

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1Δ/Δ psd2Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and psd1Δ/Δ psd2Δ/Δ mutant-derived EVs activated NF-κB, the cho1Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.     

Extracellular Vesicles of the Hyperthermophilic Archaeon “Thermococcus onnurineus” NA1T

Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, “Thermococcus onnurineus” NA1^T, were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm⁻³, were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ρ²⁵] = 1.2434 g cm⁻³) and C (ρ²⁵ = 1.2648 g cm⁻³), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1^T EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1^T were packaged into EVs, except for an ∼9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3′-terminal sequence and the absence of the 5′-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1^T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.     

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

M2 Macrophage Derived Extracellular Vesicle-Mediated Transfer of MiR-186-5p Promotes Colon Cancer Progression by Targeting DLC1

Colon cancer (CC) is one of the most common malignances in digestive tract. M2-polarized macrophages within the tumor microenvironment could facilitate CC cell growth by transferring molecules via extracellular vesicles, but the mechanisms are not fully elucidated. The current study aims to identify the possible effectors in M2 macrophage-derived extracellular vesicles (M2-EVs) and reveal related molecular mechanisms. In our study, we validated the promotion effects of M2-EVs on the proliferation and motility of CC cells, which was found to be dependent on the EVs enclosed molecules by a mild EVs digestion assay. Then we found that miR-186-5p was enriched in M2-EVs and was responsible for the tumor promoting functions of M2-EVs. Furthermore, mechanism investigation revealed M2-EVs transferring miR-186-5p inhibited DLC1 expression by targeting its 3''UTR, and restored DLC1 successfully neutralized the tumor-promoting effects of M2-EVs transferring miR-186-5p via inhibiting the β-catenin pathway. Our study revealed that M2-EVs facilitates the growth and motility of CC cells by delivering the enclosed miR-186-5p, which directly targets DLC1 mRNAs and facilitates their degradation, which could provide a potential biomarker and therapeutic target for CC.     

Brugia filariasis differentially modulates persistent Helicobacter pylori gastritis in the gerbil model

In select Helicobacter pylori-infected populations with low gastric cancer, nematode coinfections are common and both helicobacter gastritis and filariasis are modeled in gerbils. We evaluated gastritis, worm counts, tissue cytokine gene expression levels and Th1/Th2-associated antibody responses in H. pylori and Brugia pahangi mono- and coinfected gerbils. H. pylori-associated gastritis indices were significantly lower 21 weeks post-infection in coinfected gerbils (p ≤ 0.05) and were inversely proportional to worm counts (r² = ?0.62, p < 0.003). Additionally, IFN-γ, IL-1β, CXCL1, IL-4 and IL-10 mRNA levels in the gastric antrum reflected a significant host response to gastric H. pylori and as well as systemic filariasis (p ≤ 0.05). Despite increasing worm burden (p < 0.05), gastritis progressed in coinfected gerbils (p < 0.03) becoming equivalent to H. pylori-infected gerbils at 42 weeks (p = 0.7). Pro- and anti-inflammatory mediator mRNA levels were notably downregulated in B. pahangi infected gerbils below uninfected control values, suggesting hyporesponsiveness to B. pahangi. Consistent with an increasing Th1 response to H. pylori, IgG2a (p < 0.01), IL-1β (p = 0.04) and CXCL1 (p = 0.006) responses significantly increased and IL-4 (p = 0.05) and IL-10 (p = 0.04) were decreased in coinfected gerbils at 42 weeks. Initial systemic responses to B. pahangi resulted in attenuated gastritis in coinfected gerbils, but subsequent filarid-associated hyporesponsiveness appears to have promoted H. pylori gastritis.     

Iron deficiency,but not underfeeding reduces the secretion of interferon-gamma by mitogen-activated murine spleen cells

Interferon-gamma (IFN-γ), a cytokine primarily secreted by T and natural killer cells regulates cell-mediated and innate immunity. Iron deficiency, a public health problem in children impairs immune function. To determine whether reduced IFN-γ contributes to impaired immunity, we measured IFN-γ in supernatants of activated (2.5 μg/ml concanavalin A, 50 ng/ml anti-CD3 antibody) spleen cells from control (C), iron-deficient (ID), pair-fed (PF), and iron-replete mice for 3 (R3) and 14 days (R14) (11–12/group). Except for iron content, the low iron (5 ppm) and control (50 ppm) diets had identical composition. Mean indices of iron status after 51 days of feeding were as follows: C = PF ≈ R14 > R3 > ID (p < 0.01). Iron deficiency, but not pairfeeding reduced IFN-γ concentration in mitogen-treated cells by 30–43% (p < 0.05); iron repletion improved it. Reduced IFN-γ was not simply due to differences in IL-12 (IFN-γ inducer), percentage of CD3+ T cells, or impaired cell proliferation because these indices were not always decreased. It was likely due to a defect in T cell activation that leads to IFN-γ gene expression. IFN-γ positively correlated with indicators of iron status, body, and thymus weights (r = 0.238–0.472; p < 0.05). Reduced IFN-γ secretion during iron deficiency may affect response to infections.     

The association of IL-8-251T/A polymorphism with complicated malaria in Karbi Anglong district of Assam

Amongst host genetic factors, cytokine gene polymorphism can be anticipated to be an important factor as qualitative, quantitative and time of secretion play an important role in disease outcome. We have investigated association of cytokine promoter SNPs with risk of Plasmodium falciparum malaria and disease severity in a case control study in malaria endemic Karbi Anglong district of Assam, India.Frequency of IL-8-251T/A (p = 0.03 and p = 0.01) and TGF-β1-509C/T (p = 0.02 and p = 0.03) was higher in malaria in comparison to control participants and non-malarial fever controls. Interestingly, a higher frequency of mutant allele of IL-10-819T/C was observed in non-malarial fever controls compared to malaria thus suggesting its role as a distinguishing marker of the two disease groups. Higher IL-8 expression and increased frequency of IL-8-251T/A in complicated malaria (p = 0.002) was reported indicating its role in susceptibility to complicated malaria.In conclusion, our study suggests the role of mutant genotype of IL-8-251T/A as a marker of complicated malaria in our population. Surprisingly, decreased expression of TGF-β1 in uncomplicated malaria even in presence of high expressing mutant genotype was observed and needs to be investigated in context of the pool of activated cells producing the cytokine.     

Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity

AimImbalance of T-helper-cell (TH) subsets (TH1/TH2/TH17) and regulatory T-cells (Tregs) is suggested to contribute to the pathogenesis of Systemic lupus erythematosus (SLE). Therefore, we evaluated their cytokine secretion profile in SLE patients and their possible association with disease activity.MethodsSixty SLE patients, 24 rheumatoid arthritis (RA) patients and 24 healthy volunteers were included in this study. Demographic, clinical, disease activity and serological data were prospectively assessed. Plasma cytokines levels of TH1 (IL-12, IFN-γ), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-β) were measured by enzyme linked immunosorbent assays (ELISA).ResultsSLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL-12 (p < 0.001) and IL-10 (p < 0.05) but comparable levels of IL-23 and IL-4 and slight reduction (but statistically insignificant) of TGF-β levels compared to controls. IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) with disease activity. The RA group exhibited significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-β (p < 0.5) and lower IFN-γ (p < 0.001) and IL-10 (p < 0.01) than those of healthy subjects.ConclusionOur study showed a distinct profile of cytokine imbalance in SLE patients. Reduction in IFN-γ (TH1) and TGF-β1 (Treg) with the elevation in IL-6 and IL-17 (TH17) could imply skewing of T-cells toward TH17 cells. Breaking TH17/Treg balance in peripheral blood may play an important role in the development of SLE and could be responsible for an increased pro-inflammatory response especially in the active form of the disease.