Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

Endoplasmin Is a Hypoxia-Inducible Endoplasmic Reticulum-Derived Cargo of Extracellular Vesicles Released by Cardiac Cell Lines.

Cardiomyopathies are leading causes of human mortality. Recent data indicate that the cardiomyocyte-derived extracellular vesicles (EVs) released upon cardiac injury are present in circulation. This paper aimed to analyze EVs released under normal and hypoxic conditions by H9c2 (rat), AC16 (human) and HL1 (mouse) cardiac cell lines. Small (sEVs), medium (mEVs) and large EVs (lEVs) were separated from a conditioned medium by a combination of gravity filtration, differential centrifugation and tangential flow filtration. The EVs were characterized by microBCA, SPV lipid assay, nanoparticle tracking analysis, transmission and immunogold electron microscopy, flow cytometry and Western blotting. Proteomic profiles of the EVs were determined. Surprisingly, an endoplasmic reticulum chaperone, endoplasmin (ENPL, grp94 or gp96), was identified in the EV samples, and its association with EVs was validated. The secretion and uptake of ENPL was followed by confocal microscopy using GFP-ENPL fusion protein expressing HL1 cells. We identified ENPL as an internal cargo of cardiomyocyte-derived mEVs and sEVs. Based on our proteomic analysis, its presence in EVs was linked to hypoxia in HL1 and H9c2 cells, and we hypothesize that EV-associated ENPL may have a cardioprotective role by reducing cardiomyocyte ER stress.

An implanted device enables in vivo monitoring of extracellular vesicle-mediated spread of pro-inflammatory mast cell response in mice.

Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV-mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP-transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell-derived EVs were next cultured with LPS-naïve mast cells, and the induction of TNF-α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP+ mast cell-derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP+ EVs and for TNF-α production. In vitro, LPS-stimulated mast cell-derived EVs were efficiently taken up by non-stimulated mast cells, and induced TNF-α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell-derived EVs to other mast cells with subsequent induction of TNF-α expression. These data show an EV-mediated spreading of pro-inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell-derived EVs.

An improved 96 well plate format lipid quantification assay for standardisation of experiments with extracellular vesicles.

The field of extracellular vesicles (EVs) is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of EV samples have not been completely solved. Currently, EV samples are standardised on the basis of their protein content sometimes combined with determination of the particle number. However, even this combined approach may result in inaccuracy and overestimation of the EV concentration. Lipid bilayers are indispensable components of EVs. Therefore, a lipid-based quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the EV field. In this study, we set the goal to improve the previously reported sulfo-phospho-vanillin (SPV) lipid assay. We introduced an aqueous phase liposome standard (DOPC) to replace the purified lipid standards in organic solvents (used commonly in previous studies). Furthermore, we optimised the concentration of the vanillin reagent in the assay. We found that elimination of organic solvents from the reaction mixture could abolish the background colour that interfered with the assay. Comparison of the optimised assay with a commercial lipid kit (based on the original SPV lipid assay) showed an increase of sensitivity by approximately one order of magnitude. Thus, here we report a quick, reliable and sensitive test that may fill an existing gap in EV standardisation. When using the optimised lipid assay reported here, EV lipid measurements can be more reliable than protein-based measurements. Furthermore, this novel assay is almost as sensitive and as easy as measuring proteins with a simple BCA test.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice.

AIMS: The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. METHODS: Comparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. RESULTS: Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. CONCLUSIONS: Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA.

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Mast cell secretome: Soluble and vesicular components.

Mast cells are multifunctional master cells implicated in both innate and adaptive immune responses. Their role has been best characterized in allergy and anaphylaxis; however, emerging evidences support their contribution to a wide variety of human diseases. Mast cells, being capable of both degranulation and subsequent recovery, have recently attracted substantial attention as also being rich sources of secreted extracellular vesicles (including exosomes and microvesicles). Along with secreted de novo synthesized soluble molecules and secreted preformed granules, the membrane-enclosed extracellular vesicles represent a previously unexplored part of the mast cell secretome. In this review article we summarize available data regarding the different soluble molecules and membrane-enclosed structures secreted by mast cells. Furthermore, we provide an overview of the release mechanisms including degranulation, piecemeal degranulation, transgranulation, and secretion of different types of extracellular vesicles. Finally, we aim to give a summary of the known biological functions associated with the different mast cell-derived secretion products. The increasingly recognized complexity of mast cell secretome may provide important novel clues to processes by which mast cells contribute to the development of different pathologies and are capable of orchestrating immune responses both in health and disease.

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Mannose receptor and macrophage galactose-type lectin are involved in Bordetella pertussis mast cell interaction.

Mast cells are crucial in the development of immunity against Bordetella pertussis, and the function of TLRs in this process has been investigated. Here, the interaction between mast cells and B. pertussis with an emphasis on the role of CLRs is examined. In this study, two CLRs, MGL and MR, were detected for the first time on the surface of mast cells. The involvement of MR and MGL in the stimulation of mast cells by heat-inactivated BP was investigated by the use of blocking antibodies and specific carbohydrate ligands. The cell wall LOS of BP was also isolated to explore its role in this interaction. Mast cells stimulated with heat-inactivated BP or BP LOS induced TNF-α, IL-6, and IFN-γ secretion, which was suppressed by blocking MR or MGL. Inhibition of CLRs signaling during BP stimulation affected the ability of mast cells to promote cytokine secretion in T cells but had no effect on the cell-surface expression of ICAM1. Blocking MR or MGL suppressed BP-induced NF-κB expression but not ERK phosphorylation. Syk was involved in the CLR-mediated activation of mast cells by BP. Bacterial recognition by immune cells has been predominantly attributed to TLRs; in this study, the novel role of CLRs in the BP-mast cell interaction is highlighted.

The effects of Fasciola hepatica tegumental antigens on mast cell function.

Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.

Fasciola hepatica tegumental coat impairs mast cells' ability to drive Th1 immune responses.

The parasitic worm Fasciola hepatica induces strong Th2 and T-regulatory immune responses while simultaneously suppressing Th1-driven immune responses to bystander microbial infections. It also prevents the initiation of Th1-mediated autoimmune disorders in mice through the suppression of Th17 and Th1 immune responses, and this can be mimicked by parasite-derived molecules. We have isolated F. hepatica tegumental coat Ag (FhTeg) and demonstrated its suppressive effect in vivo by directly targeting dendritic cells, impairing their ability to drive Th1 responses. Mast cells are critical in promoting Th1 protective immunity during bacterial infection and in driving Th1-mediated pathological conditions in autoimmune diseases. In this article, we show that FhTeg inhibits the ability of mast cells to drive the Th1 immune response by suppressing cytokine secretion (TNF-α, IL-6, IFN-γ, and IL-10) and ICAM1 expression in mast cells stimulated with LPS or heat-inactivated Bordetella pertussis Ag. These heat-inactivated B. pertussis Ag/LPS-stimulated mast cells fail to promote Th1 immune responses in CD4(+) T cells when pretreated with FhTeg, and a role for ICAM1 in this process was demonstrated. FhTeg suppresses the activation of transcription factors in the TLR signaling pathway, which explains the decrease in cytokine production and cell surface marker expression. We demonstrated that FhTeg suppresses MAPK and NF-κB activation and enhances SOCS3 expression, which could explain its negative effect on the TLR pathways. We conclude that FhTeg targets innate immune cells, inhibiting their ability to drive Th1 immune responses.

Mast cells cultured from IL-3-treated mice show impaired responses to bacterial antigen stimulation.

OBJECTIVE AND DESIGN: This study exploits the biological activity of interleukin (IL)-3 to generate high yields of peritoneal mast cells ex vivo in order to examine pro-inflammatory immune responses in ex-vivo culture. MATERIAL OR SUBJECTS: Mast cells were obtained from the peritoneal cavity of C57BL/6 mice. TREATMENT: Mice were injected intraperitoneally twice per day for 5 days with IL-3 (40-50 µg/ml) to increase mast cell numbers. METHODS: Histological studies examined mast cell numbers in the peritoneal cavity, intestine, lung, spleen and skeletal muscle. Peritoneal mast cells cultured ex vivo (PCMCs) were stimulated for 24 h with lipopolysaccharide and Bordetella pertussis antigen and secretion of tumour necrosis factor-α, IL-6, IL-4, IL-5, IL-10 and interferon-γ into supernatant was measured by commercial ELISA. Cell surface marker expression of FcεRI, c-kit, OX40L and TLR2 was measured by flow cytometry. Mast cell degranulation was measured using a ß-hexosaminidase assay. RESULTS: IL-3 treatment increases mast cell numbers in the peritoneal cavity, spleen and muscle but not intestine and lung of C57BL/6 mice. PCMCs generated from IL-3-treated mice exhibit impaired growth, differentiation and responses to activation as measured by decreased cytokine secretion and cell surface marker expression. CONCLUSION: Mast cells cultured from IL-3-treated mice show impaired responses.

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo.

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.