Download Human mast cells release extracellular vesicle

Human mast cells release
vesicle-associated DNA
extracellular
Ganesh V Shelke, Su Chul Jang, Yanan Yin, Cecilia Lässer, Jan Lötvall
Krefting Research Centre , Internal Medicine ; Laboratory Medicine, Shanghai First People’s Hospital
Correspondence
[email protected]
[email protected]
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Disciplines
Cell Biology
Cancer
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Keywords
Exosomes
Extracellular Vesicles
DNA
Uptake
Extracellular vesicles (EVs) carry multiple bioactive molecules, including proteins and nucleic
acids. Cell-free, extracellular DNA has been reported to be present in cell cultures and in multiple body fluids, but its relative location in relation to EVs has not been described. This study
demonstrates that DNase-sensitive nucleic acids are present on the surface of EV isolates. Association of EV-DNA was revealed by increase of EV zeta potential and particle number upon
DNase treatment. Additionally, cells exposed to EVs with associated DNA show the presence of
cytoplasmic DNA traces intracellularly. In conclusion, we suggest that DNA can be associated
to the surface of EVs and can be taken up by recipient cells. DNA on EV surfaces may influence
their function in recipient cells.
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Type of Observation
Standalone
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Type of Link
Standard Data
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Submitted Feb 19th, 2016
Published Feb 21st, 2016
3x
Objective
This work aims to determine the relative location of cell-free DNA in relation to extracellular vesicles.
Introduction
Extracellular vesicles (EVs) are membrane bound structures released by cells, and found
in all body fluids. The populations of EVs are diverse, and the nomenclature includes
terms such as exosomes, ectosomes and microvesicles. Previously, it has been shown that
the subpopulations of EVs called exosomes can carry both mRNA and microRNA, which
can mediate function in recipient cells (Valadi 2007[1] ). Additionally, dsDNA has previously been proposed to be associated with EVs (Kahlert 2014[2])(Lee 2014[3] )(Balaj
2011[4] )(Thakur 2014[5] )(Guescini 2009[6] ), but its relative location to/in EVs has not
been well described. This study reports for the first time that cell-free DNA can be associated with the outside of EVs, which may influence aggregation of these EVs.
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Supported by the Velux Foundation, the University of Zurich,
and the EPFL School of Life
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DOI: 10.19185/matters.201602000034
Matters | 1 of 4
Human mast cells release extracellular vesicle-associated DNA
DNA is associated with extracellular vesicles. (A) Isolated EVs, and EVs were floated on an optiprep gradient and treated
with DNase-I or RNAse prior to DNA isolation (apoptotic bodies and microvesicles had been removed at 16000g). Nucleic
acids were visualized with agarose gel electrophoresis labeled with nucleic acid stain (GelStar). The isolated EVs were
also analyzed with NTA (Zetaviewer®) to determine the zeta potential (B) on EVs, and the particle number (C) with or
without DNase treatment. Isolated EVs were incubated with human mesenchymal stem cells for 1 or 24 hrs, and DNA
was labeled with DAPI and visualized by fluorescent microscopy at 24 hr (D), and cytoplasmic DAPI staining foci per
cell were counted (E). Student’s T-test was used to determine significant differences between groups (* p < 0.05. N = 3).
DOI: 10.19185/matters.201602000034
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Human mast cells release extracellular vesicle-associated DNA
Results and discussion
RESULTS:
On a nucleic acid-stained gel, clear DNA bands were visualized, and these were present not in samples pretreated with
DNase-I but in samples treated with RNAse (Figure 1A). The EVs fraction isolated from the density gradients had a
charge of –70 mV, which was increased to -50 mV by DNase-I treatment (Figure 1B). This result indicates that DNA
is present on the outside of the EVs, and this DNA contributes to the negative charge of the vesicles. Further, DNase
treatment increased the number of particles measured by nanoparticle tracking analysis, suggesting that the DNA to a
certain degree contributes to aggregation of the isolated EVs (Figure 1C). As a biological readout, isolated EVs associated
with extracellular DNA were taken up by human mesenchymal stem cell in a time-dependent manner (Figure 1D and
E).
DISCUSSION:
EVs carry multiple bioactive molecules, including proteins, various RNA species, and according to the present study,
DNA. In our case, the DNA was observed in EV isolates from a human mast cell line. Specifically, our study argues that
the DNase-sensitive nucleic acids are present on the outside of the EVs. Furthermore, the EV-associated DNA can be
taken up by recipient cells, which could alter cellular responses.
It is known that EVs can mediate an array of biological messages to recipient cells—including surface-to-surface antigen presentation and receptors activation—and can deliver RNA (e.g. mRNA, miRNA) cargo to recipient cells (Valadi
2007[1] )(Wolfers 2001[7] ). However, the presence and function of DNA as a cargo on the outside of EVs is less explored.
EVs have previously been associated with cell-free DNA that carries retrotransposon elements and oncogenes (Lee
2014[3] )(Balaj 2011[4] ), but overall EV-associated DNA has been extensively characterized. A recent report (Thakur
2014[5] ) emphasizes the presence of dsDNA inside of the EVs, whereas we find that a majority of DNA from the human
mast cell line is associated with outer perimeter of EVs, since it is sensitive to DNase treatment without lysing the EVs.
Our study also indicates, for the first time, that DNA covering floated EVs can lead to an increase in the net negative
charge of the vesicles. This was confirmed by reduction of net negative charge from EVs by DNase treatment. We were
also able to monitor the increase in particle numbers after DNase treatment, indirectly suggesting that EV-DNA may lead
to aggregation of EVs. These results are in line with previous observation (Petersen 2014[8] )(Heijnen .1999[9] ) made in
various electron micrographs, showing clustering of EVs, which could have occurred because of DNA on the surface of
these vesicles. The aggregation of EVs may be secondary to nonspecific aggregation of EVs during ultracentrifugation,
but our study suggests that EV-related DNA can contribute to this observation. As we observed that the extracellular
DNA floated in the density gradient, we argue strongly that it is associated with the floating vesicles with relatively low
density. Also, it is has been shown that exogenous plasmids DNA if associated with EVs are taken up more efficiently
in recipient cells (Lamichhane 2015[10] ) than free DNA, again arguing that association to EVs could be involved in
DNA uptake. Similarly, we also observed a time-dependent increase in cytoplasmic DNA foci in EV treated recipient
human mesenchymal stem cells. Overall, this study highlights the need to define the EV-associated DNA in delivering
biological function in cells that take up these EVs.
Conclusions
We conclude that cell-free DNA can be associated with the outside of EVs, which can cause aggregation of these EVs,
possibly influencing the effects of EVs in recipient cells.
Limitations
This work is based on cell-line-produced EVs, which may not reflect a biological function in vivo.
Conjectures
In the future, the detailed nature of the EV-associated DNA needs to be determined, in relation to sequences and intracellular origin. Also, the role of the EV-associated DNA in recipient cells may be studied.
DOI: 10.19185/matters.201602000034
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Human mast cells release extracellular vesicle-associated DNA
Additional Information
Methods and supplementary material
Please see https://sciencematters.io/articles/201602000034.
Funding statement
This work was funded by the Swedish Cancer Foundation, the Swedish Research Council, Assar Gabrielssons Fond, the
Swedish Heart and Lung Foundation as well as VBG-group Centre for allergy and asthma research.
Ethics Statement
Not applicable. No animal was used in the experiments. Therefore, no ethical approval is required.
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