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. 2017 Aug 15;7(1):8202.
doi: 10.1038/s41598-017-08392-1.

Antibiotic-induced Release of Small Extracellular Vesicles (Exosomes) With Surface-Associated DNA

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Free PMC article

Antibiotic-induced Release of Small Extracellular Vesicles (Exosomes) With Surface-Associated DNA

Andrea Németh et al. Sci Rep. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Effects of sustained ciprofloxacin exposure on Jurkat cells. (a,b) Viability of Jurkat cells with/without exposure to ciprofloxacin (10 µg/mL for >14 days) was analyzed by flow cytometry after staining with annexinV-FITC and propidium iodide (PI). (a) Mean values+/− S.D. (error bars) of two independent experiments are shown in the histogram plot. AxV: annexinV. (b) Representative dot plots showing the four quadrants of annexinV-FITC and PI stained Jurkat cells. (c) Exosomes (EXOs) derived from ciprofloxacin-exposed/unexposed Jurkat cells were conjugated onto latex beads and characterized by flow cytometry after staining with annexinV-FITC, anti-CD63-PE, PI or anti-histone H2B-FITC. The background fluorescence of stained latex beads is indicated by grey histograms. (d) Microvesicles (MVs) and apoptotic bodies (APOs) were labeled directly with annexinV-FITC and PI. The background fluorescence of EVs is indicated by grey histograms.
Figure 2
Figure 2
Analysis of ciprofloxacin-induced release of DNA associated with extracellular vesicles (EVs). (a) DNA content of size-based EV fractions determined with/without DNase I digestion of EVs. Concentration values represent the amount of EV-associated DNA (eluted in 3 0 µL) isolated from the conditioned medium of 2.5 × 107 Jurkat cells. Plotted values are presented as the mean+/− S.D. (error bars) of 8 independent experiments. (**P < 0.01, Friedman test, One-way ANOVA). (b) DNA amounts before and after the digestion of EVs by DNase I. The paired measurements (n = 8) for APOs, MVs and EXOs are indicated by lines (**P < 0.01, Wilcoxon signed rank test). APO: apoptotic body, MV: microvesicle, EXO: exosome (c) The presence of EXOs both in undigested and DNase I digested samples was confirmed by flow cytometry. Latex-bound EXOs were stained with annexinV-FITC and an anti-CD63-PE antibody. Dot plots are representative of three independent experiments. (d) OptiprepTM density gradient fractions of the 100,000 g pellet (containing EXOs) were re-pelleted, conjugated onto latex beads and stained with an anti-CD63-PE antibody for flow cytometry. (e) The same latex-bound OptiprepTM density gradient fractions were also stained by propidium iodide (PI). The percentages of PI positive events are shown above a threshold (horizontal line, determined by measuring labeled latex beads without conjugated density gradient fractions).
Figure 3
Figure 3
Association of DNA with EXO surface is abolished by high salt concentration. Ciprofloxacin-exposed Jurkat cell exosomes (EXOs) were conjugated onto latex beads and washed using annexinV binding buffer supplemented with 0.1 M, 1 M or 2 M NaCl. All latex-bound EXO samples were re-suspended in annexinV binding buffer, and labeled with propidium iodide (PI), annexinV-FITC and an anti-CD63-PE antibody for flow cytometry. (a) Differences of geometric mean fluorescence values between EXOs washed in high salt concentration buffers and controls are shown. Plotted values are presented as the mean+/− S.D. (error bars) of three independent experiments. *P < 0.05, Friedman test, One-way ANOVA (b) Density plots show PI, annexinV-FITC and anti-CD63-PE fluorescence of latex-bound EXOs as a function of SSC parameter. Horizontal lines indicate fluorescence threshold of labeled EXO-free latex beads.
Figure 4
Figure 4
Label-free optical biosensor analysis of surface adhesion of extracellular vesicles (EVs). (a) Photograph of an Epic microplate (384-well) is shown containing biosensors (2 × 2 mm nano-grating embedded in a high-refractive index waveguiding film) at the bottom of each well. Biosensors are imaged from the back of the plate and are visible due to diffraction. (b) Microplate wells were coated with bovine serum albumin (BSA) as a control protein or with fibronectin (FN) resulting in a shift in the resonant wavelength (Δλ). Microplate wells were equilibrated with PBS, then BSA or FN were added to the wells (indicated by the first arrows). After one hour incubation with BSA or FN, Δλ was recorded for 5 min. Then, unbound protein was washed out with PBS, and Δλ was measured again for 10 min. Then, PLL-g-PEG was used in order to block the non-specific binding sites of wells. The BSA and FN adsorption signals without addition of PLL-g-PEG (PBS only) are indicated as dashed lines, while adsorption of the blocking PLL-g-PEG in BSA- or FN-coated wells is shown by continuous line. After 30 min incubation with PLL-g-PEG, Δλ was recorded for another 5 min (starting points are indicated by the second arrows), and finally PLL-g-PEG was changed to PBS. (c) Adsorption of apoptotic bodies (APOs), microvesicles (MVs) and exosomes (EXOs) onto FN + PLL-g-PEG surfaces (continuous lines) or onto surfaces with adsorbed PLL-g-PEG only (dashed lines). (d) The Δλ values of EV adsorption onto PLL-g-PEG were subtracted from adsorption values onto FN + PLL-g-PEG, and were divided by the Δλ value of EV adsorption onto bare surfaces (as a straightforward normalization with the mass concentrations of various samples). These normalized signal values are presented as the mean+/− S.D. (error bars) of three independent experiments (**P < 0.01 between APOs and EXOs, *P < 0.05 between MVs and EXOs, Mann-Whitney U-test). (e) Comparison of EXO adsorption with/without DNase I digestion onto FN + PLL-g-PEG and onto BSA + PLL-g-PEG surfaces. Normalized signal values are presented as the mean+/− S.D. (error bars) of three independent experiments. Difference was significant in the case of FN surface (*P < 0.05, Mann Whitney U-test). PLL-g-PEG: poly(L-lysine)-graft-poly(ethylene glycol).
Figure 5
Figure 5
Characterization of extracellular vesicles (EVs) released by ciprofloxacin-exposed control, activated or apoptotic Jurkat cells. (a) Concentration values of EVs in 100 µL (isolated from the supernatant of 2.5 × 107 cells) were determined by tunable resistive pulse sensing (TRPS), and are plotted as a function of their diameter for control, activated or apoptotic cells (blue, green or red colors, respectively). Histogram plots of EVs are representative of three independent experiments. (b) Mode diameters of EV subpopulations were determined by TRPS, and are presented+/− S.D. (error bars) of three independent experiments, for control, activated and apoptotic cells. NP2000, NP800 and NP100 IZON nanopore membranes were used. (c) Transmission electron microscopy images of apoptotic bodies (APOs), microvesicles (MVs) and exosomes (EXOs) samples.
Figure 6
Figure 6
Flow cytometry analysis of extracellular vesicles (EVs) derived from ciprofloxacin-exposed control, activated or apoptotic Jurkat cells. EVs were stained by annexinV-FITC and propidium iodide (PI) for flow cytometry and analyzed before and after detergent lysis with 0.1% Triton X-100. Apoptotic bodies (APOs) and microvesicles (MVs) were labeled and measured directly, whereas exosomes (EXOs) were conjugated onto latex beads before staining. Density plots show annexinV-FITC and PI positivity of EVs derived from ciprofloxacin-exposed control, activated and apoptotic Jurkat cells. The percentages of positive events (above the threshold represented by a black line) are shown in the plots.
Figure 7
Figure 7
Assessment of DNA and DNA-binding proteins in extracellular vesicle (EV) preparations. (a-b) DNA was purified from apoptotic bodies (APOs), microvesicles (MVs) or exosomes (EXOs) released by ciprofloxacin-exposed control, activated or apoptotic Jurkat cells. (a) Nuclear (GAPDH, p53) and (b) mitochondrial (control region, RNR1) DNA sequences of DNase I-digested/non-digested EVs were amplified by PCR and analyzed by agarose gel electrophoreses. The figure displays cropped gels. Full-length, uncropped gels are shown in Supplementary Figures S11–S13. (c ) Detection of exosomal DNA derived from ciprofloxacin-exposed control, activated or apoptotic Jurkat cells using an Agilent 2100 Bioanalyzer (DNA 12,000 Kit). The electropherograms show the size distribution of purified exosomal DNA in base pairs (bp) with DNA markers at 50 bp and 17,000 bp. FU: fluorescence units. (d) Semi-quantitative mass spectrometry analysis of DNA-binding histones in EV samples. Values in the table are proportional to the amount of histones found in EVs. The presence of f lap endonuclease 1 (FEN1, also known as a mitochondrial DNA-binding nucleoid protein) was also identified by mass spectrometry.
Figure 8
Figure 8
Mass spectrometry analysis of extracellular vesicles (EVs). (a) Venn-diagrams of ciprofloxacin-exposed control, activated and apoptotic Jurkat cells and released APOs, MVs and EXOs. (b) Venn-diagrams of EVs derived from ciprofloxacin-exposed control, activated and apoptotic Jurkat cells. Percentages of mitochondrial proteins exclusively specific for apoptotic bodies (APOs), microvesicles (MVs) or exosomes (EXOs) are indicated in the graphs, respectively. mt: mitochondrial protein.

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