Abstract

   Radiation-induced bystander effects refer to the induction of
   biological changes in cells not directly hit by radiation implying that
   the number of cells affected by radiation is larger than the actual
   number of irradiated cells. Recent in vitro studies suggest the role of
   extracellular vesicles (EVs) in mediating radiation-induced bystander
   signals, but in vivo investigations are still lacking. Here, we report
   an in vivo study investigating the role of EVs in mediating radiation
   effects. C57BL/6 mice were total-body irradiated with X-rays (0.1,
   0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow
   (BM) and were intravenously injected into unirradiated (so-called
   bystander) animals. EV-induced systemic effects were compared to
   radiation effects in the directly irradiated animals. Similar to direct
   radiation, EVs from irradiated mice induced complex DNA damage in
   EV-recipient animals, manifested in an increased level of chromosomal
   aberrations and the activation of the DNA damage response. However,
   while DNA damage after direct irradiation increased with the dose,
   EV-induced effects peaked at lower doses. A significantly reduced
   hematopoietic stem cell pool in the BM as well as CD4^+ and CD8^+
   lymphocyte pool in the spleen was detected in mice injected with EVs
   isolated from animals irradiated with 2 Gy. These EV-induced
   alterations were comparable to changes present in the directly
   irradiated mice. The pool of TLR4-expressing dendritic cells was
   different in the directly irradiated mice, where it increased after
   2 Gy and in the EV-recipient animals, where it strongly decreased in a
   dose-independent manner. A panel of eight differentially expressed
   microRNAs (miRNA) was identified in the EVs originating from both low-
   and high-dose-irradiated mice, with a predicted involvement in pathways
   related to DNA damage repair, hematopoietic, and immune system
   regulation, suggesting a direct involvement of these pathways in
   mediating radiation-induced systemic effects. In conclusion, we proved
   the role of EVs in transmitting certain radiation effects, identified
   miRNAs carried by EVs potentially responsible for these effects, and
   showed that the pattern of changes was often different in the directly
   irradiated and EV-recipient bystander mice, suggesting different
   mechanisms.

   Keywords: ionizing radiation, hematopoietic system, microRNA,
   extracellular vesicles, bystander effects

Introduction

   The most intensively studied radiobiological consequence of ionizing
   radiation was for long the induction of DNA damage and cell death as
   well as the various cellular pathways activated in response to DNA
   damage in the directly irradiated cells. The discovery of non-targeted
   effects of irradiation, including genomic instability and bystander
   effects, have shifted the focus of radiobiological research from a
   purely DNA target-based orientation to a much more dynamic science
   where cellular responses, micro/macro-environmental influences, and
   systemic effects are at least as important as the dose directly
   absorbed by the cells and the organism ([47]1, [48]2).
   Radiation-induced activation of pro- or anti-inflammatory pathways is a
   radiation response mechanism equally important at systemic level as DNA
   damage response at cellular level. Therefore, molecular pathways
   connecting radiation with inflammatory and immune responses are
   intensively studied. In a recent meta-analysis, several genes and
   pathways involved in immune response following ionizing radiation (IR)
   exposure were identified, such as transforming growth factor beta
   (TGFβ) signaling pathway, interleukin pathways, nuclear factor kappa B
   (NFκB) as the key transcription factor in the activation of immune
   system by IR, as well as regulation of DNA damage response by microRNAs
   (miRNA) ([49]3). The multiple ways of the initiation of an immune
   response by radiation exposure was recently reviewed by Candeias and
   Testard. The authors highlight the importance of toll-like receptors
   (TLRs) and the direct activation of inflammatory cytokine genes by NFκB
   and p53 ([50]4).

   Radiation-induced bystander effects (RIBE) develop in cells which are
   not directly hit by IR as a result of signals received from directly
   irradiated cells. These effects can be classified as local, manifesting
   within 5 mm from the directly targeted cells and distal when bystander
   signals are transmitted to distances greater than 5 cm from the
   directly irradiated cells. These latter effects can be considered as
   systemic bystander effects ([51]5). RIBE consist of DNA damage,
   alterations in gene expression, apoptosis, cell death, or genomic
   instability ([52]6–[53]10). It has been shown that RIBE manifest even
   at low doses of radiation ([54]11) and that bystander signals can be
   transmitted both via gap junctions and soluble factors, such as TGFβ,
   IL6, IL8, tumor necrosis factor alpha (TNFα), reactive oxygen species
   (ROS), or miRNA released into the extracellular environment
   ([55]12–[56]14). A detailed overview of existing literature data about
   mediators of local and systemic bystander effects as well as mechanisms
   how RIBE develop has been recently published ([57]5). The in vivo
   studies related to immune responses elicited by direct radiation and
   bystander signals have been recently rewieved by Hekim et al. also,
   listing many important pathways mediating T-cell activation (or
   suppression), antigen-presenting cell, and natural killer (NK) cell
   activation ([58]15).

   Extracellular vesicles (EVs) are membrane-coated bodies actively
   released by various cell types. Based on their size distribution and
   biogenesis, EVs are divided into exosomes (released by multivesicular
   bodies upon cellular membrane fusion with a diameter of 50–100 nm),
   microvesicles (MVs) (formed by membrane budding with a diameter of
   20–1,000 nm), and apoptotic bodies (released during apoptosis with a
   diameter of up to 5,000 nm) ([59]16, [60]17). EVs have important roles
   in intercellular communication by transferring genetic material (in the
   form of mRNA and miRNA) and various proteins both to neighboring and
   distant recipient cells ([61]18), thus influencing their function.
   Mounting evidences suggest that EVs may be involved in RIBE
   ([62]19–[63]22) albeit all of these evidences are restricted to in
   vitro studies.

   The bone marrow (BM) is a particularly radiosensitive organ where apart
   from the hematopoietic stem cells and progenitor cells, there is also
   the stroma composed of fibroblasts, endothelial cells, mesenchymal stem
   cells, osteoblasts, osteoclasts, adipocytes, and chondrocytes. A close
   and dynamic cooperation exists between the hematopoietic stem cell
   compartment and BM stroma, which maintain and adapt to the needs of
   hematopoiesis and tissue turnover ([64]23). At higher doses where
   direct effects dominate, the damage of the stem cells determines both
   the level of BM damage and the long-term health consequences. At lower
   doses, where radiation-induced direct cell death is moderate and
   bystander effects are prevalent, bystander signaling between the two
   compartments might significantly influence BM damage, with an impact on
   long-term health outcomes.

   In the present study, we have investigated the role of BM-derived EVs
   in mediating systemic RIBE in vivo. EVs isolated from the BM of
   irradiated mice were transferred intravenously into healthy naïve
   animals. The effects of EV transfer were followed on the BM cells and
   splenocytes of EV-recipient mice (called bystander mice) (Figure
   [65]1). We have found that transfer of EVs from irradiated mice induced
   various effects in the recipients. Alterations in the recipient mice
   resembled the alterations exhibited in the directly irradiated animals,
   suggesting that EVs could transmit biological information from
   irradiated to unirradiated cells. We also analyzed the miRNA cargo of
   the EVs prepared from the BM of directly irradiated mice and identified
   a panel of differentially expressed miRNA suggesting their involvement
   in mediating RIBE.

Figure 1.

   [66]Figure 1
   [67]Open in a new tab

   Schematic representation of the workflow of the study. C57Bl/6 mice
   were irradiated with different doses of ionizing radiation (0–2 Gy).
   Mice were sacrificed 24 h later; spleen and bone marrow (BM) were
   collected. Extracellular vesicles (EVs) were isolated from the BM
   supernatant. Bystander effects were monitored by injecting the
   BM-derived EVs in non-irradiated healthy mice, and 24 h later, the same
   organs were harvested as from the directly irradiated animals. DNA
   double-strand break analysis was performed by γ-H2AX assay from the
   spleen cells, chromosomal aberration were evaluated from the BM cells.
   BM and spleen cells were characterized phenotypically. EVs from BM of
   directly irradiated animals were subjected to miRNA profiling.

Materials and Methods

Animal Model and Irradiation

   Nine- to fourteen-week-old male C57/BL6 mice were used in all
   experiments. Mice were kept and investigated in accordance with the
   guidelines and all applicable sections of the Hungarian and European
   regulations and directives. This study was carried out in accordance
   with the recommendations of the 1998 XXVIII Hungarian law about animal
   protection and welfare. All animal studies were approved, and
   permission was issued by Budapest and Pest County Administration Office
   Food Chain Safety and Animal Health Board. Mice were total-body
   irradiated with 0 (control), 0.1, 0.25 and 2 Gy X-rays using THX-250
   therapeutic X-ray source (Medicor, Budapest, Hungary). For each dose,
   12–15 mice were used. Mice were selected from at least five different
   litters, which were mixed prior to irradiation or bystander injections,
   so that each experimental group randomly contained mice aged between 9
   and 14 weeks.

Isolation of Murine BM Cells and Splenocytes

   Bone marrows were isolated from the femur and tibia of mice by flushing
   out the tissue from the diaphysis of the bones and suspended in
   phosphate-buffered saline (PBS). BM single-cell suspension was made by
   mechanical disaggregation of the tissue. Intact, viable cells were
   pelleted by centrifugation at 500 g, 4°C for 10 min. Part of the
   pelleted BM cells was processed freshly for phenotypical
   characterization by flow cytometry while another part was suspended in
   heat-inactivated fetal bovine serum containing 10% dimethylsulphoxide,
   frozen in liquid nitrogen, and sent to Oxford Brookes University for
   chromosomal analysis. The BM supernatant was used for EV isolation.

   Spleens were mechanically disaggregated and cell suspensions were
   collected and pelleted in PBS. Red blood cells were removed by
   incubation of the pellets in 5 ml lysis buffer containing 1.66%
   ammonium chloride for 5 min. Cells were washed with PBS and passed
   through a 40-µm cell strainer to obtain single-cell suspension.

   Live BM and spleen cells were counted by trypan blue exclusion. Cells
   were used for subsequent immune phenotyping of different
   subpopulations, apoptosis, and γ-H2AX staining.

   Bone marrow cells and spleens of irradiated and bystander mice were
   processed individually.

Isolation, Validation, and In Vivo Transfer of EVs

   Extracellular vesicles were prepared from BM supernatant of control and
   irradiated animals by pooling the BM supernatant from a minimum of
   eight mice/radiation dose. EVs were isolated 24 h after irradiation by
   the ExoQuick-TC kit (System Biosciences, Palo Alto CA, USA), following
   the manufacturer’s instructions. Briefly, the supernatant was pooled
   and incubated overnight at 4°C with ExoQuick-TC solution followed by
   centrifugation at 1,500 g for 30 min. EV pellets were suspended in
   200 µl PBS. A GE Healthcare PD SpinTrap G-25 desalting column (GE
   Healthcare, Life Sciences, WI, USA) was used to remove ExoQuick
   polymers from the EV solution.

   The hydrodynamic size of EVs was determined by the dynamic light
   scattering (DLS) method using an Avid Nano W130i DLS instrument (Avid
   Nano, High Wycombe, UK).

   For transmission electron microscopy, EV samples kept in 3% PFA were
   applied to copper grids and negatively stained with a 0.5% uranyl
   acetate (v/v) solution for 2 min. Grids were air dried for 10 min and
   viewed using a Hitachi H-7650 transmission electron microscope (Hitachi
   Ltd., Tokyo, Japan) operated at 100 kV.

   Protein content of EVs was measured by Bradford protein assay kit
   (Thermo Fisher Scientific, Waltham, MA, USA) using a Synergy HT
   (Biotek, Winooski, USA) plate reader.

   For Western blot analysis of exosome-specific protein markers, EVs were
   lysed with RIPA lysis buffer containing 2% protease inhibitors
   (Sigma-Aldrich, Darmstadt, Germany). Equal amounts of protein lysates
   from the EVs prepared from BM of mice irradiated with different doses
   were loaded and electrophoresed on 10% sodium dodecyl
   sulfate-polyacrylamide (SDS-PAGE) gel and transferred to PVDF membranes
   (Bio-Rad, Hercules, CA, USA). Murine BM whole cell lysate treated in
   the same way was used as control. As a protein standard, Prism Ultra
   Protein Ladder (Abcam) was used. Anti-mouse CD9, TSG101, and calnexin
   antibodies (Abcam) were diluted as suggested by the supplier, and
   lysates were incubated at room temperature (RT) for 1.5 h, followed by
   1-h incubation with horseradish peroxidase-conjugated goat anti- rabbit
   secondary antibody (Abcam). Membranes were washed in Tris-buffered
   saline-tween buffer three times, and protein bands were visualized
   using 3,3′-diaminobenzidine substrate (Sigma-Aldrich), by chromogenic
   method.

   Extracellular vesicle-associated acetylcholinesterase activity was
   determined in EV solution using the Acetylcholinesterase (AchE) Assay
   Kit (Abcam, Cambridge, UK) over a time period of 30 min by following
   absorbance at 410 nm with a Synergy HT plate reader.

   For setting up the bystander animals, EVs isolated from the directly
   irradiated animals were injected in the tail vein of healthy
   unirradiated mice, using 10 µg of EVs suspended in 100 µl PBS. Mice
   were sacrificed 24 h after EV injection. BM and spleen from the
   bystander animals were isolated as described above for the directly
   irradiated animals and used for immune phenotyping and DNA damage
   assay.

Immunostaining of Murine Splenocytes for γ-H2AX Assay

   γ-H2AX assay was performed from the freshly isolated splenocytes of the
   directly irradiated and bystander animals both by immunocytochemistry
   and flow cytometry. For each sample 10^6 cells in 500 µl PBS were
   seeded on 13-mm round coverslips placed in 24 well plates. Plates were
   centrifuged at 35 g (500 rpm) for 5 min, supernatant was removed, and
   cells were fixed in 0.5 ml 2% paraformaldehyde (PFA) at RT for 5 min.
   Wells were washed with PBS under low-speed shaking three times 5 min.
   Permeabilization was performed at RT for 15 min using 0.5 ml 0.25%
   Triton-X 100 solution with 0.1% glycine. After subsequent washing and
   3% bovine serum albumin (BSA) blocking (30 min, RT), incubation with
   primary antibody against γ-H2AX [phospho-Histone-H2A.X (Ser139) (20E3)
   rabbit monoclonal antibody (mAb), Cell Signaling Technology, Leiden,
   The Netherlands] was performed at RT for 40 min. This was followed by
   staining with Alexa 588-conjugated goat anti-mouse secondary antibody
   (Abcam) at RT for 30 min. After three consecutive washing steps,
   coverslips were removed from the plate and mounted onto a microscope
   slide using one drop of Fluoroshield mounting medium with DAPI (Abcam).
   For quantitative analysis, foci were manually counted using a Zeiss
   Axio Imager A1 phase-contrast fluorescent microscope (Carl Zeiss
   microscopy, GmbH, Oberkochen, Germany) equipped with a 100× objective.
   Images were analyzed by the Zen2012 software (Carl Zeiss microscopy,
   GmbH). At least 100 randomly chosen cells or 50 foci per slide were
   counted.

   For the analysis of γ-H2AX by flow cytometry, splenocytes were fixed in
   4% PFA at 37°C for 10 min. Permeabilization was done in 90% ice-cold
   methanol for 30 min. Labeling with primary and secondary antibodies was
   performed as above. The proportion of γ-H2AX-positive cells was
   determined using a FACSCalibur flow cytometer (Beckton Dickinson, NJ,
   USA). Analysis was performed using the Cell-Quest Pro data acquisition
   and analysis software (Beckton Dickinson).

Quantification of Chromosomal Aberrations

   Frozen BM cell pellets were thawed, washed two times with MEM-α medium,
   and cells were pelleted again for chromosome analysis by centrifugation
   at 180 g for 8 min at RT. Supernatants were removed, and cell pellets
   were resuspended prior to addition of fresh MEM-α media (Thermo Fisher
   Scientific) and 10 µg/ml demecolcine (Sigma-Aldrich). Tubes were then
   placed for 1 h in a humidified 5% CO[2] incubator at 37°C followed by
   centrifugation for 10 min at 200 g RT. Supernatants were discarded, and
   the cell pellets were each resuspended in 5 ml of 74 mM potassium
   chloride solution (VWR International, Radnor, USA) and incubated for
   30 min at 37°C in a water bath. To each tube, 3 ml of “½ strength
   hypotonic solution” [1.94 mM Tri-sodium citrate solution (VWR) and
   3.75 mM potassium chloride solution (VWR)] was added, and further
   incubated for 8 min. Cells were fixed in 3:1 Carnoys fixative (Fisher
   Scientific, Hampton, New Hampshire, USA) for 13 min. Samples were
   centrifuged at 200 g for 10 min at RT, pellets resuspended again in
   fixative and incubated for 30 min at RT prior to centrifugation at
   200 g for 10 min at RT, and the procedure was repeated once more with
   20 min incubation. Cells were kept at −20°C overnight.

   Slides were prepared from the fixed samples as follows: samples were
   centrifuged at 180 g for 10 min, supernatants were aspirated, and
   pellets resuspended in approximately 2 ml of fresh 3:1 fixative.
   Single-use fine-tip minipastettes (Alpha Laboratories Ltd., Eastleigh,
   Hampshire, UK) were used to pipette each cell suspension up and down
   before dropping a single drop onto the center of individual labeled
   degreased microscope slides. This process of layering cells was
   repeated until there was a reasonable coverage of cells on each
   microscope slide. Depending on the sample’s mitotic index, two to four
   slides were prepared from each sample. Samples were then air dried at
   RT for 24 h prior to staining with 6.7% Giemsa Stain improved R66
   solution Gurr^® (VWR) in buffer solution (pH 6.8). Slides were air
   dried before addition of cover slips secured with Entellan^® new rapid
   mounting media (VWR) and coded for analysis. Where possible, 200 well
   spread metaphases were analyzed from each sample using a light
   microscope and 100× objective.

   The Fisher’s exact test was performed, each irradiated/bystander group
   were compared to their respective control. Groups with p-values less
   than 0.05 were considered statistically significant.

Immune Phenotyping of Splenocytes and BM Cells

   The following directly labeled anti-mouse monoclonal antibodies were
   used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for
   lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic
   population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE
   and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail
   (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for
   hematopoietic stem cells, all purchased from BioLegend (BioLegend, San
   Diego, CA, USA).

   The phenotypical analysis of splenocytes was performed using the
   following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for
   helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE,
   I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic
   cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect
   proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was
   used.

   Single-cell suspensions of splenocytes or BM cells were incubated with
   the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C
   for 20 min for cell surface staining. For intracellular staining
   (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer
   (eBioscience), according to the manufacturer’s instructions.
   Measurements were performed with a FACSCalibur flow cytometer as
   described above.

Analysis of Apoptosis in Irradiated and Bystander Splenocytes

   Apoptosis was detected by the TUNEL assay using the Mebstain Apoptosis
   Kit Direct (MBL, Nagoya, Japan). Briefly, splenocytes were kept in
   250 µl ice-cold PBS and 750 µl 75% ethanol at 4°C for 20 min. Cells
   were washed, pelleted, and resuspended in the residual PBS. Fixation
   was done with 1 ml 1% PFA at RT for 30 min. Fixed cells were kept at
   4°C overnight and then pelleted, and a mix of 27 µl of terminal
   deoxy-nucleotidil transferase (TdT) buffer/1.5 μl of FITC-dUTP/1.5 μl
   TdT enzyme per sample was added to the pellet. FACS analysis was
   performed after incubating the samples at 37°C for 60 min.

Profiling of miRNA Isolated from BM-Derived EVs

miRNA profiling

   Extracellular vesicles were prepared from BM of control and irradiated
   mice by pooling the BM supernatant of five mice/radiation
   dose/experiment. Three independent experiments were performed.

   The EVs prepared were sent for analysis to Exiqon Services (Exiqon
   Services, Vedbaek, Denmark), where RNA isolation, miRNA profiling with
   a polymerase chain reaction (PCR) panel, and data pre-processing were
   performed.

   Total RNA was extracted by Exiqon from the EVs using the Qiagen
   miRNeasy^® Mini Kit (Qiagen, Hilden, Germany). Briefly, EVs were lysed
   in Qiazol lysis reagent then the lysate was incubated with chloroform
   at RT for 2 min. The supernatant was treated with 100% ethanol and
   centrifuged using a Qiagen RNeasy^® Mini spin. The Qiagen RNeasy^® Mini
   spin column was rinsed with the provided buffers then transferred to a
   new microcentrifuge tube, and the lid was left uncapped for 1 min to
   allow the column to dry. Total RNA was eluted with 50 µl of RNase-free
   water.

   MicroRNA analysis with RT-PCR array was also performed by Exiqon.
   Briefly, 19 µl RNA was reverse transcribed in 95 µl reaction volume
   using the miRCURY LNA™ Universal RT microRNA PCR, polyadenylation, and
   cDNA synthesis kit (Exiqon). cDNA was diluted 50× and assayed in 10-µl
   PCR reaction volume according to the protocol of the kit; each miRNA
   was assayed once by qPCR on the miRNA Ready-to-Use PCR, Mouse&Rat panel
   I + II using ExiLENT SYBR^® Green master mix. Negative controls
   excluding template from the reverse transcription reaction were
   performed and profiled similarly to the samples. The amplification was
   performed in a LightCycler^® 480 Real-Time PCR System (Roche, Basel,
   Switzerland) in 384 well plates. The amplification curves were analyzed
   using the Roche LC software, both for determination of quantification
   cycles (Cq) (by the second derivative method) and for melting curve
   (T[m]) analysis.

   The amplification efficiency was calculated by Exiqon using algorithms
   similar to the LinReg software. All assays were inspected for distinct
   melting curves, and the T[m] was checked to be within known
   specifications for the assay. Furthermore, assays must have been
   detected with three Cqs less than the negative control, and with
   Cq < 37 to be included in the data analysis. Data that did not pass
   these criteria were omitted from any further analysis. Cq was
   calculated as the second derivative.

   Using NormFinder, the best normalizer was found to be the average of
   assays detected in all samples. All data were normalized to the average
   of assays detected in all samples (average − assay Cq). The heat map
   diagram and the principal component analysis (PCA) were performed on
   all samples and on the top 50 miRNA with highest SD. The normalized Cq
   values have been used for the analysis.

Data Analysis of miRNA Arrays

   Data analysis of the miRNA arrays, based on normalized Cq values
   (determined by Exiqon) was performed by our group. For defining
   differentially expressed miRNA, differences were calculated pairwise as
   fold changes compared to the miRNA expression from non-irradiated
   (0 Gy) samples. The average fold changes of the three independent
   experiments were calculated. Student’s paired t-test was applied to
   these data for significance analysis.

   To uncover the potential biological function of miRNAs differentially
   expressed in EVs both in 0.1 Gy and 2 Gy irradiated animals, a multiple
   miRNA effect analysis using DIANA-miRPath v.3.0 software ([68]24) was
   performed. The DIANA-microT-CDS target prediction algorithm was
   employed to predict miRNA targets. This was combined with Kyoto
   Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)
   databases. A target prediction threshold of 0.8 with p-value of 0.05
   and false discovery rate correction was applied. A list of the
   predicted target genes of miRNAs altered by IR and a list of KEGG
   pathways ranked by significance was obtained in this way. Next, seven
   pathways closely connected to our investigated functional endpoints and
   considered by us as the most important ones were chosen from the KEGG
   pathway list and mapped those genes from the list of predicted target
   genes into the selected KEGG pathways, which were targeted by more than
   one differentially expressed miRNAs.

   To identify the processes co-regulated by these genes, a global network
   of functional coupling was constructed using FunCoup 3.0 software with
   the focus on finding new couplings between search terms. Within this
   software, an expansion algorithm was used with genes as a group,
   prioritizing common neighbors (meaning that all links to all the genes
   of interest are considered and genes that are most strongly linked to
   other genes of interest are prioritized). A confidence threshold of 0.8
   and expansion depth of one step including 20 nodes per expansion step
   was applied during analysis.

Statistical Analysis

   Data are presented as mean ± SD. In most of the cases Student’s t-test
   was applied to determine statistical significance, using GraphPad Prism
   version 6.00 for Windows (GraphPad Software,[69]^1 La Jolla, CA, USA),
   if not stated otherwise. Data were considered statistically significant
   if p-value was lower than 0.05.

Results

BM-Derived EVs Are Composed of Exosomes and MVs

   The measured hydrodynamic mean diameter of the EVs was 169 nm
   (±SD = 83), 252 nm (±SD = 136), and 226 nm (±SD = 106) in mice treated
   with sham, 0.1 Gy, and 2 Gy irradiation, respectively. Differences in
   the mean diameter of EVs were statistically not significant, indicating
   that irradiation did not alter the size distribution of the EVs
   (Figures [70]2A–C).

Figure 2.

   [71]Figure 2
   [72]Open in a new tab

   Characterization of bone marrow-derived extracellular vesicles (EVs).
   (A–C) Size distribution of the EVs isolated 24 h following irradiation
   with 0 Gy (A), 0.1 Gy (B), and 2 Gy (C), determined by measuring the
   hydrodynamic size using the dynamic light scattering method. (D)
   Transmission electron microscopy imaging of EVs. Representative image
   of EVs isolated from control (0 Gy) mice. (E) Western blot analysis of
   EVs for calnexin, TSG101, and CD9. Lanes 1 and 7 show the protein
   ladder, lane 2 is the cell lysate, lane 3 is an unirradiated (0 Gy)
   sample isolated with Exoquick-TC, lanes 4–6 are 0, 0.1, and 2-Gy
   samples isolated with Exoquick-TC and filtered through PD SpinTrap G-25
   column (F) Acetylcholinesterase enzyme activity of EVs from samples
   irradiated with different doses was assessed by an enzyme activity
   assay. OD was measured at 412 nm. Data are the mean ± SD of three
   independent experiments.

   Electron microscopic analysis indicated the presence of vesicular
   structures in the isolates, many of which had a typical “cup-shaped”
   aspect characteristic for exosomes (Figure [73]2D).

   The EVs were further characterized by Western blot analysis following
   minimal required criteria suggested by Lötvall et al. for EV
   identification ([74]25): a minimum of two EV-specific protein markers
   expected to be present in EV isolates and an endosomal protein not
   expected to be present in EVs were determined. EVs from both control
   and irradiated mice were positive for two markers commonly used for
   exosome identification: the tetraspanin CD9, a protein highly enriched
   in EVs ([75]26), and the TSG101, involved in multivesicular biogenesis
   ([76]27), and were negative for calnexin, an endoplasmic reticulum
   marker (Figure [77]2E). The coexistence of these criteria is considered
   as EV markers and identifies our isolated samples as EVs.

   The AchE activity was also measured in the isolated EVs. Although AchE
   activity is not considered as an absolute specific EV marker, if
   present, it can further strengthen their identity. AchE activity was
   present in comparable amounts in the EV isolates from control and
   irradiated animals confirming the presence of exosomes and MVs in all
   samples (Figure [78]2F).

In Vivo Transfer of EVs from Irradiated Mice Induces γ-H2AX Foci Formation in
the Spleen of Recipient Mice

   The frequency of DNA double-strand breaks (DSBs) generated by in vivo
   transfer of EVs was investigated in the spleen of bystander animals and
   compared to DSBs generated in total-body irradiated mice. DSB analysis
   was performed by the γ-H2AX assay using both a fluorescent microscopy
   and a flow cytometry protocol. The fluorescent microscopy protocol is
   considered a more accurate and more specific method than evaluating the
   frequency of event-positive cells by flow cytometry ([79]28). However,
   the latter method is much quicker, allows the quantification of much
   higher number of cells, and in this way, increases the statistical
   power in cases where the number of alterations is low ([80]29). As
   expected, a dose-dependent increase of DNA damage was detected in
   directly irradiated animals. In bystander mice, which received EVs from
   irradiated animals γ-H2AX foci levels also increased both in terms of
   average foci/cell (Figures [81]3A,C) and the frequency of
   γ-H2AX-positive cells (Figures [82]3B,D). However, the increase was
   more moderate than in the directly irradiated animals, and no strict
   dose-dependency was observed, since the detected damage levels after
   low- and moderate-dose irradiation were comparable to high-dose
   irradiation (Figure [83]3). These data indicate that BM-derived EVs
   originating from irradiated animals could mediate the activation of the
   DNA damage response pathway in the splenocytes of EV-injected bystander
   animals and that RIBE peaked at low doses.

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   DNA double-strand breaks in directly irradiated and bystander animals
   measured by γ-H2AX assay. (A) Microscopic image of splenocytes of
   directly irradiated and bystander animals immunostained for γ-H2AX.
   Arrows indicate the location of γ-H2AX^+ foci, cell nuclei stained with
   DAPI are shown in blue, γ-H2AX^+ foci stained with Alexa488 are shown
   in green. (B) Flow cytometry plots of splenocytes stained for γ-H2AX.
   The gates indicate the percent of γ-H2AX^+ cells within the
   splenocytes. (C) Histogram representing the average number of γ-H2AX^+
   foci per cells counted by fluorescent microscopy (N = 7–10). (D)
   Histogram representing the percent of γ-H2AX^+ cells within the
   splenocytes measured by flow cytometry (N = 7–10). Bars represent
   mean ± SD, significance was tested by Student’s t-test (*p < 0.05,
   **p < 0.01, ***p < 0.001).

EV Transfer from Irradiated Mice Induces Chromosomal Aberrations in Recipient
Animals

   As expected, the frequency of chromosomal aberrations increased in the
   BM cells of directly irradiated mice. In bystander mice which received
   EVs from directly irradiated animals, the frequency of chromosomal
   aberrations also increased, but to a lesser extent. In the directly
   irradiated mice, the highest level of chromosomal aberrations was
   detected at the highest dose, while in the bystander mice it peaked
   around 0.25 Gy (Figure [86]4A). Most aberrations detected were
   chromatid in nature (Figure [87]4B). EV-recipient bystander groups
   overall showed a greater proportion of chromatid aberrations compared
   to directly irradiated mice.

Figure 4.

   [88]Figure 4
   [89]Open in a new tab

   Chromosomal aberrations in bone marrow cells isolated from irradiated
   and control mice. (A) Chromosomal aberrations were scored in 200
   metaphase spreads 24 h after irradiation or extracellular vesicles
   transfer. Bars represent mean ± SEM, significance was tested by
   Fisher’s exact test. Each irradiated/bystander group was compared to
   its respective control. Groups with p-values less than 0.05 were
   considered statistically significant (*). (B) Total aberrations were
   scored regardless of their nature and plotted as fractions of the
   total.

EV Transfer from Irradiated to Bystander Mice Induces Quantitative Changes in
the Cellular Composition of BM and Spleen

Alterations in BM

   Direct as well as EV transfer-induced bystander effects were studied in
   more detail in the BM stem and progenitor cell compartments. Namely,
   alterations in the hematopoietic stem cells (Lineage-Sca-1^+cKit^+),
   lymphoid progenitors (CD45^+CD90.2^+), myeloid progenitors
   (Gr1^+CD11b^+), megakaryocytes, and megakaryocyte progenitors
   (CD41^+CD61^+), as well as erythroid progenitors (CD71^+Ter119^+) were
   studied.

   In directly irradiated mice, the absolute number of the hematopoietic
   stem cells decreased to 38, 34, and 21 after 0.1, 0.25, and 2 Gy
   irradiation, respectively, when compared to unirradiated animals
   (Figures [90]5A,B). In the EV-recipient animals the stem cell numbers
   also decreased, but changes were milder and moderately depended on
   dose. In the 0.1-Gy bystander group, changes were statistically not
   significant, while in the 0.25- and 2-Gy bystander mice, the number of
   hematopoietic stem cells decreased to almost identical levels (65 and
   60% surviving cells) (Figures [91]5A,B).

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   Immune phenotyping of bone marrow (BM) cells isolated from irradiated
   and bystander animals. BM cells isolated from directly irradiated and
   bystander mice were stained with fluorescently labeled antibodies and
   were analyzed by flow cytometry. Histogram (A) represents the cell
   number of hematopoietic stem cells in mouse BM. Dot plots from the flow
   cytometric analysis (B) represent the distribution of hematopoietic
   Sca-1 and c-Kit (CD117) double positive, Lineage (CD3^+, Gr1^+,
   CD11b^+, CD45R^+, Ter119^+) negative stem cells. The plots show the
   gated Lineage negative cells in which Sca1^+c-Kit^+ cells were
   evaluated. Histogram (C) shows the number of lymphoid progenitors in
   mouse BM. Dot plots from the flow cytometric analysis (D) show the
   distribution of CD45 and CD90.2 double positive lymphoid progenitor
   cells. Bars represent mean ± SD (N = 7–12), significance was tested by
   Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

   Beside stem cells, the lymphoid progenitors were another radiosensitive
   population, since their number decreased to 70 and 15% after 0.25 and
   2 Gy, respectively, in directly irradiated mice. However, in this case,
   EV could not transmit the effect to recipient mice (Figures [94]5C,D).
   Megakaryocyte progenitors in BM of directly irradiated animals
   exhibited a small decrease in cell number. Although changes were
   statistically not significant, the tendency showed a dose-dependent
   decrease (Figure S1A in Supplementary Material). Myeloid and erythroid
   progenitor cell numbers were not affected either by irradiation or EV
   transfer (Figures S1B,C in Supplementary Material).

Alterations in the Spleen

   Lymphocytes constitute the major cellular fraction within the murine
   spleen (approximately 85%), which is also a rich source of DCs.
   Radiation-induced direct and bystander changes were monitored by
   following the absolute number and proliferative capacity of the
   different lymphocyte subpopulations (CD4^+ and CD8^+ T lymphocytes,
   CD19^+ B cells, and NK cells), as well as the number and activation
   status of splenic DCs.

   Regarding the direct effect of irradiation on the splenocyte
   subpopulations, a strong difference in the radiosensitivity of the
   various cellular subsets was observed. Low doses had no significant
   effect, but irradiation with 2 Gy reduced CD4^+ T cell, CD8^+ T cell,
   and B cell pool to 60, 45, and 39% of control values, respectively
   (Figures [95]6A,C,E). NK cell numbers were not affected by irradiation
   (Figure [96]6G). Interestingly, strong bystander responses were
   detected in CD4^+ and CD8^+ T cell populations of animals injected with
   EVs from irradiated mice, while the effect was absent in B and NK cells
   (Figures [97]6A,C,E,G).

Figure 6.

   [98]Figure 6
   [99]Open in a new tab

   Immune phenotyping of splenic lymphocytes isolated from irradiated and
   bystander animals. Splenocytes isolated from directly irradiated and
   bystander mice were stained with fluorescently labeled antibodies and
   were analyzed by flow cytometry. Histograms represent the cell numbers
   of the corresponding subpopulations calculated per 10 mg spleen: CD4^+
   T cells (A) and proliferating CD4^+ T cells (B); CD8^+ T cells (C) and
   proliferating CD8^+ T cells (D); B cells (E) and proliferating B cells
   (F); natural killer (NK) cells (G) and proliferating NK cells (H). Bars
   represent mean ± SD (N = 5); significance was tested by Student’s
   t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

   The basal proliferation rate was about 9.5, 9, 24 and 26% for CD4^+,
   CD8^+, B, and NK cells, respectively, and EV injection per se did not
   alter this proliferation rate (Figures [100]6B,D,F,H).
   Radiation-induced changes in the proliferative capacity of splenocytes
   reflected their radiosensitivity, decreasing after 2 Gy in all
   lymphocytes. Bystander responses were similar, albeit milder than in
   the directly irradiated animals (Figures [101]6B,D,F,H).

   Splenic DCs were identified by their CD11c and MHCII double positivity.
   In contrast to lymphocytes, the number of DCs did not change in the
   directly irradiated animals. Bystander responses were also absent for
   all doses (Figure [102]7A). TLR4 expression on DC cell surface is a
   sign of DC activation by lipopolysaccharide (LPS) or LPS-like
   endogenous danger signals, such as high mobility group binding protein
   1 (HMGB1) ([103]30). Since radiation-induced cellular damage is
   associated with danger signal release, we investigated
   radiation-induced changes in the fraction of TLR4-expressing DCs. A
   significantly increased fraction of TLR4-expressing DCs was detected
   after direct irradiation with 2 Gy. Surprisingly, EV-induced bystander
   responses showed a completely different pattern of TLR4 expression,
   since the proportion of TLR4-expressing DCs within the total DC
   population was very strongly reduced after treatment with EVs derived
   from irradiated animals irrespective of the dose (Figures [104]7B,C).

Figure 7.

   [105]Figure 7
   [106]Open in a new tab

   Immune phenotyping of splenic dendritic cells (DCs) isolated from
   irradiated and bystander animals. Splenocytes isolated from directly
   irradiated and bystander mice were stained with fluorescently labeled
   antibodies and were analyzed by flow cytometry. Histogram (A) shows the
   number of CD11b^+ MHCII^+ splenic DCs per 10 mg spleen. Histogram (B)
   shows the relative ratio of TLR4^+ dendritic cells within the total
   splenic DC population. Irradiated and bystander samples were compared
   to their corresponding unirradiated controls. Dot plots from the flow
   cytometric analysis (C) represent the distribution of TLR4^+ cells
   within the DC population. The plots show the gated CD11c^+ MHCII^+
   positive cells in which TLR4 expression was determined. Bars represent
   mean ± SD (N = 5), significance was tested by Student’s t-test
   (*p < 0.05, **p < 0.01, ***p < 0.001).

EV Transfer from Irradiated to Bystander Mice Does Not Induce Apoptosis in
Splenocytes

   Since phenotypical analysis indicated a strong radiation response of
   splenic lymphocytes both in the directly irradiated and EV-recipient
   animals, which could be only partially explained by the reduced
   proliferation capacity of the cells after irradiation, we investigated
   potential alterations in apoptosis frequency in total splenocytes by
   the TUNEL assay. As presented in Figure S2 in Supplementary Material,
   the fraction of apoptotic cells increased strongly in the directly
   irradiated animals after irradiation with 2 Gy. However, EV-induced
   bystander responses were completely absent, indicating that EV transfer
   did not have any apoptosis-inducing effect.

Analysis of microRNA Profile of EVs Derived from the BM of Irradiated Mice

Similar miRNAs Are Affected after Both Low- and High-Dose Irradiation

   The average number of miRNAs that could be identified in EVs derived
   from BM of unirradiated, control mice was 500 per sample. It was not
   characteristic for irradiation to induce the appearance or
   disappearance of miRNAs in the EVs with very few exceptions; miRNAs,
   such as miR-124, miR-346, miR-449c, and miR-381, were present, while
   miR-695 and miR-761 were absent in the samples irradiated with 2 Gy.
   Raw data were uploaded to STOREDB database,[107]^2 accession number
   [DOI:10.20348/STOREDB/1062], dataset 1101. According to the PCA,
   samples seemed to cluster based on the radiation group they belonged
   to, with a better separation of the 2 Gy samples.

   When comparing the miRNA content of the EVs of irradiated and control
   mice, 20 miRNAs were found to be differentially expressed in the 0.1 Gy
   group (Table S1 in Supplementary Material) and 90 miRNAs in the 2 Gy
   group (Table S2 in Supplementary Material) using a t-test with a cutoff
   p-value <0.05. Out of these, eight miRNAs were affected after both low-
   and high-dose irradiation: five miRNAs (mmu-miR-33-3p, mmu-miR-200c-5p,
   mmu-miR-140-3p, mmu-miR-744-3p, and mmu-miR-669o-5p) were downregulated
   and three miRNAs (mmu-miR-152-3p, mmu-miR-199a-5p, and mmu-miR-375-3p)
   were upregulated. Changes in the level of these miRNAs were dose
   dependent, as shown in Figure [108]8.

Figure 8.

   Figure 8
   [109]Open in a new tab

   microRNAs (miRNAs) differentially expressed in both 0.1 and 2 Gy
   extracellular vesicles (EVs) compared to EVs from control animals. A
   miRNA profiling of EVs isolated from bone marrow of control mice and
   mice irradiated with 0.1 or 2 Gy was performed by a qPCR panel array.
   miRNAs with significantly modulated expression relative to control are
   presented in the graph. Data are the mean ± SD of three independent
   experiments. Significance was tested by Student’s t-test (*p < 0.05).

miRNA Target Prediction and Pathway Analysis Shows a Direct Link between
miRNA Expression Pattern and EV-Induced Changes in the Hematopoietic System
after Irradiation

   In order to create a link between the differentially expressed miRNAs
   and EV-induced changes in the hematopoietic system of the bystander
   animals, a functional analysis using DIANA miRPath software followed by
   a network analysis using FunCoup 3.0 software was performed.

   Analysis of the target genes of the 20 differentially expressed miRNAs
   in the 0.1-Gy samples revealed that these miRNAs targeted 33 different
   KEGG pathways (Table S3 in Supplementary Material), whereas the 90
   differentially expressed miRNAs derived from the 2-Gy samples targeted
   60 KEGG pathways with a high degree of significance (p ≤ 0.05) (Table
   S4 in Supplementary Material).

   A more detailed target prediction and pathway analysis of the eight
   miRNAs modulated in both 0.1 and 2 Gy irradiated samples was performed
   by applying a GO and KEGG Pathway Enrichment Analysis. While the GO
   pathway annotates different genes and gene products to certain gross
   biological terms, such as biological process and subcellular
   localization ([110]31), the KEGG pathway database is a collection of
   diagrams representing complex pathway maps of molecular interactions
   and networks ([111]32). The top GO processes, predicted to be
   influenced by these eight miRNAs, were associated with development and
   differentiation, metabolic and biosynthetic processes, cell growth,
   motility, and cell death. It also showed that all miRNAs within these
   pathways were located in the following cellular compartments: nuclear
   chromosome, cytoplasmic stress granule, and cytoplasmic membrane-bound
   vesicle (Table S5 in Supplementary Material), indicating not only a
   concentration of IR-induced damage at chromosomal level but also
   highlighting the vesicular origin of the miRNAs.

   Using the KEGG database, 27 pathways were predicted to be influenced by
   the differentially expressed miRNAs, many of them dealing with
   mechanisms connected to cellular radiation response, DNA repair [such
   as Hippo, Hedgehog, Forkhead box O (Foxo), Phosphoinositide-3-kinase
   (PI3K), TGFβ signaling pathways], as well as pathways connected to the
   hematopoietic system [signaling pathways regulating pluripotency of
   stem cells, Wnt signaling pathway, human T cell lymphotrophic virus
   (HTLV) infections] (Figure [112]9; Table S6 in Supplementary Material).

Figure 9.

   [113]Figure 9
   [114]Open in a new tab

   Analysis strategy for evaluating the effects of differentially
   expressed microRNAs (miRNAs). The set of miRNAs differentially
   expressed from both 0.1 Gy vs. control and 2 Gy vs. control was
   analyzed for predicted target genes and predicted pathways using the
   DIANA miRPath software. Seven pathways (highlighted in gray) considered
   to be important for the endpoints of the study were further analyzed.
   Messenger RNAs potentially regulated by more than one differentially
   expressed miRNA were mapped from these seven pathways, followed by a
   network analysis of these mRNAs using FunCoup 3.0 software.

   To find the putative responsible mRNAs driving the observed functional
   effects caused by EV transfer (DNA damage and phenotypical changes in
   central and peripheral hematopoietic system), seven pathways were
   chosen for further study out of the 27 identified (highlighted in
   Figure [115]9). Messenger RNAs potentially regulated by more than one
   differentially expressed miRNA were mapped from these seven pathways.
   Twelve mRNAs co-regulated by six miRNAs were found, which were involved
   in one or more of the selected pathways, as shown in Figures [116]9 and
   [117]10. We also noticed that most of the products of these mRNAs were
   involved in multiple pathways.

Figure 10.

   [118]Figure 10
   [119]Open in a new tab

   A hypothetical network of KEGG pathways predicted to be altered by
   mRNAs targeted by the microRNAs (miRNAs) differentially expressed in
   the EVs in both 0.1 and 2 Gy samples. (A) Black circles represent
   differentially expressed miRNA, yellow boxes are their predicted target
   genes and colored boxes represent the pathways including these genes.
   (B) The full name and annotation of target mRNAs.

   A gene coupling network was constructed by connecting these 12 mRNAs
   using the FunCoup software. Since FunCoup is a collection of
   genome-wide functional couplings, which integrates evidence types
   derived from high-throughput genomics and proteomics data, such as
   protein–protein interaction, mRNA co-expression, protein co-expression,
   shared transcription factor binding, and co-miRNA regulation, by shared
   miRNA targeting ([120]33, [121]34), it is suitable to reveal new
   functional links not identified solely by the KEGG pathway analysis.
   The most enriched signaling pathways detected with this approach were
   strongly related to the hematopoietic and immune system, such as T cell
   signaling, B cell signaling, NK-mediated cytotoxicity, chemokine
   signaling, Fc epsilon signaling, insulin signaling, and Jak–Stat
   signaling (Figure [122]9; Table [123]1).

Table 1.

   Significantly enriched pathways according to FunCoup network analysis.
   Kyoto Encyclopedia of Genes and Genomes signaling pathway Number of
   genes p-Value
   T cell receptor signaling pathway 6 5.97E−4
   B cell receptor signaling pathway 5 5.97E−4
   Insulin signaling pathway 6 5.97E−4
   ErbB signaling pathway 5 5.97E−4
   Fc epsilon RI signaling pathway 5 5.97E−4
   Neurotrophin signaling pathway 5 2.73E−3
   TGF-beta signaling pathway 4 6.68E−3
   Chemokine signaling pathway 5 1.15E−2
   Jak–STAT signaling pathway 4 2.76E−2
   Wnt signaling pathway 4 2.76E−2
   Natural killer cell-mediated cytotoxicity 4 3.84E−2
   MAPK signaling pathway 4 1.02E−1
   [124]Open in a new tab

   Number of genes refers to the number of mRNAs involved in the
   corresponding pathway.

Discussion

   Radiation-induced bystander effects have important consequences in
   radiation protection, since due to this phenomenon not only the
   directly irradiated cells exhibit biological damage but a significantly
   larger number of cells are also affected, increasing the likelihood of
   radiation-induced adverse health effects ([125]35). Therefore,
   significant effort has been done to understand the mechanisms governing
   this phenomenon.

   Recent works have indicated that EVs released from irradiated cells may
   play a role in mediating RIBE. Al-Mayah et al. showed that treatment of
   bystander MCF-7 breast cancer cells with exosomes isolated from media
   of irradiated cells increased the level of genomic damage ([126]20),
   and this effect persisted for more than 20 population doublings in the
   progeny of bystander cells ([127]19). Mutschelknaus et al. demonstrated
   that exosomes derived from irradiated head and neck cancer cell lines
   increased both the proliferation and survival of recipient cells
   ([128]36). We should note, however, that these evidences have been
   shown exclusively under in vitro conditions.

   In the present work, we designed an in vivo model to study the ability
   of EVs to mediate bystander effects, where EVs extracted from the BM of
   total-body irradiated mice were injected intravenously into naïve mice
   and EV-transmitted effects were followed in the BM and spleen of the
   EV-recipient animals. The reason for choosing the hematopoietic system
   for our studies was that both the BM and the spleen are highly
   radiosensitive tissues, where radiation-induced bystander signals have
   been identified as important modulators of radiation effects ([129]37,
   [130]38). Since it was shown by several research groups that BM was an
   important tissue milieu where MVs-mediated signals were able to
   modulate the phenotype of the cells ([131]39–[132]41), we intended to
   test the hypothesis that EVs could be at least in part responsible for
   local and/or systemic RIBE. Formerly, we have investigated the in vivo
   biodistribution of BM-derived EVs upon intravenous injection and
   demonstrated their stable presence both in the spleen and BM 24 h after
   injection ([133]42). The EVs used for the current experiments had a
   mean diameter of 200 nm and a cup-shaped aspect and were highly
   enriched in the TSG101 and tetraspanin CD9 proteins, while lacking
   cellular markers of endosomal origin, indicating that the EV isolates
   were composed of exosomes and most probably MVs as well ([134]26,
   [135]43). While formerly the exosomes were considered the main and
   unique EV types involved in intercellular communication, recent
   publications have proven also the involvement of MVs in this process.
   MVs, similarly to exosomes, have a rich mRNA, miRNA, and protein cargo
   ([136]44). Recently, Wen et al. have demonstrated that a combination of
   exosomes and MVs had a stronger effect in transferring biological
   processes from one cell to the other than either fraction alone
   ([137]45).

   In order to evaluate the role of EVs in mediating radiation effects,
   first we investigated whether EVs could transmit systemically
   radiation-induced DNA and chromosomal damage to unirradiated BM and
   spleen cells. The most characteristic type of DNA damage caused by IR
   is DNA DSB, a highly cytotoxic form of DNA damage, which, if not
   repaired in short time, can lead to cell death or genomic instability
   ([138]46). The phosphorylation of the histone H2AX in the vicinity of a
   DSB is considered a specific marker for this type of DNA lesion
   ([139]47). The phosphorylated H2AX molecules are induced during the
   repair process of DSB and can be observed as distinct foci in nuclei of
   the cells in the neighborhood of the damage. The sensitivity of the
   method to detect even very low doses of radiation exposure was reported
   in several publications, which also proved that the assay is dose
   dependent ([140]48–[141]50). In line with these findings, our data
   showed a correlation of γ-H2AX-positive cells with the applied
   radiation dose within the spleens of the directly irradiated animals.

   γ-H2AX foci evaluation was also used for characterization of RIBE both
   in vitro ([142]51, [143]52) and in vivo ([144]53). Sokolov et al.
   demonstrated that γ-H2AX co-localized with proteins involved in DNA
   damage response in bystander human fibroblast cultures ([145]52). Here,
   we have demonstrated that BM-derived EVs from irradiated mice induced
   phosphorylation of the H2AX protein in EV-recipient bystander animals.
   The role of EV in mediating radiation-induced DNA damage in
   non-irradiated cells has not been reported yet. However, Dutta et al.
   showed that EVs isolated from the cell culture supernatant of human
   breast cancer cell lines were able to induce phosphorylation of key
   proteins (ataxia-teleangiectasia mutated (ATM), H2AX, Chk1, and p53)
   involved in DNA damage response in primary mammary epithelial cells in
   vitro by transmitting signals that led to ROS production and a
   consequential oxidative stress in the EV-recipient cells ([146]54,
   [147]55). The role of EVs in inducing oxidative stress and mediating
   redox-regulated signaling processes in EV-recipient cells has been
   shown by several other recent reports as well ([148]55, [149]56).
   Fontaine et al. proved the implicit role of EVs in this process, since
   the increased oxidative stress in the vascular wall of patients after
   coronary surgery disappeared if using EV-depleted plasma ([150]56). It
   has been shown that EVs from preeclamptic women were directly taken up
   by endothelial cells leading to iNOS synthesis and activation of NFκB
   ([151]57). Lee et al. found that hyperoxia-induced oxidative stress in
   lung epithelial cells led to increased EV production which in turn was
   taken up by macrophages leading to macrophage activation and increased
   production of NFκB-regulated pro-inflammatory molecules ([152]44). It
   is known that ROS are mainly responsible for X-ray-induced DNA damage
   and activation of the DNA damage response pathways and the above
   publications prove that EVs are able to transmit oxidative stress in
   recipient cells. Thus, although we have not determined ROS levels in
   EV-recipient cells, it is logical to assume that the development of
   complex DNA damage consisting in increased IR-specific chromosomal
   aberrations and activation of the DNA damage response pathway in naïve
   mice receiving EVs from irradiated animals was mediated via
   redox-regulated signaling. This conclusion is supported by the fact
   that mice receiving EVs from non-irradiated mice showed background
   levels of DNA damage. Furthermore, as detailed later in this section,
   several pathways involved in DNA damage repair have been regulated by
   miRNA differentially expressed in EVs originating from the irradiated
   animals. While the above cited references point to a specific effect of