Small extracellular vesicles derived from mesenchymal stromal cells mitigate intestinal toxicity in a mouse model of acute radiation syndrome.


 Background: Human exposure to high doses of radiation resulting in acute radiation syndrome and death could rapidly escalate to a mass casualty catastrophe in the event of nuclear accidents or terrorism. The primary reason is that there is presently no effective treatment option, especially for radiation-induced gastrointestinal syndrome. This syndrome results from disruption of mucosal barrier integrity leading to severe dehydration, blood loss and sepsis. In this study, we tested whether small extracellular vesicles/exosomes derived from mesenchymal stromal cells (MSC) could reduce radiation-related mucosal barrier damage and reduce radiation-induced animal mortality. Methods: Human MSC-derived small extracellular vesicles/exosomes were intravenously administered to NUDE mice, 3, 24 and 48 hours after lethal whole-body irradiation (10 Gy). Integrity of the small intestine epithelial barrier was assessed by morphologic analysis, immunostaining for tight junction protein (claudin-3) and in vivo permeability to 4 kDa FITC-labeled dextran. Renewal of small intestinal epithelium was determined by the quantification of epithelial cell apoptosis (TUNEL staining) and proliferation (Ki67 immunostaining). Statistical analyses were performed using one-way ANOVA followed by a Tukey test. Statistical analyses of mouse survival were performed using the methods of Kaplan-Meier and Cox. Results: We demonstrated that MSC-derived small extracellular vesicles/exosomes treatment reduced by 85% the instantaneous mortality risk in mice subjected to 10 Gy whole-body irradiation and thus increased their survival time. This effect could be attributed to the efficacy of MSC-derived small extracellular vesicles/exosomes in reducing mucosal barrier disruption. We showed that MSC-derived small extracellular vesicles/exosomes improved renewal of the small intestinal epithelium by stimulating proliferation and inhibiting apoptosis of the epithelial crypt cells. MSC-derived small extracellular vesicles/exosomes also reduced radiation-induced mucosal permeability as evidenced by the preservation of claudin-3 immunostaining at the tight junctions of the epithelium. Conclusions: MSC-derived small extracellular vesicles/exosomes promote epithelial repair and regeneration and preserve structural integrity of the intestinal epithelium in mice exposed to radiation-induced gastrointestinal toxicity. Our results suggest that the administration of MSC-derived small extracellular vesicles/exosomes could be a treatment modality to limit acute radiation syndrome.


Introduction
Accidental or intended catastrophic nuclear/radiological events represent a real threat of mass casualty catastrophe. In such events, exposed victims would be considered "at increased risk" of developing exposure-related morbidity and/or mortality, called acute radiation syndrome (ARS). Whole-body irradiation (WBI) doses can be divided into potentially sublethal (≤ 2 Gy), lethal (between 2 and 10 Gy) and supralethal (≥ 10 Gy). For doses between 2 and 6 Gy, the hematopoietic (HP) injury is expected to be the major contribution to the mortality of victims that occurs within weeks after exposure. In this case, acute radiation exposure triggers the death of hematopoietic stem cells and progenitor cells leading to myelosuppression and increased susceptibility to infection, hemorrhage and anemia. After exposure to higher doses between 6 and 10 Gy, victims develop both irreversible HP and gastrointestinal (GI) injuries. Radiation-induced GI syndrome is characterized by death of GI stem/progenitor cells, drastic functional dysregulation of the intestinal epithelium and loss of digestive barrier integrity. Victims suffer from abdominal pain, diarrhea, dehydration, intestinal bleeding and sepsis, and mortality occurs within 2 weeks after exposure. Because the time window of opportunity for intervention is very short, between 24 and 48 hours after exposure, most drugs under investigation are radioprotectors or radiomitigators. Nevertheless, in 2006, a European consensus was established for the treatment of accidental radiation-induced HP syndrome [1,2]. This treatment involves acute administration of a cytokine combination to stimulate residual hematopoiesis. In the case of irreversible medullar aplasia, bone marrow transplantation could be used. Although these treatments were efficacious for HP syndrome management, they were not able to rescue GI syndrome in experimental models [3,4] Consequently, GI syndrome remains intractable to clinical intervention and is lethal for patients exposed to high doses of radiation.
An innovative therapy using the administration of mesenchymal stromal cells (MSC) has been proposed for treatment of GI alterations in a context of ARS syndromes [5,6]. MSC have been reported to have pleiotropic properties, and have been tested in more than 1000 clinical trials for the treatment of a wide range of diseases (http://www.clinicaltrials.gov), including of the bone marrow and GI tract. In 2006, it was proposed that MSC exert their therapeutic effects through secretion of bioactive factors [7,8]. An emerging consensus highlights that most of the paracrine physiological functions of MSC are related to their potential to release extracellular vehicles (EVs), namely MSC-derived small EVs/exosomes and/or microvesicles [9,10]. Similarly, the therapeutic benefits of MSC for radiation-induced intestinal toxicity might also be attributed to the release of MSC-derived exosomes which were already implicated as the mediator of MSC protection in necrotizing enterocolitis of the intestine [11].
Exosomes are bilipid membrane nanovesicles (30-100 nm) produced by intracellular endosomes and released into the extracellular environment after fusion with the plasma membrane. Exosomes contain proteins such as growth factors, adhesion molecules, heat shock proteins, cytoplasmic enzymes and signal transduction proteins, but also functional messenger RNA (mRNA) and microRNA. MSC-derived exosomes, like MSC themselves, are very attractive as they might be useful clinical tools for therapeutic cargo delivery to injured cells and as key mediators of signaling within the stem cell niche.
In this context, the aim of this study was to test the effect of MSC-derived small EVs including exosomes on radiation-induced GI toxicity. We used a model of mice subjected to lethal WBI in order to mimic the overlapping of multiorgan failure observed in ARS. To assess the therapeutic benefit of MSC-derived small EVs/exosomes, we chose a WBI dose of 10 Gy, inducing, as we reported, myelosuppression and high transient rupture of the intestinal barrier. We demonstrated that short-term MSC-derived small EV/exosome treatment significantly delayed time to death in the WBI animals and this delay could be attributed to the maintenance of intestinal barrier integrity.

Material And Methods
All experiments were performed in compliance with the Guide for the Care and Use of

Irradiation protocol
Male NUDE mice (Janvier SA, Le Genest St Isle, France) 6/8 weeks of age were received and housed in a temperature-controlled room (21 ± 1 °C). They were allowed free access to water and fed standard pellets. Mice were anesthetized by ketamine and xylazine preparation 2:1 (v/v) mixture diluted in 0.9% NaCl, injected at 0.1 mL/g and a single WBI dose was delivered by a medical accelerator (Alphée). Alphée is an accelerator-type radiation source (maximal energy 4 MeV with an average energy of about 1.5 MeV; 30 kA).
Preparation and administration of MSC-derived small EVs/exosomes All isolations and characterizations were performed as previously described [12,13], but with some modifications. Briefly, immortalized E1-MYC 16.3 human embryonic stem cellderived MSCs were cultured in DMEM (GE Healthcare, USA) with 10% fetal bovine serum (FBS) (Thermofisher Scientific, Waltham, MA, USA). To obtain small EVs/exosomes, 80% confluent cells were grown in a chemically defined medium for 3 days and conditioned medium was harvested as previously described (ref: PMID 17565974). The conditioned medium was clarified of cell debris, size fractionated and concentrated 50X by tangential flow filtration using a membrane with a molecular weight cut-off (MWCO) of 100 kDa (Sartorius, Gottingen, Germany). Small extracellular vesicle yield was assayed by protein concentration using a NanoOrange Protein Quantification Kit (Thermofisher Scientific).
Each batch of small extracellular vesicle preparation was qualified for particle size distribution (See below: Nanoparticle Tracking Analysis) and presence of exosomeassociated markers (See below Transmission Electron Microscopy).
The MSC-derived small EVs/exosomes were lyophilized by Paracrine Therapeutics using a proprietary technique, stored at − 20 °C and re-constituted with water for use. A total of 600 µg of MSC-derived small EVs/exosomes was intravenously administered in three

Immunohistochemistry
Hydrated sections were dipped into permeabilization solution consisting of 0.1% Triton X-100 in PBS and were rinsed in a distilled water bath for 5 min. Then, endogenous enzymes were blocked using 3% hydrogen peroxide (H 2 O 2 ) in methanol for 10 min and washed again in a 50 mM Tris buffer containing 9 g/L NaCl (TBS). To expose masked epitopes, tissues were incubated for 30 min in 10 mmol/L buffered citrate, pH 6.0. Non-specific In vivo intestinal permeability assay In vivo intestinal permeability was assessed using fluorescein dextran (FITC-Dextran 4, Sigma-Aldrich) as previously described [14]. Mice were orally gavaged with 0.75 mg/g body weight of 4 kDa FITC-labeled dextran and blood samples were obtained from the retro-orbital venous plexus 5 h after this administration. Blood samples were centrifuged for 10 min at 5000 rpm and plasma was taken and frozen at -20 °C and analyzed the following day. Intestinal permeability to 4 kDa FITC-labeled dextran was determined by measuring the fluorescence intensity in plasma at 485 nm/525 nm using an automatic Infinite M200 microplate reader (Tecan, Lyon, France).

Statistical analysis
Data are given as mean ± S.E.M. (standard error of the mean). Results were compared between groups by one-way ANOVA followed by a Tukey test using GraphPad 7.0 software (GraphPad, San Diego, CA). Results were also compared between groups through normal or Poisson regression models according to the nature of the parameter of interest, continuous response (villus height and intestinal permeability) or count data (apoptosis and proliferation), respectively.
Mouse survival curves were calculated by the Kaplan-Meier method and the P-value was determined by a log-rank test possibly adjusted for multiple comparisons. The Cox survival model was used in the assessment of the association between MSC-derived exosome treatment and risk of death [15]. The coefficients in a Cox regression relate to hazard, which quantifies an increase in the risk or a protective effect according to the sign of the fitted coefficients (positive or negative respectively). Significance analyses were set at ***p ≤ 0.0001, ** p ≤ 0.001, *p ≤ 0.05 vs control, and at § § § p ≤ 0.0001, § § p ≤ 0.001, § p ≤ 0.05 vs the 10 Gy WBI group. The regression and survival analysis were conducted using MATLAB Version: 8.2.0.701 (R2013b) and the graphical representations were generated using GraphPad 7.0 software (GraphPad, San Diego, CA).

Characterization of the NUDE mouse model: WBI-induced severe hematopoieticinjury is associated with dose-dependent small intestine damage.
In this part of the study using a model of severe injury-induced death, we determined the limiting doses of WBI that induce rupture of the gut barrier. Mice were subjected to decreasing lethal WBI doses between 15 and 10 Gy. Reduction in WBI doses was associated with an increase in mouse survival time (figure 1). Maximum mouse survival time was 7 days for 15 Gy, 8 days for 13 or 12 Gy and 9 days for 10 Gy. Moreover, the risk of instantaneous death after 10 Gy was reduced by a factor of 3.75 compared to 15 Gy (p≤0.001, Cox model) and by 2.75 compared to 13 Gy (p≤0.05, Cox model). No difference in the risk of instantaneous death was observed between mice subjected to 12 or 10 Gy.
Reducing doses also led to less weight loss, 5 days after irradiation (in supplementary data 1, p≤0.001 for all tested doses vs control mice). There was similar weight loss in mice receiving 15 or 13 Gy (in supplementary data 1, 28 and 30% loss vs control mice) and in mice receiving 12 or 10 Gy (in supplementary data 1, 20% loss vs control mice).
However, we observed significant differences in weight loss between 15 or 13 Gy vs 12 or 10 Gy (in supplementary data 1, for 15 Gy vs 12 and 10 Gy p≤0.001 and for 13 Gy vs 12 and 10 Gy p≤0.05 and p≤0.001, respectively). For all WBI doses tested, histological analysis showed similar severe damage in bone marrow that was indicative of myelosuppression. Transplantation of total bone marrow in whole body-irradiated NUDE mice did not prevent death (data not shown). Indeed, at all doses of WBI, death seemed to be triggered mainly by intestinal injury. Therefore, we measured crypt cell viability as the first criterion of intestinal injury. Three days after WBI, we showed a dose-dependent effect on the percentage of surviving crypts in the small intestine. Only 10% of the crypts were viable after the highest dose (15 Gy

MSC-derived small EVs/exosomes extend life of irradiated NUDE mice
MSC-derived small EVs/exosomes induced significant therapeutic efficacy as shown by their ability to delay 10 Gy WBI-induced death (figure 4, log-rank test p<0.0001). Five days after WBI when 50% of mice had died mostly from intestinal toxicity, 100% of mice treated with MSC-derived small EVs/exosomes were still alive. MSC-derived small EVs/exosomes delayed death at the lethal dose of 50% (LD50) in mice by 3.5 days compared to untreated WBI mice. Consistent with these observations, the risk of instantaneous death induced by 10 Gy WBI was reduced by a statistically significant 85% (Cox model hazard ratio=0.15, p≤0.0001). EVs/exosomes were able to limit WBI-induced disruption of the small intestinal barrier.

MSC-derived small EVs/exosomes stimulate the renewal of the small intestine and improve the regenerative process in irradiated NUDE mice
We first analyzed the time-dependent effect (1, 2 and 3 days after WBI) of MSC-derived small EVs/exosomes on the level of both apoptotic and proliferating cells in the small intestinal crypts as an index of the regenerative capacity of the epithelium. Villus height as an index of epithelial thickness and therefore of structural integrity was assessed to demonstrate treatment efficacy in epithelium rescue.

Apoptosis analysis (figure 6a):
The physiological level of apoptotic cells per crypt assessed by TUNEL assay in control mice was very low. The average value quantified was 1.50 ± 0.25% apoptotic cells per crypt. One day after 10 Gy WBI, we observed a significant 9-fold increase in apoptotic cells compared to the basal level (p≤0.0001). This increase was reduced on days 2 and 3, but remained significant at 6-and 3-fold higher than the basal level, respectively (p≤0.0001 both). Administration of MSC-derived small EVs/exosomes significantly reduced radiation-induced apoptosis of epithelial crypt cells 1 and 2 days post-exposure (3.7% in irradiated and small EV/exosome-treated mice vs 13.2% in irradiated mice, p≤0.0001, and 2.2% in irradiated and small EV/exosome-treated mice vs 8.4% in irradiated mice p≤0.0001, respectively). At 2 days, MSC-derived small EVs/exosomes provided a prompt return to the basal level of epithelial apoptotic cells (2.2% in irradiated and small EV/exosome-treated mice vs 1.5% in control mice, p=0.23).

Crypt cell proliferation analysis (figure 6b):
The estimated basal proliferation (proportion of Ki67-positive cells among the analyzed ones) was 27.9 ± 2.4% in control mice. One day and 2 days after WBI, this basal proliferation fell by approximately a third to 20.5 ± 3.80% (p≤0.001 vs control mice) and 19.0 ± 3.6% (p≤0.0001 vs control mice), respectively. Three days after WBI, proliferating

Structural analysis:
As shown in figure 7, the villus height measured in control mice was 223.2 ± 5.0 µm. Ten Gy WBI at 3 days led to partial epithelial atrophy, corresponding to a significant reduction of villus height to 133.5 ± 5.1 µm (p≤0.0001 vs control mice). Villus height in irradiated mice after administration of MSC-derived small EVs/exosomes was 159.8 ± 9.2 µm, corresponding to a significant 20.0% rise compared to the average value obtained in irradiated mice (p=0.016). This part of the study demonstrated the rapid action of MSCderived small EVs/exosomes in preventing loss of structural mass in the small intestine, possibly by increasing cellular proliferation and reducing apoptosis.

Discussion
The manifestation of ARS is multifactorial and involves several overlapping complex mechanisms. Therefore, the development of therapeutics for this syndrome is challenging.
Many studies have shown that MSC-derived small EVs have repair and regenerative effects on many injured organs like heart, kidney, liver, skin and also the intestine [16].
Therefore, we hypothesized that MSC small cellular vesicles with their pleiotropic potency would also be highly efficacious in the management of ARS, a complex injury. In accordance with this hypothesis, we provided the first proof of concept that MSC-derived small EVs are a promising therapeutic for mitigation of radiation-induced toxicity.
It has been previously demonstrated that MSC-derived small EVs with their complex cargo of proteins, RNA and lipids have the capacity to participate simultaneously in a wide spectrum of biochemical and cellular activities [17]. The benefit of the MSC-derived small EVs seems to be directly correlated with their ability to improve and alleviate the repair or injurious processes simultaneously as they occur during tissue repair and recovery. This capacity is critical in treating complex injuries and so is advantageous for ARS management. In support of this proposal, MSC-derived small EVs have been reported to have multiple activities, such as immunomodulation [18], anti-apoptosis and pro-survival [19], decrease of oxidative stress [20], promotion of angiogenesis and re-epithelization [9]. Moreover, several recent studies have reported that MSC-derived small EVs exert their therapeutic effects through the concomitant modulation of several processes, such as increased ATP synthesis, activation of survival kinase signaling and decrease of oxidative stress [20]. Furthermore, their effect on osteochondral defects appears through the activation of multiple pathways by enhancing proliferation, decreasing apoptosis and modulating immune activity using different components in their cargo [21].
The terms "MSC-derived small EVs" and "microvesicles" refer to two different EV types.
Isolation of different EV types by their biogenesis is currently not practical or possible because currently we lack specific biomarkers. Size and density of EVs are commonly used for their isolation, but cannot distinguish among the different EV types, whose biophysical parameters overlap. Therefore, all MSC-derived EV preparations described to date are probably heterogeneous mixtures of different EV types of unknown biogenesis. In this study, we established that at least a fraction of the EVs in our preparation is derived from the endosome and so these EVs are MSC-derived small EVs [26]. However, at least two other EV types have been reported [27]. Since the MSC-derived EVs in our preparation are between 50-200 nm (in supplementary data 4), the term "MSC-derived small EVs" in this paper is synonymous with ''MSC-derived exosomes" as per recent recommendations [28,29].
Here, we report that the administration of MSC-derived exosomes increases through several processes the length of survival of mice that developed multiorgan injuries after exposure to 10 Gy WBI. As GI syndrome is the critical limitation in combatting ARS effectively, we chose to focus on assessing the efficacy of MSC exosomes in rescuing radiation-induced GI mucosal barrier integrity. Barrier impairment could lead to lifethreatening sepsis. Since sepsis is a major cause of death in GI syndrome [30] and the GI mucosal barrier is highly sensitive to radiation, preservation of this barrier is a major therapeutic target [31]. Here we show that the effectiveness of MSC exosomes against radiation-induced intestinal toxicity is mediated by their ability to maintain intestinal barrier integrity by enhancing mucosal renewal and limiting intestinal permeability.
One of the manifestations of radiation-induced small intestinal toxicity is loss of the selfrenewal ability of the epithelium and the subsequent mucosal atrophy associated with dysregulation of the apoptosis/proliferation balance. This can lead to mucosal barrier alteration. In this study, we observed that mucosal atrophy in mice after exposure to 10 Gy WBI was minimized by MSC-derived exosomes. Increase in mucosal thickness is likely mediated by the ability of MSC-derived exosomes to protect epithelial cells from apoptosis and to promote their proliferation. Our results demonstrate that, after 10 Gy WBI, epithelial renewal occurs faster in mice treated with MSC-derived exosomes than in untreated mice. These results are consistent with a previous in vitro observation that irradiation enhances internalization of MSC-derived exosomes in epithelial cells leading to an increase of recipient cell viability [32]. The protection of other epithelial cell types by administration of MSC-derived exosomes has already been described for cutaneous wound healing [33], kidney toxicity rescue [34] and liver regeneration [35].
Another manifestation of radiation-induced GI toxicity is the enhancement of permeability in the GI mucosa after barrier disruption. Ten Gy WBI led to rapid and drastic increase in gut permeability which in our experiments may result from barrier impairment in both small intestinal and colonic mucosa. The colon is a part of the gut that is the primary source of pathogens and toxins, meaning that it is sensitive to stress-induced endotoxemia and bacteremia. Shukla et al showed that the colonic mucosal barrier is highly sensitive to radiation, more than the barrier formed by the small intestinal epithelium [31]. After 10 Gy WBI, although a critical epithelial cell mass is preserved in the small intestine, we observed a partial reduction and disruption of claudin-3, a transmembrane protein of tight junctions, which may reflect barrier dysfunction in this part of the gut in our model. MSC-derived exosomes prevent transient increase of radiation-induced gut mucosal permeability probably by maintaining tight junction functionality, as our results show for claudin-3. MSC-derived exosomes also preserved the gut barrier integrity in a model of necrotizing enterocolitis, an effect associated with their ability to reduce the incidence and severity of the enterocolitis [11]. In part, by reducing/preventing the first steps of radiation-induced intestinal barrier disruption and probably bacterial translocation and inflammatory response, MSC-derived exosomes might provide therapeutic benefits characterized by their ability to delay radiation-induced death. Our results on crypt cell apoptosis/proliferation and epithelial permeability of the irradiated/exosome-treated small intestine show that preservation of gut barrier integrity is one of the decisive parameters that need to be controlled to reduce radiation toxicity.
Nevertheless, the therapeutic benefit of MSC-derived exosomes could also be expected to involve partial protection of the HP system. Recent studies have demonstrated that MSC derived-EVs given after lethal WBI enhance long-term survival. This effect is associated with recovery of HP cells, particularly white blood cells, which probably occurs through MSC-EVs targeting hematopoietic stem cells [36]. Consistent with these studies, we also observed a reduced short-term WBI-induced myelosuppression which was evidenced by hemorrhage reduction and the presence of hematopoietic cells in bone marrow slices from MSC exosome-treated mice (in supplementary data 5). In order to optimize therapy for the management of ARS, the potential effects of MSC-derived exosomes on radiation-induced toxicity in the HP system should be thoroughly investigated and characterized.
The quality of ARS management also depends on the expediency of medical support provided to exposed victims. Schoefinius et al showed that MSC-derived EVs accelerated the kinetics of thrombocyte recovery after lethal WBI compared to MSC themselves [36].
In our study, we also observed that small intestinal mucosa self-renewed more rapidly in mice treated with MSC-derived exosomes compared to mice treated with MSC themselves (data not shown). We noted fast benefits of MSC-derived exosomes, as early as 24 h after WBI, which positions MSC-derived exosomes as a very good candidate for ARS management as they can act quickly on both the HP and GI systems. The datasets generated/analyzed during the current study are available.

Ethics approval and consent
All experiments were performed in compliance with the Guide for the Care and Use of