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), suggesting an inability of epithelium to repair and regenerate (figure 2a, p≤0.001 vs control mice). In support of this assumption, we also reported severe structural epithelial alterations, shown in figure 2b and in supplementary data 2a respectively by villus atrophy area (reduction of villus height, p≤0.001 vs control mice and crypt depth tendency vs control mice), and in supplementary data 2b by ulceration areas (confirmed by the reduction of crypt/villus density, p≤0.001 vs control mice). This was concomitant with a 2.5-fold increase in intestinal permeability (figure 3, 5.21 +/- 0.68 in control mice vs 13.51 +/- 2.9 in 15 Gy irradiated mice, p<0.001 vs control mice). Fifteen Gy WBI induced severe and irreversible small intestinal disorders and death at between 4 to 7 days.
At the lowest, 10 Gy dose, WBI did not affect crypt viability (figure 2a). Nevertheless, we observed transient (only at 3 days, data not shown at 5 days) disruption of the intestinal barrier as shown by significant villus atrophy (figure 2b, 223.2 +/- 6.6 µm in control mice vs 134.8 +/- 3.86 µm in 10 Gy-irradiated mice, p<0.001 vs controls) and a 1.9-fold increase of intestinal permeability (figure 3, 5.22 +/- 0.68 mg/mL 4 kDa FITC-labeled dextran in control mice vs 9.68 +/-1.63 mg/mL 4 kDa FITC-labeled dextran in 10 Gy-irradiated mice, p<0.05 vs control mice). Ten Gy WBI induced short-term small intestinal disorders that although reversible led to death at between 4 to 9 days.
Significant therapeutic benefit of MSC-derived small EVs/exosomes in irradiated NUDE mice developing hematopoietic and intestinal injury overlap
Based on the dose-effect observations, we opted for a dose of 10 Gy WBI for the rest of the experiments. This dose of irradiation generated ARS with severe small intestinal injury, but preserved sufficient surviving crypts to support therapeutic intervention to repair and regenerate the crypts, and provides a first proof of concept for the therapeutic efficacy of MSC-derived small EVs/exosomes in ARS management.
Based on assays evaluating dose-dependent effects of MSC-derived small EVs/exosomes (in supplementary data 3), we chose to administer a total of 600 µg of MSC-derived small EVs/exosomes in three injections of 200 µg, at 6, 24 and 48 h after WBI.
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).
MSC-derived small EVs/exosomes reduce gut barrier dysfunction after WBI in NUDE mice.
Measurement of intestinal permeability and immunostaining of some transmembrane proteins of tight junctions were used as indexes of gut barrier function. We demonstrated that 10 Gy WBI induced a transient 1.8-fold enhancement of gut permeability (figure 5a, p≤0.0001). Three days post-WBI, MSC-derived small EVs/exosomes prevented radiation-induced increased gut permeability as shown in figure 5a, with no significant difference in plasma fluorescein dextran concentrations between irradiated and small EV/exosome-treated and control mice (figure 5a, 5.68 mg/mL and 4.88 mg/mL, respectively). Claudin-3 plays an important role in the safeguarding of gut barrier function. It is a transmembrane protein of tight junctions localized predominantly at the intercellular junctions of the gut epithelium (figure 5b 1,4). Among a set of other proteins of tight junctions such as ZO-1 and occludin (data not shown), only claudin-3 immunostaining in the small intestine decreased drastically 3 days after 10 Gy WBI (figure 5b 2,5). Reduction of junctional claudin-3 level after WBI could in part explain enhancement of intestinal permeability. Treatment with MSC-derived small EVs/exosomes maintained a significant level of claudin-3 immunostaining despite a reduction in expression compared to the basal level. Importantly, claudin-3 remained localized at the membrane junction, signifying preservation of tight junctions (figure 5b 3,6). In conclusion, MSC-derived small 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 crypt cells returned to the basal level (28.9% in irradiated mice vs 27.9% in control mice, p=0.49). These results suggested that 3 days after WBI, healing of the small intestinal through crypt cell proliferation was initiated.
MSC-derived small EVs/exosomes promoted a 1.4-fold increase in proliferating cells compared to the basal level at 1 day (39.3% in irradiated and small EV/exosome-treated mice vs 27.9% in control mice, p=0.0004) and 1.2-fold at 2 days with borderline significance (34.4% in irradiated and small EV/exosome-treated mice vs 27.9% in control mice, p=0.07). This MSC small EV/exosome-induced proliferation process returned to the basal level 3 days after WBI (25.1% in irradiated and small EV/exosome-treated mice vs 27.9% in control mice, p=0.13). Therefore, MSC-derived small EVs/exosomes induce a rapid but transient acceleration in crypt cell proliferation, and possibly promote epithelial renewal.
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 MSC-derived small EVs/exosomes in preventing loss of structural mass in the small intestine, possibly by increasing cellular proliferation and reducing apoptosis.