The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice

Background

The intestine is particularly sensitive to moderate-high radiation dose and the development of gastrointestinal syndrome (GIS) leads to the rapid loss of intestinal mucosal integrity, resulting in bacterial infiltration, sepsis that comprise patient survival. There is an urgent need for effective and rapid therapeutic countermeasures. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, anti-inflammatory and regenerative properties. We studied the therapeutic impact of SVF and its action on the intestinal stem cell compartment.

Methods

Mice exposed to the abdominal radiation (18 Gy) received a single intravenous injection of stromal vascular fraction (SVF) (2.5 × 10⁶ cells), obtained by enzymatic digestion of inguinal fat tissue, on the day of irradiation. Mortality was evaluated as well as intestinal regeneration by histological analyses and absorption function.

Results

The SVF treatment limited the weight loss of the mice and inhibited the intestinal permeability and mortality after abdominal irradiation. Histological analyses showed that SVF treatment stimulated the regeneration of the epithelium by promoting numerous enlarged hyperproliferative zones. SVF restored CD24⁺/lysozyme⁻ and Paneth cell populations in the ISC compartment with the presence of Paneth Ki67⁺ cells. SVF has an anti-inflammatory effect by repressing pro-inflammatory cytokines, increasing M2 macrophages in the ileum and anti-inflammatory monocyte subtypes CD11b⁺Ly6c^lowCX3CR1^high in the spleen.

Conclusions

Through the pleiotropic effects that contribute to limiting radiation-induced lethality, SVF opens up attractive prospects for the treatment of emergency GIS.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-021-02373-y.

Keywords: Stromal vascular fraction, Intestine, Irradiation, Regeneration

Mice

Male C57BL/6JRj mice were purchased from Janvier (Le Genest Saint Isle, France) and the animals were housed in the IRSN animal facilities, which are accredited by the French Ministry of Agriculture for performing experiments on rodents. The animal experiments were performed in compliance with French and European regulations on the protection of animals used for scientific purposes (EC Directive 2010/63/EU and French Decree 2013–118). All experiments were approved by the Ethics Committee #81 and authorised by the French Ministry of Research (under the reference APAFIS#10434-2017062814437026-v1). In this study, the experimental protocol was in line with the standard support treatment recommended for the patient presenting an acute radiation syndrome [16]. Thus, the antibiotic Avemix (8g/L) will be associated with all treatments and during the experimentation.

Irradiation procedure

Eight-week-old C57BL/6JRj mice were irradiated under anaesthesia (with a continuous flow 1.5% isoflurane in oxygen) on a medical linear accelerator (Elekta synergy®) delivering 4MVp X-rays (mean photon energy about 1.3 MeV). Reference dosimetry measurements were performed using a 0.125 cc cylindrical ionisation chamber calibrated in dose to water in a mouse equivalent tissue phantom placed on a plexiglass support. A dose rate about 2.5 Gy/min in dose to water was used. A localised 2-cm larger abdominal irradiation window containing intestine was chosen to limit the upper thorax, head and neck and lower and upper extremity exposure. The mice were exposed to 16, 18, 20 and 22 Gy. The dose of 18 Gy was further selected as the sublethal optimal irradiation dose for SVF-related effects. The uncertainty of the dose rate measurement was about 5% at k = 2.

Isolation of SVF

Stromal vascular fraction (SVF) cells were isolated from inguinal fat pad adipose tissue samples of 12-week-old C57BL/6JRj mouse donors. From the modified methods [17], the fat pads were excised, finely cut and incubated in a digestion medium containing 0.1% type I collagenase (Sigma-Aldrich, France) and 1% penicillin-streptomycin in MEM-α milieu by gentle shaking for 20 min at 37°C. The samples were briefly mechanically disrupted using the gentleMACS Dissociator (Miltenyi Biotec) and then digested again in MEM-α 0.1% type I collagenase medium followed by mechanical disruption using the gentleMACS Dissociator. To remove mature adipocytes and undigested tissue, the cell suspension was filtered sequentially through 100- and 70-μm cell strainers and centrifuged (400g, 10 min) to spin down stromal vascular fraction cell pellets. The pellets were resuspended in phosphate-buffered saline (PBS) for i.v. injection.

Histology

Intestinal permeability in vivo

In vivo intestinal permeability measurement was assessed at 7 days following irradiation, using 4000 Da FITC-dextran (Sigma-Aldrich, France). In short, FITC-dextran (0.60 mg/g body weight) was administrated by gavage and 4 h after the mice were euthanised and plasma obtained by cardiac puncture. Standard curves were obtained by diluting the FITC-dextran in the PBS-diluted plasma to determine the serum levels of FITC-dextran in the different treatment groups. The FITC concentration in the plasma was then measured with a microplate Luminometer (Mithras LB940, Berthold) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Splenic cell isolation

To isolate the splenic cells, the spleen was smashed with a syringe plunger and the cells were collected in PBS buffer followed by passage through 40-μm cell strainers and centrifuged (400×g, 10 min). The red blood cells were lysed with ACK lysis buffer (Thermo Fisher Scientific, France).

Flow cytometric analysis

Flow cytometry was performed using the BD FACSCanto II and analysed with FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded through Fixable Viability Dye eFluor staining (eBioscience). Nonspecific antibody binding was blocked with an anti-CD16/32 (Mouse Fcγ block clone 2.4G2). The cells were incubated for 30 min in 100 μl PBS with conjugated antibodies (BD Biosciences, France): anti CD45 (V500 conjugated; clone 30-F11), CD11b (BV421 conjugated; clone M1/70), anti CD3 (FITC conjugated; clone 145-2C11), anti CD45R/B220 (PE-Cy7 conjugated; clone RA3-6B2), anti Ly6C (FITC conjugated; clone AL-21), anti CD31 (PE conjugated; clone MEC13.3), anti CD34 (Alexa Fluor 647 conjugated; clone RAM34), anti CD146 (FITC conjugated; clone ME-9F1), anti CD29 (PE conjugated; clone HM B1.1), anti Sca-1 (PE-Cy7 conjugated; clone D7), anti CD44 (PE-Cy5.5 conjugated; clone IM7), anti CD105 (Alexa Fluor 647 conjugated; clone MJ7/18), anti SSEA (BV421 conjugated; clone MC631), anti CX3CR1 from Biolegend (France) (PE-Cy7 conjugated; clone SA011F11), anti CCR2 (Alexa Fluor 647 conjugated; clone SA203G11) and CD90 from southern Biotech (FITC conjugated; clone SA203G11). Isotype-matched antibodies (BD Biosciences) were used for control staining. All antibodies were used at 1:100 dilution. The concentration of cell suspensions was adjusted to 1 × 10⁶ cells per 100 μl.

Spreading assay and immunostaining

Spleen mononuclear cells MNCs isolated from all the experimental groups were resuspended in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin and seeded on round 12-mm diameter coverslips in a 24-well plate coated with 5μg/ml recombinant mouse VCAM-1 (R&D Systems) for 2 h at 37°C. After two washes with PBS, the cells were fixed with 4% paraformaldehyde for 10 min at +4°C. The cells were rinsed three times with PBS and then incubated in PBS/0.1% Triton X-100 (Sigma-Aldrich) for 10 min. After three rinses with PBS and incubation with 2% BSA to block nonspecific staining, the cells were stained with 0.165 μM Alexa Fluor 546 Phalloidin (Thermo Fisher Scientific, France) for 60 min at 37°C. The primary antibody anti-CX3CR1 (Abcam) was used for immunostaining. After the incubation, slides were washed with PBS-T and probed with fluorescence-conjugated secondary antibodies for 1 h at RT. The cell nuclei were counterstained by Vectashield mounting medium with DAPI (Vector). Cells with flat morphology or lamellipodia were positively scored for spreading.

Monocyte/macrophage polarisation

The spleen cells were washed three times and cultured in a 12-well tissue culture in RPMI-1640 supplemented with 10% FBS and antibiotics (penicillin/streptomycin, Invitrogen) and 100 ng/ml of M-CSF (R&D Systems, France) at 37°C in a humidified atmosphere containing 5% CO₂ for 7 days to induce full macrophage differentiation and maturation. Macrophage polarisation was obtained by removing the culture medium and culturing cells for an additional 24 h in RPMI-1640 supplemented with 10% FBS and antibiotics and 100 ng/ml LPS (derived from E. coli 0111:B4, Invivogen) plus 20 ng/ml IFN-γ (for M1 polarisation, M(LPS-IFN-γ)) or 20 ng/ml IL-4 (for M2 polarisation, M(IL-4)) (R&D Systems). The cells were harvested for RT-qPCR analysis.

Real-time PCR analysis

Total RNA was extracted from the ileum and spleen cells with the RNeasy Mini kit (Qiagen), and cDNA was prepared with the SuperScript RT Reagent Kit (Applied Biosystems). Real-time PCR was performed on an ABI Prism 7900 Sequence Detection System. All Taqman primers and probes came from Life Technologies (France) (Supplemental table). The data were analysed using the 2^−ΔΔCt method, with normalisation to the Ct of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene.

Statistics

The data are expressed as the mean ± SEM. We used one-way or two-way analyses of variance (ANOVA) and then a Bonferroni post-test to determine the significance of the differences. p values less than 0.05 were considered statistically significant.

Characteristics of radiation-induced GIS after abdominal irradiation

In order to limit bone marrow exposure, abdominal irradiation was performed [19]. The current standard endpoint for the study of acute GIS is animal lethality within 10 days of radiation exposure (LD50/10) (LD: lethal dose for 50% at 10 days). In this way, to define the sublethal dose, we administered an increasing dose up to 16 to 22 Gy to obtain a GIS characterised by a strong and rapid weight loss and death between 7 and 10 days. Exposure to 16, 18, 20 and 22 Gy was lethal in 0, 70, 75 and 100% of the mice within 14 days with a DL0/10, DL65/10, DL75/10 and DL100/10, respectively (Fig. 1a). Lethality was strongly correlated with weight loss, with about a 30% loss observed 7 days after irradiation in groups of animals irradiated at 18 and 20 Gy (Fig. 1b). The dose that induces a GIS was established on the basis of the number of regenerating crypts forming microcolonies 3.5 days after irradiation. It is a marker of intestinal integrity objectified by the presence of 10% of crypts per intestinal section with at least 8–10 cells per crypts and is correlated with survival. This time corresponds to the maximum expected difference between restoration and loss of crypts/villi. At day 3.5, histological analysis (HES) showed a change in tissue architecture observed from the 18-Gy dose with drastic reductions in villi length, hypoplasia of crypts and destruction of epithelial architecture (Fig. 1c). The analysis of the density of regenerating intestinal crypts was 66, 17, 8 and 1% at doses of 16, 18, 20 and 22 Gy, respectively, compared with non-irradiated mice (Fig. 1d). In parallel, representative images of TUNEL-positive cells characterising the irreversible cell death showed an increase in the frequency of TUNEL-positive cells in the mucosa with an escalating dose from 16 to 22 Gy compared with non-irradiated mice (Fig. 1e). Taken together, from the results of survival experiments and the histological analysis, a dose of 18 Gy was the sublethal dose inducing a GIS.

SVF improves survival following a sublethal dose of irradiation and mitigates GIS

SVF was prepared from animal donors and characterised by flow cytometry. The gating strategy and subpopulation determination are shown in supplemental figure (S1). The viability of cells was >95%. Expression of mesenchymal, vascular, pericytic and haematopoietic markers as well their combinations showed that the percentage of the mesenchymal marker was >60% for CD90, CD44 and CD29; >50% for Sca-1; and >10% for CD105 and 0.3% for SSEA3. The SVF also contained haematopoietic cells with >60% CD45⁺CD3⁺, 2% CD45⁺CD11b⁺ and >20% CD45⁺CD11b⁻ B220⁺. Interestingly, SVF also contained a small amount (<1%) of endothelial mature cell (CD31⁺CD34⁻CD146⁺) and endothelial progenitor (CD31⁺CD34⁺CD146⁺).

Weight loss monitoring was first used to establish the concentration of SVF cells to be injected. At a concentration of 0.5 × 10⁶, SVF cells had no effect on weight loss on day 7 (about 25%) compared with the irradiated group. Seven days after irradiation, a loss of only 10% was observed in the groups of animals injected with 1 and 2.5 × 10⁶ (Fig. 2a). The SVF concentration of 2.5 × 10⁶ cells will be injected in future experiments. Treatment with SVF of 2.5 × 10⁶ cells limits weight loss and allows them to regain weight faster than the irradiated mice (Fig. 2b). In terms of mortality, while 65% of the untreated-irradiated mice died within 10 days, the SVF treatment saved 80% of mice from lethality (Fig. 2c).

High irradiation doses induce apoptosis of the crypt epithelial cells, resulting in a decrease in regenerating crypt colonies at 3.5 days and ultimately villi denudation by day 7 post-irradiation. The number of regenerative crypts microcolonies 3.5 days after irradiation was significantly increased in mice treated with SVF (44.5 ± 5.3/μm; p<0.001) compared with the irradiated mice (17.5 ± 2.2/μm) (Fig. 2d), indicating an intestinal regenerative response. Moreover, mice treated with SVF had a significant decrease in the number of Tunel⁺ cells (5-fold; p<0.001), especially near the position of the crypt cells 7 days after irradiation, compared with irradiated mice; the number and the localisation (far away from the crypt cells) of Tunel⁺ cells were similar to the non-irradiated mice (Fig. 2e).

Disruption of the intestine structural integrity alters its barrier function, making it permeable to luminal content. Dextran is unable to cross the intestinal epithelium when it is intact [20]. Intestinal permeability was quantified 7 days after irradiation with FITC-labelled dextran in the blood 4 h after gavage. Abdominal irradiation significantly increased intestinal permeability (17.5 ± 2.6μg/ml versus 1.4 ± 0.1μg/ml for the control; p<0.001). Treatment with SVF restores the impermeability (3.2±1.5 μg/ml) compared to the non-irradiated mice (Fig. 2f). The permeability was characterised by tight junction loss. Indeed, immunostaining of the tight junction protein zonula occludens-1 (ZO-1) showed that ZO-1 in the control group was expressed in the cytomembrane of epithelial cells along the villi. Seven days post-irradiation, the expression of ZO-1 was considerably reduced when SVF treatment restored its expression (Fig. 2g). These results indicate that SVF treatment induced a functional restoration of intestinal epithelial integrity.

SVF increases intestinal regeneration

The maintenance of the epithelial barrier is also mediated by epithelial cell adhesion molecules such as EpCam, which also plays a role in cell proliferation, migration and differentiation [21]. In non-irradiated mice, EpCam expression is observed at the crypts and villi and localised on the plasma membrane. Seven days after irradiation, a decrease in EpCam expression was observed as well as a disturbance in its localisation, with a mislocalisation in cytoplasm (Fig. 3a). SVF treatment is associated with a recovery of EpCam expression both in the stem cell compartment and on the surface of the mucosa.

Decreased EpCam expression may also impact stem cell proliferation/differentiation [21]. In the control group, Ki-67 immunostaining showed that the majority of proliferating cells were located in the transient amplification cell compartment and did not reside in the base of the crypt. Irradiation leads to a decrease in the number of Ki-67⁺ cells in the ISC compartment. SVF treatment results in a significant increase in the number of Ki-67-positive cells in the ISC compartment, the transient amplification cell compartment and the extending regenerative epithelium, resulting in an enlarged and expanded hyperproliferative cluster area (Fig. 3b).

Mesenchymal-derived factors are essential for maintaining intestinal epithelial stem cell homeostasis. In particular, the double-positive stromal cells Gp38⁺CD34⁺ in the pericryptal localisation implicated in the production of niche factors are localised near the crypts in close contact with Lgr5⁺ stem cells [22]. The double immunostaining Gp38/CD34 showed that irradiation negatively impacts these stromal niches 7 days after irradiation and that the treatment by SVF enables them to be restored (Fig. 3c).

SVF stimulates the restoration of the IECS

CD24 is a marker of the lower crypt IECS [23] and therefore used to identify LGR5⁺ and Paneth cells. In non-irradiated mice, we observed the presence of 2.5 CD24⁺ lysozyme⁻ cells per crypt, the corner-shaped cell population between Paneth cells (Fig. 4a). An 80% loss of these cells was observed at day 7 after irradiation and SVF treatment restored this cell population. Paneth cells contribute to intestinal homeostasis by synthesising peptides and antimicrobial proteins such as lysozyme and stem cell niche factors [2] and further promoting crypt base formation [24]. By using lysozyme staining to identify Paneth cells, we observed that the number of lysozyme⁺ cells decreased significantly on day 7 after irradiation (p<0.001) compared with the control mice (Fig. 4b). In the control mice, the lysozyme was normally efficiently packaged in well-defined secretory granules in the Paneth cells. In the irradiated mice, the Paneth cells exhibited altered lysozyme allocation patterns, with the majority showing reduced (lowly granular) or diffuse lysozyme staining. After SVF treatment, the number of Paneth cells per crypt increased compared with the irradiated mice (p<0.001), which was also higher than in the control mice. Interestingly, we also observed that the number of lysozyme⁺ cells in the “upper crypt” increased in mice treated with SVF, suggesting an aberrant ISC niche architecture. The co-localisation of MUC2 (goblet cell marker) and lysozyme (Paneth cell marker) staining characterised an “intermediate” goblet/Paneth cells and were definited as immature or bipotential progenitor cells [25]. Double immunostaining showed that the presence of Paneth cells at the “upper crypt” was co-staining with Muc2 after SVF treatment, indicating the presence of the “intermediate” cells (Fig. 4c). To assess whether the observed proliferative cluster was related to IECS, we performed a Ki67/CD24 co-staining. In the control mice, rare Ki67⁺/CD24⁺ cells were present in the crypts, with the majority of proliferating cells being located in the transit amplification compartment (TA) and not in the base of the crypt (Fig. 5a). On the other hand, in the proliferative cluster induced by SVF treatment, the CD24⁺ cells were Ki67⁺; an expansion of the TA zone was also observed. In addition, to determine which population was responsible for increased crypt cell numbers, a double Ki67/lysozyme staining was carried out and showed the presence of Ki67⁺/lysozyme⁻ and Ki67⁺/lysozyme⁺ cells 7 days after irradiation in the irradiated mice treated with SVF (Fig. 5b), indicating the proliferation of ISC and in particular the Paneth cells. It has recently been reported that hyperproliferative crypts show a high expression of HSP60, the mitochondrial molecular chaperone involved in the control of ISC homeostasis, and that HSP60-deficient crypts display a loss of stemness and cell proliferation, resulting in an aberrant Paneth cell phenotype [26]. Here, we observed an increase in the HSP60⁺ cells in the regeneration zone after treatment of SVF (Fig. 5c), indicating the absence of mitochondrial impairment. Together, these results showed that treatment with SVF restored the CD24⁺/lysozyme⁻ and Paneth cell populations and increased the proliferation of the TA compartment (Ki67⁺/CD24⁺) as well as the proliferation of Paneth cells (lysosyme⁺/Ki67⁺).

SVF limits the inflammatory process by shifting the monocyte-macrophage phenotype

Macrophages have a central role in the epithelial barrier and in the maintenance of mucosal homeostasis. The spleen is the source of the main subsets of macrophage-generating monocytes [27, 28], and we therefore evaluated the percentage of pro-inflammatory and anti-inflammatory monocytes in the spleen 7 days after irradiation and the impact of SVF treatment on the subsets of splenic monocytes. Firstly, the migration potential of splenic monocytes was assessed using the spreading assay performed on a rmVCAM-1 matrix at day 7 after irradiation with and without SVF treatment. Unspread cells were defined as round cells, while spread cells were defined as cells with extended pseudopodia. The percentage of cells adopting the spreading morphology was quantified using fluorescein-conjugated phalloidin, which binds to filamentous actin (Fig. 6a). In the non-irradiated mice, actin filament staining was weak in the splenic monocytes and significantly increased in the irradiated mice (2.7-fold compared with the control; p<0.001). Treatment with SVF significantly reduced the intensity of phalloidin staining to 42% compared with the irradiated mice. This is associated with morphological changes characterised by a normalisation of cell diameter, suggesting an impact on the pro-inflammatory phenotype of these cells [29]. Flow cytometry analysis of pro-inflammatory and anti-inflammatory monocytes subsets showed a 2.5-fold (p<0.01) increase in the percentage of Ly6c^high pro-inflammatory monocytes defined as CD11b⁺Ly6c^highCCR2^high in the irradiated mice compared with the control mice (Fig. 6b). Treatment with SVF did not reduce the percentage of Ly6c^high monocytes. On the contrary, the anti-inflammatory monocytes subset CD11b⁺Ly6c^lowCX3CR1^high of mice treated with SVF showed a 2-fold (p<0.001) higher percentage of anti-inflammatory Ly6c^low monocytes than the irradiated mice, which was confirmed by CX3CR1 immunostaining of the splenic monocytes (p<0.001) (Fig. 6c). In addition, the splenic monocytes could differentiate into pro-inflammatory M1 or anti-inflammatory M2 macrophages and contribute to tissue repair [30]. To address this issue, macrophages generated by 7 days of culture with M-CSF were stimulated with LPS-IFΝγ (M1 conversion) or IL-4 (M2 conversion) and the level of mRNA associated with macrophage polarity was analysed by real-time PCR. Our results showed that irradiation stimulated the expression of genes linked to the M1 marker (i.e., iNos, MMP9) without any effect on the expression of genes linked to the M2 marker (Fig. 6d). SVF treatment, on the other hand, reduced the mRNA levels of M1 (iNos, MMP9) markers and increased the mRNA levels of M2 markers (CX3CR1, CD206, Arg1, IL-10). Interestingly, the SVF treatment limited the levels of mRNA coding for excessive remodeling markers such as TGF-β 1 or TNC.

In the ileum, the double CD68/CD206 staining showed the absence of CD206⁺ cells in the irradiated mice and the SVF treatment led to an increase in CD68⁺/CD206⁺ cells (Fig. 7a). This anti-inflammatory effect in the intestine by the SVF treatment is supported by real-time PCR results with a significant suppression of mRNA levels coding for IL1-1β and IL-6 compared with the irradiated mice (p<0.001) (Fig. 7b). Taken together, these results suggest that SVF treatment changes the polarity of monocyte/macrophage to an anti-inflammatory phenotype, which may contribute to improving the GIS.

Ethics approval and consent to participate

All experiment procedures in this study were approved and performed in accordance with the guidelines of the Animal Ethics Committee from the French Ministry of Agriculture (EC Directive 2010/63/EU and French Decret 2013–118).

Consent for publication

Not applicable in this section.

Competing interests

The authors declare that they have no competing interests.

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Supplementary Materials

Data Availability Statement

All relevant data and material to reproduce the findings are available in the manuscript.