In the present study, we evaluated the therapeutic potential and molecular mechanisms of faecal microbiota transplantation (FMT) in a rodent model of lethal irradiation and demonstrated that transplantation of enteric microbes from healthy mice remarkably mitigated radiation‐induced GI syndrome in a sex‐dependent fashion. More importantly, FMT retained the mRNA and long non‐coding RNA (lncRNA) expression profiles of the small intestine. Thus, our findings provide new insights into the function and underlying protective mechanism of intestinal microbiota transplant in the context of radiation‐induced toxicity in a preclinical experimental setting.

The GI tract has the unique property of harbouring numerous microbes within the lumen, most of which are bacteria that have co‐evolved with the host in a mutualistic relationship (Kamada et al , 2013 ). Recently, investigations focusing on gut microbiota have experienced a renaissance, and growing evidence supports a pivotal role of intestinal microbes as key regulatory elements in their host's physiologic and pathologic status (Ostaff et al , 2013 ). For example, intestinal microbes govern metabolic function and energy balance, and flora disequilibrium is deemed to contribute to the development of numerous metabolic diseases (Amar et al , 2011 ; Nieuwdorp et al , 2014 ). Intestinal microbes are continuously shaping the development of their host's immune system, directly modulating the innate and adaptive immune responses (Sommer & Backhed, 2013 ; Barroso‐Batista et al , 2015 ). Epidemiological studies reveal that the intestinal microbiome engages in the pathogenesis of inflammatory bowel disease (IBD) with characteristic shifts in the composition of the intestinal microbiota, reinforcing the view that IBD results from altered interactions between intestinal microbes and the mucosal immune system (Kostic et al , 2014 ). Germ‐free mice and faecal transplant research demonstrate that changes in the microbiota are necessary and sufficient for both low‐grade inflammation and metabolic syndrome (Chassaing et al , 2015 ). Moreover, marked shifts in bacterial communities in the gut are inextricably intertwined with the development of diet‐associated cancer (Schulz et al , 2014 ; Feng et al , 2015 ; Gorjifard & Goldszmid, 2015 ), suggesting that enteral bacteria might be used as diagnostic biomarkers for many cancers. However, the relationship between enteric bacteria and the radiosensitivity of hosts is unknown; moreover, whether intestinal microbes can be used to mitigate radiation‐induced injury remains undocumented.

Radiation exposure in a mass casualty setting is a serious military and public health concern (Taniguchi et al , 2014 ). Exposure to a high dose of irradiation in a short time is associated with bone marrow toxicity (haematopoietic syndrome) and gastrointestinal (GI) toxicity (GI syndrome), which are collectively known as acute radiation syndrome (ARS; Kirsch et al , 2010 ; Leibowitz et al , 2014 ). ARS may facilitate an intractable pathologic process and even cause eventual death (Lee et al , 2014 ). In addition, mounting clinical evidence has shown the bone marrow and small intestine epithelium to be the major sites of injury during radiation therapy, owing to their higher sensitivity to ionizing radiation (Ciorba et al , 2012 ). Radiation‐mediated toxicity, especially radiation‐induced gastrointestinal injury, is a medical problem that urgently needs effective therapy.

Angiogenesis also shows a positive relationship with tumour development. Radiotherapy is one of the most successful and widely used cancer therapies (Svensson et al , 2006 ). To investigate whether FMT can be used to improve prognosis in tumour radiotherapy, we further examined whether FMT accelerated tumour cell proliferation in vivo . Accordingly, C57BL/6 mice were injected with HT29 (or A549) tumour cells subcutaneously (Fig EV5 A and B) and treated with faecal microorganisms for 10 days. We found that gut microbe treatment did not significantly change the animals' body weight (Fig EV5 C–F) and the volume and weight of the tumours in animals receiving exogenous cancer cells (Fig 7 C–F), suggesting that intestinal microbiota transplant might be employed as a radioprotector to improve prognosis in cancer radiotherapy. Together, our data demonstrate that FMT improves angiogenesis without facilitating tumour growth.

Angiogenesis has been regarded as a protective effector against radiation‐induced toxicity. Thus, we further investigated whether intestinal microbiota transplantation improved angiogenesis in the system. Intriguingly, immunohistochemical (IHC) staining revealed that the expression of F8, a marker for angiogenesis, was elevated in gut microbe‐treated mice compared with that of controls (Fig 7 A). qPCR assays further validated that gut microbe transplant up‐regulated the expression level of Vegf mRNA in small intestine (Fig 7 B), suggesting that FMT accelerates angiogenesis in the small intestine of irradiated male and female mice.

To further decipher the protective mechanism of FMT, we performed gene ontology (GO) analysis using small intestine tissues from saline and sex‐matched FMT treatment groups. Interestingly, compared with the saline treatment group, the top 30 terms exhibiting significant expression alterations were mostly involved in innate and adaptive immunity in male mice treated with sex‐matched FMT (Fig 6 A). However, sex‐matched FMT altered the expression of genes involved in metabolism in irradiated female mice, including genes required for cellular lipid catabolic processes and fatty acid beta‐oxidation (Fig 6 B), suggesting that different sex‐specific intestinal microbiota dictates their divergent physiologic status. In addition, we assessed the mRNA expression profile of genes involved in immunity and metabolism by cluster analysis and found remarkable changes in the immune and metabolic gene expression profiles between the gut microbe‐treated experimental group and the saline‐treated group (Fig 6 C–F), indicating that the hosts are responsive to intestinal microbiota treatment. We also compared the gene expression profile of small intestine tissues from irradiated mice between saline treatment and sex‐mismatched (or sex‐mixed) groups. GO analysis revealed that sex‐mismatched (or sex‐mixed) FMT‐enriched top 30 terms were quite different from those altered by sex‐matched FMT in male ( Appendix Fig S2A–D ) and female mice ( Appendix Fig S3A–D ), which further validated that the effect of sex‐specific intestinal microbiota on hosts was different. Given that lncRNAs have been reported to have an important role in immunity and metabolism modulation, we examined the lncRNA expression patterns related to the mRNAs in our study and observed that lncRNA expression was retained in sex‐matched FMT‐treated mice ( Appendix Figs S4–S7 ), indicating that the intestinal microbiota transplant affects the host's mRNA and lncRNA expression profiles to mitigate radiation‐induced toxicity.

Principal component and β‐diversity analysis were used to measure the shift of intestinal bacterial composition structure in sex‐mismatched FMT (C) and sex‐mixed FMT (D) female mice after irradiation at days 5 and 10, n = 4. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

Principal component and β‐diversity analysis were used to measure the shift of intestinal bacterial composition profile in sex‐mismatched FMT (A) and sex‐mixed FMT (B) male mice after irradiation at days 5 and 10, n = 4. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

Principal component and β‐diversity analysis were used to measure the shift of the intestinal bacterial composition structure in saline‐treated (C) and sex‐matched FMT (D) female mice after irradiation at days 5 and 10, n = 4. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

Principal component and β‐diversity analysis were used to measure the shift of the intestinal bacterial composition profile in saline‐treated (A) and sex‐matched FMT (B) male mice after irradiation at days 5 and 10, n = 4. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

Next, principal component analysis (PCA) was used to further determine the role of FMT in changing the intestinal bacterial flora profile. As shown in Fig 5 A, the gut bacterial composition profile substantially changed after radiation exposure at day 10 in male mice and was inhibited by gut microbial therapy (Figs 5 B and EV4 A and B). As expected, irradiation exposure shaped the intestinal bacterial composition of female mice, and microbial therapy altered the composition of faecal microbial flora in these animals (Figs 5 C and D, and EV4 C and D). Specifically, irradiation exposure caused a down‐regulation of the relative abundance of Bacteroidetes (or Firmicutes) at the phylum level in male mice (or in female mice) at day 10, but FMT reversed this down‐regulation (Figs 5 E–H and EV4 E–H). Together, our observation demonstrates that gut microbiota treatment preserves the gut bacterial composition in both male and female mice after exposure to radiation.

To elucidate the mechanism of FMT mitigating radiation‐induced toxicity, we performed 16S rRNA sequencing to analyse the bacterial taxonomic composition in faecal microbiota‐treated and saline‐treated animals. Irradiation caused persistent dysbiosis in the gut bacterial ecosystem at days 5 and 10, and FMT effectively erased these changes; in male mice, for example, irradiation continually decreased the frequency of Bacteroides (or Lactobacillus ) at the genus level (Fig 4 A), but male + TBI treatment increased (or stabilized) the frequency of Bacteroides (or Lactobacillus ) at day 5 (Fig 4 B). In contrast, female + TBI treatment led to a reduction in Bacteroides and a bloom in Lactobacillus at day 5, whereas female/male + TBI treatment led to opposite alterations in the flora (Fig EV3 A and B). At day 10, the frequency of Bacteroides and Lactobacillus dropped in all gut microbe‐treated male mice (Figs 4 A and B, and EV3 A and B). Moreover, in the saline and female treatment groups, the frequency of Prevotella at the genus level decreased progressively (Figs 4 C and EV3 C), whereas the frequency of Prevotella was increased in male and female/male treatment cohorts (Figs 4 D and EV3 D). In female mice, the frequency of Bacteroides was elevated at day 5 and then declined to a lower level at day 10 in almost all animals (Figs 4 E and F, and EV3 E and F). Notably, female + TBI treatment recovered the frequency of Bacteroides to the level of controls at day 10 (Fig 4 F). In addition, at day 10, the saline, male and female/male treatment groups showed a reduction in the Prevotella frequency at the genus level (Figs 4 G and EV3 G and H), but the female treatment group exhibited the reverse outcome (Fig 4 H). Thus, our observation indicates that FMT has a profound influence on the radiation‐altered gut bacterial composition.

On the basis of the aforementioned observations, we further untangled the impact of FMT on GI function and integrity in animals receiving faecal microbiota transferred from the same gender after irradiation exposure. Day 21 after 6.5 Gy TBI, haematoxylin and eosin (H&E) staining revealed a dramatic decrease in the number of intact intestinal villi, which was rescued by gut microbiota treatment (Figs 3 A and EV2 A). The Alcian Blue periodic acid‐Schiff (AB‐PAS) and periodic acid‐Schiff (PAS) staining further showed that FMT thickened the mucus layer and increased the number of goblet cells obviously which injured by irradiation exposure (Figs 3 A and EV2 A) in male and female mice. In addition, the total amount of formed stool gathered from the cage of the saline‐treated group was much lower than that of the FMT group (Figs 3 B and C, and EV2 B and C). Moreover, FMT decreased the radiation‐heightened FITC–dextran level in peripheral blood (Figs 3 D and EV2 D), suggesting that FMT improves GI tract function and epithelial integrity in irradiated animals. Using quantitative polymerase chain reaction (qPCR), we further validated the finding that the expression of Muc2 , Glut1 ( Slc2a1 ), Pgk1 , intestinal trefoil factor ( TFF3/ITF1 ) and multidrug resistance protein 1 ( MDR1 ) (Figs 3 E–I and EV2 E–I), which all participate in epithelial integrity maintaining after toxic stimuli, reached about threefold higher levels in the small intestine tissues of both irradiated male and female mice after FMT. Together, our observations demonstrate that gavage of gut microbiota improves GI tract function and epithelial integrity after irradiation.

To better understand the effect of the gut flora on the haematopoietic system, we checked the size of the spleens from each group. The size of the spleens was reduced after irradiation, but restored in the female + TBI, male + TBI and female/male + TBI treatment groups (Figs 2 F and G, and EV1 C and D). Moreover, these treatment regimens also elevated WBC counts slightly in the peripheral blood (Fig EV1 E and G) without affecting blood haemoglobin levels (Fig EV1 F and H). Because TBI ablates all marrow and FMT only mitigates TBI‐induced haematopoietic toxicity slightly, we further performed FMT combined with bone marrow transplant (BMT; Fig 2 H). After 8 Gy irradiation exposure, the survival rate of mice treated with FMT combined with BMT was much higher than that of mice treated FMT or BMT alone (Figs 2 I and EV1 I), suggesting that FMT combined with BMT might significantly mitigate irradiation‐induced toxicity.

The Shannon diversity index and β‐diversity of intestinal bacteria between male and female mice without irradiation were assessed by 16S high‐throughput sequencing, n = 4. Statistically significant differences are indicated: Student's t ‐test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

Divergent factors, such as genetic predisposition, diet and inflammation states, can differently affect enteric bacterial flora (Smith et al , 2015 ; Schaubeck et al , 2016 ). Therefore, we compared the diversity of gut bacterial composition between male and female C57BL/6 mice using 16S rRNA sequencing before irradiation. Interestingly, we found that the bacteria taxonomic proportions in stool of male and female mice were different (Fig 2 A). Compared with female mice, male animals harboured a higher frequency of Bacteroides but a lower frequency of Prevotella at the genus level (Fig 2 B and Appendix Fig S1E ). Accordingly, we performed sex‐matched or mismatched FMT according to the following scheme: female faecal microbiota transplants to male or female mice (female + TBI); male faecal microbiota transplants to male or female mice (male + TBI); and a mixture of faecal microbiota from male and female transplants to male or female mice (female/male + TBI, the weight of male and female stool was 1:1); saline was used as a control (saline + TBI). The C56BL/6 mice were separated into their respective cohorts and gavage with faecal microbiota or saline after TBI, as depicted in Fig 2 C. Notably, the faecal microbiota treatments overtly increased the survival rate and body weight of male and female mice after 6.5 Gy TBI (Figs 2 D and E, and EV1 A and B). In particular, the optimal results were obtained when the gender of donors matched with that of recipients, indicating that FMT is an effective therapy against radiation‐induced death in a mouse model and its efficiency is determined by the gender matched between the donor and recipient. We also repeated the experiments using older donors, but the three kinds of FMT all failed to increase the survival rate of irradiated male and female mice (data not shown).

The Shannon diversity index and β‐diversity of intestinal bacteria in male (C) and female (D) mice were examined by 16S high‐throughput sequencing after 6 weeks of antibiotics treatment, n = 4 per group. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

The observed species number and Shannon diversity index of intestinal bacteria in male (A) and female (B) mice were examined by 16S high‐throughput sequencing after irradiation at days 5 and 10, n = 4 per group. Statistically significant differences are indicated: Wilcoxon rank sum test. The top and bottom boundaries of each box indicate the 75 th and 25 th quartile values, respectively, and lines within each box represent the 50 th quartile (median) values. Ends of whiskers mark the lowest and highest diversity values in each instance.

To determine the relationship between the intestinal bacterial pattern and radiosusceptibility, we used 16S rRNA sequencing to analyse the enteric bacterial profile in the mice faeces after irradiation exposure on days 5 and 10. As shown in Fig 1 A and B, 6.5 Gy gamma ray altered the diversity and composition of enteric bacteria in male but not in female C57BL/6 mice. Antibiotic treatment has been reported to be capable of shifting the intestinal bacterial communities in mice (Theriot et al , 2014 ; Brown et al , 2016 ). Accordingly, mice were administered drinking water containing ampicillin or streptomycin for 6 weeks, and then, we performed 16S rRNA sequencing analysis and obtained that enteric bacterial composition of antibiotic‐fed mice (Abt mice) was quite different from that of controls (drinking water without antibiotics; Fig 1 C and D); however, the gut microbiota of control mice did not change overtly during the 6 weeks ( Appendix Fig S1A and B ). After 6.5 Gy total body irradiation (TBI), our observations revealed that the survival rate of Abt mice was significantly higher than that of controls, indicating that changes in the intestinal bacterial communities are able to influence the radiosensitivity of both male and female mice (Fig 1 E and F). A decrease in peripheral white blood cell (WBC) counts has been observed in irradiated animals (Deng et al , 2015 ). Although we observed that ampicillin and streptomycin conferred protection against sub‐lethal irradiation, these antibiotics did not heighten peripheral WBC counts ( Appendix Fig S1C and D ), indicating that antibiotics could not ameliorate radiation‐induced haematopoietic syndrome.

Discussion

The gastrointestinal tract is inhabited by a dense population of organized and highly specialized microbial flora that collectively modulates host immunity and metabolism. Compositional and functional changes in commensal microbiota are thought to be involved in the pathogenesis of many diseases (Petrof & Khoruts, 2014; Vanhoecke et al, 2015). Recently, epidemiological and clinical studies on symbiotic microbiota have experienced a renaissance (Vasconcelos et al, 2016). Understanding how the enteric microbiota affects health and disease requires a paradigm shift from focusing on individual pathogens to an ecological approach that considers the community as a whole (Lozupone et al, 2012). These intestinal microbes boost biofilm formation by facilitating microbial co‐aggregation and the production of biosurfactants and bacteriocins, which selectively kill other microorganisms to maintain microbiota stability, enhance gut barrier function through interacting with epithelia and modulate the host immune function (Borody & Khoruts, 2012). It is thus possible that pharmacological modification of the intestinal microbiome can be a therapeutic strategy for the treatment of many human diseases (Owyang & Wu, 2014). For example, targeted restoration of the intestinal bacteria is beneficial in treating recalcitrant or recurring C. difficile infection (Lawley et al, 2012). Strategically, FMT is the most direct and radical way to alter the composition of a human's enteric microbiota to improve the quality of life in patients with inflammatory bowel disease (Moayyedi et al, 2015; Wei et al, 2015), non‐alcoholic fatty liver disease (Le Roy et al, 2013), metabolic syndrome (Vrieze et al, 2012) and neuropsychiatric disorders (Xu et al, 2015). Thus far however, whether FMT could be used as a therapeutic method to ameliorate radiation‐induced toxicity remains unknown. Given the changes in the microbiota vary with time, we performed FMT to treat irradiated mice for 10 days and observed that FMT preserved the radiation‐impaired enteric bacterial composition during these 10 days. We observed that multiple FMT doses increased the survival rate of irradiated mice suggesting that FMT administration may provide protection against radiation‐induced toxicity. Therefore, our findings support the hypothesis that FMT might emerge as a potential therapeutic option for radiation‐induced death.

Given that multiple factors, such as age and genetics (Lozupone et al, 2012), can drive gut microbiota alteration, the use of FMT should follow guidelines. Clinical studies noted that donors with any gastrointestinal complaints, metabolic syndrome, autoimmune diseases or allergic diseases should be excluded (Borody & Khoruts, 2012). Here, our high‐throughput sequencing showed a difference in the intestinal bacterial composition between male and female C57BL/6 mice. Thus, we achieved sex‐matched and mismatched FMT and discovered that the optimal therapeutic efficacy of FMT was obtained when the sex of donors matched recipients. Although sex‐matched, sex‐mismatched and sex‐mixed FMT shaped the gut bacterial composition of irradiated mice efficiently, the frequency of specific bacteria changed by the three kinds of FMT was different, suggesting that the colonization of key bacterial strains might contribute to the efficient of FMT‐mitigated radiation‐induced toxicity. Together, our observations bolster the position that establishing formal guidelines for FMT is imperative to developing therapies or clinical trials.

Given that the clinical components of acute radiation sickness include intricate haematopoietic syndrome and intractable GI syndrome (Waselenko et al, 2004), we examined the role of intestinal microbes on mitigating radiation‐induced bone marrow toxicity and GI toxicity. The reduction in the number and/or size of the splenic germinal centre and memory CD4+ T cells in germ‐free mice (Cerf‐Bensussan & Gaboriau‐Routhiau, 2010) suggested an important role of gut microbiota in the development of the peripheral immune system. Likewise, we found that FMT was able to restore the size of spleens in irradiated mice, indicating that FMT might similarly strengthen the immune system of radiation‐exposed mice. Owing to gut microbiota treatment elevated WBC counts in the peripheral blood slightly, we further preformed FMT combined with BMT to remedy TBI‐induced toxicity. Notably, comparing with FMT or BMT alone, administration of FMT combined with BMT overtly increased the survival rate of mice after 8 Gy irradiation exposure, suggesting that bone marrow toxicity might be the main limiting factor for therapeutic effect of FMT. On the basis of our investigations, we concluded that FMT improved GI tract function and intestinal epithelial integrity after irradiation, suggesting that FMT could be a potential therapy for radiation‐induced GI toxicity. In agreement with the role of angiogenesis in radioprotection (Okunieff et al, 1998), we found that FMT was capable of up‐regulating Vegf level in the small intestine of irradiated mice. Because VEGF also facilitates the development of tumorigenesis (Carmeliet & Jain, 2011), we determined whether enteric microbiota transplant could accelerate the growth of cancer cells following subcutaneous implantation of either lung or colon cancer cells into C57BL/6 mice. Our data showed that FMT failed to enhance the proliferation of the carcinoma cells. Therefore, as a potential therapeutic approach to mitigate radiation‐induced toxicity, FMT might be used in tumour radiotherapy to improve the prognosis.

At the molecular level, microarray analysis revealed a marked difference in the mRNA expression profile in small intestine tissues between FMT‐treated and saline‐treated mice. Our observation showed that the gavage of sex‐matched faecal microbiota had distinguishable effects on immunity and metabolism (Marcobal et al, 2013; Bromberg et al, 2015), suggesting that different sex‐specific gut microbiota performs divergent physiologic status. Given that lncRNAs, which have been primarily studied in the context of genomic imprinting, cancer, and cell differentiation, are now emerging as important regulators of immunity and metabolism (Cui et al, 2015a,b; Yao et al, 2016), we analysed the related lncRNA expression pattern in parallel. As expected, FMT caused an alteration in the spectrum of lncRNA expression, indicating that gut microbe transplant not only preserves intestinal bacterial composition in hosts but also retains the mRNA and lncRNA expression profile. LncRNAs are able to interact with chromatin at several thousand different locations across multiple chromosomes and govern large‐scale gene expression programmes (Guttman & Rinn, 2012; Vance & Ponting, 2014), and they have been regarded as pivotal modulators of physiologic or pathologic status (Ponting et al, 2009). They also regulate gene expression profiles at the post‐transcriptional level through influencing the stability of mRNA (Gong & Maquat, 2011; Kretz et al, 2013). Our findings now suggest that the gut microbe transplant‐mediated lncRNA expression profile fluctuations might play important roles in mitigating radiation‐induced injury, which warrants further study.

In conclusion, our work demonstrates that gavage of gut microbes alleviates and protects against radiation‐induced injury in a mouse model. Specifically, FMT is able to heighten the survival rate of irradiated mice. Moreover, FMT improves GI tract function and epithelial integrity to ameliorate irradiation‐induced GI toxicity. Mechanistically, intestinal microbiota transplant preserves the bacterial communities and retains mRNA as well as the lncRNA expression profile in the hosts. Thus, our findings provide new insights into the function of FMT. Clinically, our observations underpin the suggestion that FMT might be employed as a novel therapeutic method for irradiation‐induced injury to improve the prognosis of patients after radiotherapy.