www.nature.com/ismecomms ARTICLE OPEN Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation 1,2 1 1,3 1,4 1 2 1 Charlotte Segers ✉, Mohamed Mysara , Jürgen Claesen , Sarah Baatout , Natalie Leys , Sarah Lebeer , Mieke Verslegers and Felice Mastroleo 1 © The Author(s) 2021 Pelvic radiotherapy is known to evoke intestinal mucositis and dysbiosis. Currently, there are no effective therapies available to mitigate these injuries, which is partly due to a lack of insight into the events causing mucositis and dysbiosis. Here, the complex interplay between the murine host and its microbiome following pelvic irradiation was mapped by characterizing intestinal mucositis along with extensive 16S microbial profiling. We demonstrated important morphological and inflammatory implications within one day after exposure, thereby impairing intestinal functionality and inducing translocation of intraluminal bacteria into mesenteric lymph nodes as innovatively quantified by flow cytometry. Concurrent 16S microbial profiling revealed a delayed impact of pelvic irradiation on beta diversity. Analysis of composition of microbiomes identified biomarkers for pelvic irradiation. Among them, members of the families Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae were differentially affected. Altogether, our unprecedented findings showed how pelvic irradiation evoked structural and functional changes in the intestine, which secondarily resulted in a microbiome shift. Therefore, the presented in vivo irradiation-gut-microbiome platform allows further research into the pathobiology of pelvic irradiation-induced intestinal mucositis and resultant dysbiosis, as well as the exploration of mitigating treatments including drugs and food supplements. ISME Communications (2021) 1:24 ; https://doi.org/10.1038/s43705-021-00024-0 INTRODUCTION intestinal mucositis and dysbiosis, as well as the lack of an Gut dysbiosis is generally defined as the qualitatively and appropriate research model system. For instance, cell (co-)culture quantitatively altered composition of intestinal microbiota, which set-ups based on intestinal epithelial cells are largely incomplete offers an advantage for emergence and outbreak of pathogens to capture the complexity of the intestinal tissue, the gut and has a cascading impact on the immune system.1 In fact, microbiome and the underlying pathobiology of irradiation- dysbiosis is well known to develop following radiotherapy and induced enteropathy.7–10 Additionally, total body irradiation is was shown to be associated with the severity of irradiation- typically used as experimental modality, which however evokes a induced intestinal mucositis.2,3 In accordance, germ-free mice concomitant hematopoietic syndrome aggravating irradiation- were reported to be more resistant to lethal (16 Gy) total body induced mucositis.11 In this regard, the importance of the gut irradiation as compared to conventionally-housed specific microbiome in the response to local pelvic irradiation has not pathogen-free mice.4 However, whether dysbiosis represents a been addressed in sufficient detail.12–15 cause or a consequence of irradiation-induced intestinal mucositis In this study, we present novel insights in the structural and is not well understood. functional implications of intestinal mucositis and the consequent Currently, there are no effective therapies available to mitigate development of dysbiosis in a mouse model for pelvic irradiation irradiation-induced intestinal mucositis in human patients. There- by using an innovative multi-level approach. Within one day, fore, given their safe character, food supplements including pelvic irradiation initiated intestinal mucositis as shown by probiotics, prebiotics, and vitamins are being investigated for their histology and hallmarks of inflammation. Then, a functionally potential to provide radioprotection and re-establish an orthobio- disrupted barrier resulted in bacterial translocation investigated tic (i.e., balanced composition providing beneficial effects to the through the innovative use of flow cytometry. Consequently, host) gut microbiome.5 For example, the probiotic preparation extensive 16S microbial profiling revealed delayed changes in the VSL#3 reduced the severity and incidence of irradiation-induced gut microbiota composition. Our research thus presents an in vivo diarrhea in a Phase II clinical trial.6 The major limitation in the irradiation-gut-microbiome test platform to study the pathobiol- search for new food supplements is the lack of mechanistic ogy and new potential treatments for pelvic irradiation-induced understanding of the events underlying irradiation-induced intestinal mucositis and secondary gut microbial dysbiosis. 1Interdisciplinary Biosciences group, Belgian Nuclear Research Centre SCK CEN, Mol, Belgium. 2Department of Bioscience Engineering, University of Antwerp, Antwerp, Belgium. 3Department of Epidemiology and Data Science, Amsterdam UMC, VU University Amsterdam, Amsterdam, The Netherlands. 4Department of Biotechnology, University of Ghent, ✉ Ghent, Belgium. email: [email protected] Received: 25 February 2021 Revised: 12 May 2021 Accepted: 25 May 2021 C. Segers et al. 2 MATERIAL AND METHODS colon. They were collected in sterile, ice cold phosphate buffered Mice saline and mashed manually using a 10 mL syringe plunger All animals were housed at the specific pathogen free animal through a 40 µm nylon cell strainer (VWR, Belgium). Homogenized facility of SCK CEN in accordance with the Ethical Committee mesenteric lymph nodes were analyzed for total cell count by the Animal Studies of Medanex Clinic (EC MxCl 2018-093) and were innovative use of flow cytometry, as an alternative to bacterial approved by the SCK CEN animal welfare committee. All animal plate counting. This allowed for a more complete bacterial experiments were carried out in compliance with the Belgian counting by avoiding arbitrary growth selection based on (an-) laboratory animal legislation and the European Communities aerobic cultivation or medium used. Briefly, samples were diluted Council Directive of 22 September 2010 (2010/63/EU). (1:5 000; to obtain an event rate between 200 and 2 000 events/µl) Five weeks-old, male C57Bl/6JRj mice were purchased from in 0.2 µm filtered phosphate buffered saline, and additionally Janvier (Bio Services, The Netherlands) and housed individually in filtered using a 5 µm syringe filter to remove eukaryotic cells. ventilated cages under standard laboratory conditions (12‐hr Then, SYBR Green I dye (at a final concentration of 1:10 000; light/dark cycle). Upon arrival, mice were acclimatized for two Invitrogen, USA) was added and samples were incubated for 20 weeks. All animals had access to food and water ad libitum in the min at 37 °C. Stained bacterial suspensions were then processed formulation of DietGel® 76A (Bio Services, The Netherlands) on a BD Accuri C6 flow cytometer (BD Biosciences, Belgium) and supplemented with 10% maltodextrin (Applichem, Germany). detected in the FL-1 channel (533/30 nm) after excitation with the To minimize confounding factors across experimental cohorts, blue laser (488 nm, 20 mW). The device was calibrated according only male mice were used, housed in the same room and to manufacturer’s instructions. Eventually, all samples were controlled for aging effects by choosing age-matched mice.16 In analyzed using the BD Accuri C6 software (v.1.0.264.21). addition, potential other confounding factors including care taker (s), order of handling and differences in weight were investigated Fecal DNA extraction and 16S rRNA gene sequencing and excluded for its impact on the microbiome. Fecal samples were longitudinally collected before, as well as one day, three days and seven days after exposure, to characterize Irradiation protocol acute (at PID1) and delayed (at PID3 and PID7) effects. At all The effects of pelvic irradiation were studied by applying a sub- sampling points, seven mice were included in each group. Total lethal dose of 12 Gy as previously described.17,18 Eight weeks-old fecal DNA was extracted using the DNeasy PowerSoil Pro Kit mice were anesthetized by intraperitoneal injection of 50 mg/kg (Qiagen, The Netherlands), according to manufacturer’s instruc- 1234567890();,: ketamine hydrochloride (Nimatek, Eurovet Animal Health, The tions. Extracted DNA concentrations were quantified by the Netherlands) and 0.25 mg/kg medetomidine hydrochloride (Domi- QuantiFluor dsDNA system (Promega, the Netherlands). High- tor, Orion Corporation, Finland). To minimize distress caused by throughput amplicon sequencing of the V3-V4 hypervariable dried out eyes, a 2 mg/kg carbomerum gel (Vidisic, Bausch+Lomb, region was performed on BaseClear’s Illumina MiSeq (V3 Belgium) was applied. A maximum of seven mice were placed in a chemistry) platform according to manufacturer’s guidelines. custom-made disk-shaped Plexiglas box (20 cm diameter, 5 cm Herein, a positive control in the form of a mock DNA sample, height), with each individual animal in a prone position and their and negative controls in the form of blank extraction and blank lower body parts towards the center. The box was covered by a library samples were included as recommended.19 lead shield (5 mm thick) except for the center of the cover (9 cm diameter) allowing local, pelvic irradiation of the mice (Supple- Sequencing data processing and diversity analysis mentary Fig. 1A). During transportation to the SCK CEN irradiation Paired-end sequencing data were analyzed through